development and validation of a lc/ms/ms method...
TRANSCRIPT
ISSN 0973-4945 CODEN ECJHAO
E-Journal of Chemistry
httpwwwejchemnet 2012 9(2) 899-911
Development and Validation of a LCMSMS Method
for the Determination of Duloxetine in Human Plasma
and its Application to Pharmacokinetic Study
D CHANDRAPAL REDDY
1 2 A T BAPUJI
1 V SURYANARAYANA RAO
3 V
HIMABINDU 2 D RAMA RAJU
1 SYED SYEDBA
1 and H L V RAVIKIRAN
1
1Department of Clinical Pharmacology
APL-Research Centre Hyderabad 500090 AP India 2Institute of Science and Technology
JNT University Kukatpally Hyderabad 500085 AP India 3Department of Chemistry SKD University
Anantapur 515 055 AP India
dcpreddy007yahoocoin
Received 8 August 2011 Accepted 4 October 2011
Abstract A selective high sensitive and high throughput liquid
chromatography-tandem mass spectrometry (LC-ESI-MSMS) method has been
developed and validated for the chromatographic separation and quantitation of
duloxetine in human EDTA plasma using fluoxetine (IS) as an internal standard
Analyte and IS were extracted from human plasma by liquid-liquid extraction
using MTBE-n Hexane (8020)The eluted samples were chromatographed on
X-terra RP8 (50 mm46 mm 5 microm particle size) column by using mixture of
30 mM ammonium formate (pH-50005) and acetonitrile as an isocratic mobile
phase at a flow rate of 040 mLmin and analyzed by mass spectrometer in the
multiple reaction monitoring (MRM) using the respective mz 29808rarr1540 for
duloxetine and 31002rarr14807 for IS The linearity of the response
concentration curve was established in human plasma over the concentration
range 0100-100017 ngmL The lower detection limit (LODSNgt3) was 004
ngmL and the lower limit of quantization (LOQSNgt10) was 0100 ngmL
This LC-MSMS method was validated with Intra-batch and Inter-batch
precision of 521-702The Intra-batch and Inter-batch accuracy was 9714-
10350 respectively Recovery of duloxetine in human plasma is 8031 and
ISTD recovery is 8109 The main pharmacokinetic parameters were Tmax (hr)
= (7251581) Cmax (ngmL) (44594 0t = (984702526502)
and AUC0 (1027147
Keywords Duloxetine UPLC-MSMS Human plasma Extraction efficiency Bioequivalence study
and pharmacokinetic
D C REDDY et al 900
Introduction
Duloxetine HCl (DLX) (Figure 1) is chemically 2(+)-(S)-N-methyl-(gamma)-(1-naphthyloxy)-2
thiophenepropylamine hydrochloride1 Duloxetine hydrochloride is a newer selective
serotonin and norepinephrine reuptake inhibitor (SSNRI) used for major depressive
disorders2-3
The empirical formula is C18H19NOSHCl and having a molecular weight of
33388 It is used for the treatment of naturopathic pain associated with peripheral
neuropathy especially diabetic polyneuropathy for which it is first-line and as an add-on
treatment in stress urinary incontinence instead of surgery4-5
also indicated for the
management of fibromyalgia6-7
It restores the balance of neurotransmitters in the brain like
serotonin and norepinehrine8 Moreover it is also being used in the treatment of peripheral
neuropathy caused by certain anti cancer drugs9
Figure 1 Chemical structure for duloxetine HCl
In the references few bioanalytical methods are reported for the determination of
duloxetine in human plasma by LC-MSMS10
HPLC analysis of duloxetine in human
plasma with SPE11
capillary electrophories with laser-induced fluorescence detection12
in
blood using HPLC with spectrometric detection and column switching13
and LC-MS (SIM
mode)14
The following are the advantages of the proposed method over those reported earlier (1)
Greater sensitivity is achieved (0100 ngmL) even with low plasma volumes and method is
well suited for pharmacokinetic analysis (2) Employing a single-step liquid-liquid
extraction procedure minimizes the chances of errors saves considerable time and simplifies
the sample preparation procedure (3) Because of the use of less plasma volume (0300 mL)
the volume of the sample to be collected for time point from subjects during the study is
reduced significantly-this allows inclusion of additional points (4) The rapid sample
analysis turnaround time of 300 minutes makes it an attractive procedure in high-throughput
bioanalysis of duloxetine in human plasma The chromatographic conditions were optimized
and the results of validation in terms of Specificity linearity precision accuracy extraction
efficiency dilution integrity and Stabilities were provided The devised method was used in
duloxetine clinical study which was conducted in accord with USFDA guidelines Typical
bioavailability including AUC0t (the area under plasma concentration-time curve) and Cmax
(the maximum plasma concentration) AUC0 (Area under the concentration time-curves
from time zero to infinity) parameters were compared
Experimental
Reference standards of Duloxetine (Potency (ww 995) and Fluoxetine (999) were
procured from Aurobindo pharma Ltd (Hyderabad India) Methanol and Acetonitrile were
of HPLC Grade purchased from JT Baker (Philipsburg USA) Analytical-grade
Ammonium formate was purchased from sdfine chemicals (Mumbai India) tert-butyl
Development and Validation of a LCMSMS Method for the Determination 901
methyl ether and n-Hexane were of HPLC Grade purchased from Merck specialties (Mumbai
India) and Formic acid (AR Grade) was purchased from (RFCL Chemicals New Delhi
India) Polypropylene vials (Torsens products Pvt Ltd Kolkata India) Water used for the
LC-MSMS analysis was prepared using a Milli Q water purification system procured from
Millipore (Bangalore India) Human plasma was procured from Cauvery Diagnostics and
blood bank Hyderabad India)and was stored at -20degC until use
Liquid Chromatographic Conditions
A waters Acquity UPLC system (Milford MA USA) consisting of binary solvent manager
sample manager and column manager was used for setting the reverse-phase liquid
chromatographic conditions The separation of Duloxetine and Fluoxetine (IS) was
performed on X-terra RP8 (50mmtimes46mm (length inner diameter) with 5 microm particle size)
and was maintained at 35degC in column oven The mobile phase consists of 30mM
Ammonium formate (pH 500005) and acetonitrile in 1090 (vv) ratio For isocratic
elution the flow rate of the mobile phase was kept at 040 mLmin The total
chromatographic run time was 30 min The sample manager temperature was maintained at
10degC and the pressure of the system was 1200 psi
Mass Spectrometric Conditions
Ionization and detection of analyte and IS was carried out on a triple quadrupole mass
spectrometer WATERS Quattro Micro (Milford MA USA) equipped with electro spray
ionization and operating in positive ion mode Quantization was performed using multiple reaction
monitoring (MRM) mode to monitor Parent rarr Product ion (mz) transitions for Duloxetine
29808 rarr 1540 and 31002 rarr 14807 for IS respectively (Figure 2 shows Figure 2 (A) Product
ion mass spectra of Duloxetine (mz 29808rarr1540 scan range 80-320 amu) and Figure 2 (B)
Product ion mass spectra of Fluoxetine (mz 31012rarr14807 amu) scan range 50-350 amu)
Figure 2 (A) Product ion mass spectra of Duloxetine (mz 29808rarr1540 scan range
80-320 amu)
D C REDDY et al 902
Figure 2 (B) Product ion mass spectra of Fluoxetine (mz 31012rarr14807 amu) scan
range 50-350 amu
The source dependent parameters maintained for Duloxetine and Fluoxetine were
capillary 350 kV extractor 00V RF lens 02V source temperature 100degC desolvation
temperature 400degC cone gas flow 80plusmn10 Lh desolvation gas flow 800plusmn10 Lh The
optimum values for compound dependent parameters (MRM file parameters) like cone
voltage and collision energy set were 10 V and 60 eV for the analyte and 150 V and
180 eV for IS respectively The dwell time easy set at 500 ms Mass Lynx software version
41 was used to control all parameters of UPLC and MS
Standard Stock Calibration Standards and Quality Control Sample Preparation
The standard stock solution of 1 mgmL of duloxetine and fluoxetine was prepared by
dissolving requisite amount in methanol Calibration standards and quality control (QC)
samples were prepared by spiking (2 total volume of blank plasma) blank plasma with stock
solution Calibration curve standards were made at 0100 0200 0500 10002 and 25004
50009 80014 and 100017 ngmL respectively while quality control samples were prepared
at four levels viz 77752 ngmL (HQC high quality control) 42064 (MQC middle quality
control) 0300 ngmL (LQC low quality control) 0100 ngmL (LLOQQC lowest level quality
control) Stock solution (1 mgmL) of the internal standard was prepared by dissolving 10 mg
of Fluoxetine in 10 mL of methanol An aliquot of 500 microL of this solution was further diluted
to 100 mL in the same diluent to obtain solution of 5000 microgmL All the solutions (standard
stock calibration standards and quality control samples) were store at 2-8degC until use
Protocol for Sample Preparation
Prior to analysis all frozen subjects samples calibration standards and quality control samples
were thawed and allowed to equilibrate at room temperature To an aliquot of 300 microL of spiked
plasma sample 50 microL internal standard was added and vortexed for 20 s Further 100 microL of
50 mM Ammonium formate was added and vortexed 20 s To these samples 25 mL of
Development and Validation of a LCMSMS Method for the Determination 903
extraction solvent (MTBE n Hexane 2080 vv) was added and samples were extracted on
extractor at 32 times g for 10 10 min centrifugation of the samples was done at 3200 times g for
10 min at 10degC 20 mL supernant was separated and evaporated to dryness under nitrogen at
50degC plusmn 05degC for 15 psi and 15 min The dried samples were reconstituted with 300 microL of
mobile phase and 20 microL was injection in the chromatographic system
Method Validation
The method validation was performed as per USFDA guidelines15
System suitability
experiment was performed by injecting six consecutive injections using aqueous standard
mixture of Duloxetine and internal standard at the start of each batch during the method
validation The carryover effect of the auto sampler was evaluated by injecting a sequence of
injections solutions of aqueous standard Mobile phase standard blank extracted standard
equivalent to highest standard in the calibration range As per the acceptance criteria the
response in blank should not be greater than 20 of LLOQ response16
The linearity of the method was determined by analysis of five linear curves containing
eight non-zero concentration The ratio of area response for drug and IS was used for
regression analysis Each calibration curve was analyzed individually by using least square
weighed (1x2)
linear regression The lowest standard on the calibration curve was accepted
as the lower limit of quantitation (LLOQ) if the analyte response was at least five times
more than that of the drug free (blank) extracted plasma The deviation of than that of drug
free (blank) extracted plasma The deviation of standards other than LLOQ from nominal
concentration should not to be more than plusmn150
The selectivity of the method towards endogenous plasma matrix components was
assessed in twelve batches (7 normal of K2 EDTA plasma 2 haemolysed 2 lipidemic and
and 1 heparinised) of blank human plasma This was done to estimate the extent to which
endogenous plasma components contribute towards interference at the retention time of
analytes and IS The cross talk of MRM for analytes and IS was checked using highest
standard on calibration curve and working solution of IS
For determining the intra-day accuracy and precision replicate analysis of plasma
samples of Duloxetine was performed on the same day The run consisted of a calibration
curve and six replicates of LLOQ LQC MQC and HQC samples The inter-day accuracy
and precision were assessed by analysis of three precision and accuracy batches on three
consecutive validation days The precision of the method was determined by calculating the
percent coefficient of variation (CV) for each level The deviation at each concentration
level from the nominal concentration was expected to be within plusmn150 except LLOQ for
which it should be within plusmn200
The relative recovery matrix effect and process efficiency were assessed as
recommended by Matuszewski et al17
All three parameters were evaluated at Std-1 Std-3
Std-5 Std-6 and Std-8 levels in six replicates Relative recovery (RE) was calculated by
comparing the mean area response of extracted samples (spiked before extraction) to that of
unextracted samples (spiked after extraction) at each CC level The recovery of IS was
similarly estimated Absolute matrix effect (ME) was assessed by comparing the mean area
response of unextracted samples (spiked after extraction) with mean area of neat standard
solutions The overall lsquoprocess efficiencyrsquo (PE) was calculated by comparing the mean
area response of extracted samples (spiked before extraction) to that with mean area of neat
standard solutions at each CC level The assessment of relative matrix effect was based on
direct comparison of the MSMS responses (peak areas) of the analytes spiked into extracts
originating from different lots of plasma The variability in these responses expressed as
CV was considered as the measure of relative matrix effect
D C REDDY et al 904
Stability experiments were carried out to examine the analyte stability in stock solutions
and in plasma samples under different conditions Short term stability at room temperature
and long term stability of spiked solution stored at ndash 70ordmC was assessed by comparing the
area response of stability sample of analyte and IS with the area response of sample prepared
from fresh stock solutions The solutions were considered stable if the deviation from
nominal value was within plusmn10 Autosampler wet extract stability bench top stability dry
extract stability and freeze- thaw stability were performed at LQC and HQC using six
replicates at each level The samples were considered stable if the deviation from the mean
calculated concentration of freshly thawed quality control samples was within plusmn15
To authenticate the ruggedness of the proposed method it was done on two precision
and accuracy batches The first batch was analysed by different analysts while the second
batch was analysed on different column and different LC-MSMS Dilution integrity
experiment was conducted by diluting the stock solution prepared as spiked standard at
concentration of 200034 ngmL for Duloxetine The precision and accuracy for dilution
integrity standards at 15th
and 110th
determined by analyzing the samples against
calibration curve standards
Study Design
A pharmacokinetic study was conducted on 12 healthy adult male human subjects under
fating conditions (n = 12) following oral administration of 60 mg delayed release capsules
Each volunteer was judged to be in good health through medical history physical
examination and routine laboratory tests Written consent was taken from all the volunteers
after informing them about the objectives and possible risks involved in the study An
independent ethics committee constituted as per Indian council of Medical Research (ICMR)
approved the study protocol The study was conducted strictly in accordance with guidelines
laid down by international conference on Harmonization and USFDA18
A single oral dose
of 60 mg drug was given to the volunteers with 240 mL of water Blood samples were
collected at 00 (pre-dose) 100 200 300 400 450 500 550 600 650 700 750
800 1000 1200 1600 2000 2400 3600 4800 and 7200 h after oral administration of
the dose for test formulation in labeled K2 EDTA- vaccuettes Plasma was separated by
centrifugation(3200 times g 10ordmC 10 min) and kept frozen at ndash 70ordmC until analysis During
study volunteers had a standard diet while water intake was free
Results and Discussion
Method Development
Chromatographic resolution of Duloxetine and IS was initiated under isocratic conditions to
obtain adequate response sharp peak shape and a short analysis time Thus separation was
tried using various combinations of methanolacetonitrile acidic buffers and additives like
formic acid on different reversed-phase columns with 5microm particle size viz Chromolith
Hypersil X-terra Kromasil Intertsil and Grace ACE Cyano (150 mm and 250 mm times 46 mm)
Chromolith RP-18 (50 mm times 46 mm) Kromasil (50 mm and 100 mm times 46 mm) and
Gemini C-18 (50 mm times 46 mm) to find the optimal column that produced the best
sensitivity efficiency and peak shape The analytes showed poor separation and
reproducibility for proposed linear range except for x-terra RP-8 column that offered superior
peak shape baseline separation desired linearity and reproducibility The mobile phase
consisting of 30 mM ammonium formate adjusted the pH 500005 with formic acid and
methanol (3070 vv) ratio and having 30 mM ammonium formate pH around 50-55 were
found most suitable for eluting Duloxetine and IS at 150 and 148 min respectively Also
Development and Validation of a LCMSMS Method for the Determination 905
the reproducibility of retention times for the analytes expressed as CV was le2 for 100
injections on the same column
The inherent selectivity of MSMS detection was also expected to be beneficial in
developing a selective and sensitive method The present study was conducted using ESI as
the ionization source as it gave high intensity for drug and IS as they have similar sites for
protonation Initially the extraction of Duloxetine and IS was carried out via protein
precipitation with common solvents like acetonitrile methanol and acetone but the
sensitivity and reproducibility were poor in all the solvents with frequent clogging of the
column which required online flushing of the column Liquid-liquid extraction technique
was also tested to isolate the drugs from plasma using diethyl ether dichromethane ethyl
acetate methyl tert butyl ether and isopropyl alcohol (alone and in combination) as
extracting solvents However the recovery was inconsistent with some ion suppression
(greater than 15 CV) in most of these solvent systems Further use of 100 microL of 50mM
ammonium formate to extraction in methyl tert butyl ether n Hexane (8020 vv) gave
consistent recoveries for the analytes especially at the LLOQ level with minimum matrix
interference A general internal standard was used to minimize any analytical variation due
to solvent evaporation integrity of the column and ionization efficiency of analytes
Fluoxetine was used as an internal standard (IS) in the present study which had similar
chromatographic behavior and was quantitatively extracted with the proposed extraction
procedure Also there was no effect of IS on analyte recovery sensitivity or ion
suppression
System Suitability and Auto Sampler Carryover
Throughout the method validation the CV of system suitability was observed below 40
at the retention time of duloxetine and the IS Carryover evaluation was performed in each
analytical run so as to ensure that it does not affect the accuracy and the precision of the
proposed method There was negligible carryover (le4 of the LLOQ response) observed
during auto sampler carryover experiment No enhancement in the response was observed in
double blank after subsequent injection of highest calibration standard (aqueous and
extracted) at the retention time of analytes and IS
Linearity and Lower Limit of Quantification (LLOQ)
The calibration curves were linear over the concentration range of 0100100017 ngmL
with correlation coefficient r2 ge 09963 for duloxetine respectively The equations for means
(n=5) of five calibration curves for duloxetine The standard deviation value for slope
intercept observed were 09921 09952 09951 09997 09996 and 000008 000007
00005 00006 00005 and 00005 for duloxetine respectively The accuracy and precision
(CV) observed for the calibration curve standards ranged from 9114 to 10459 and 082
to 1154 respectively The lowest concentration (LLOQ) in the standard curve for both the
isomers was measured at a signal-to-noise ratio (SN) of ge 100 Figure 3 shows the
representative LC-MSMS chromatograms of (A) calibration curve of duloxetine (B)
Double blank plasma with out IS (C) Blank plasma with IS and (D) Duloxetine and
fluoxetine at LLOQ
D C REDDY et al 906
Figure 3 (a) Calibration curve of Duloxetine
Figure 3 (b) Double blank plasma (without IS)
Figure 3 (c) Blank plasma with IS
Res
ponse
Res
idual
Development and Validation of a LCMSMS Method for the Determination 907
Figure 3 (d) Duloxetine and Fluoxetine at LLOQ (mz) 298081540
Selectivity Accuracy and Precision
To establish the selectivity of the method for interference due to endogenous plasma
components from haemolysed lipidemic heparinised and K2 EDTA blank plasmas the
change in the area ratio (analyteIS) at LLOQ level was within 4-8 while the precision
(CV) in their measurement varied from 21 to 56 representative MRM ion
chromatograms extracted (A) blank human plasma (double blank) (B) blank plasma
fortified with IS (mz 31002rarr14807) duloxetine at LLOQ (mz 29808rarr1540) the
selectivity of the method The extraction procedure together with mass detection gave very
good selectivity for the analysis of both the drug and IS in the blank plasma No endogenous
interferences were found at the retention times of analytes and IS
The intra- and inter batch precision and accuracy were established from validation runs
performed at HQC MQC LQC and LLOQ QC levels The intra- and inter batch precision
ranged from 074 to 976 for duloxetineThe accuracy values were within 9714-10449
for both the analytes in intra- and inter batches The precision and accuracy values for intra-
and inter day experiments in plasma are shown in Table 1(A)
Table 1 (A) Comparison of intra- and inter-batch precision and accuracy for Duloxetine
QC ID Nominal
concentration ngmL
Intrabatch
n
Mean
concentration
observed ngmL
CV Accuracy
LLOQQC
LQC
MQC
HQC
0100
0300
42060
77750
6
0104
0307
41255
81240
770
556
379
503
10350
10229
9808
10449
QC ID Nominal
concentration ngmL
Intrabatch
n
Mean
concentration
observed ngmL
CV Accuracy
LLOQQC
LQC
MQC
HQC
0100
0300
42060
77750
24
0103
0308
40863
79684
685
702
521
552
10325
10259
9714
10248
D C REDDY et al 908
(B) Stability of Duloxetine under various conditions (n=6)
Storage conditions Nominal
Concentration ngmL
Mean calculated
conc (ngmL) plusmn SD
Mean
accuracy
Bench top stability
(After 567 h at ~ at 25degC
LQC
HQC
0300
77750
0298 plusmn 00090
75382 plusmn 26005
9928
9695
Freeze thaw stability
(3 Cycles)
LQC
HQC
0300
77750
0298 plusmn 00060
79717 plusmn 49832
9939
10253
Dry extract stability
(2582 h below 10degC)
LQC
HQC
0300
77750
0299 plusmn 00087
83442 plusmn 52259
9967
10732
Wet extract stability
(2643 h below 10degC)
LQC
HQC
0300
77750
0309 plusmn 00136
76943 plusmn 34201
10294
9896
Auto sampler stability
(2188 h 10degC)
LQC
HQC
0300
77750
0297 plusmn 00112
73886 plusmn 43496
9889
9500
Long term stability in plasma
at -70degC
(2366 days at -70degC)
LQC
HQC
0300
77750
0298 plusmn 00060
71398 plusmn 54829
9939
9183
Recovery and Stability Results
The relative recovery absolute matrix effect and process efficiency data at LQC MQC and
HQC levels is presented The recovery for drug and IS in human plasma was 8031 and
8109 Further the extent of matrix effect in different lots of plasma (spiked after
extraction) was within the acceptable limit as evident from the precision (CV) values in
Table 2
Stock solutions for short term stability of Duloxetine and IS were stable at room
temperature for minimum period of about 6 h and between 2 and 8degC for about 7 days
Duloxetine in control human plasma (bench top) at room temperature was stable at least for
567 h at ambient temperature and for minimum of three freeze and thaw cycles Auto
sampler stability of the spiked quality control samples maintained at 10degC was maintained
up to 2188 h Long-term stability of the spiked quality control samples stored at -70degC was
determined up to 2366 days The accuracy values for different stability experiment in
plasma are shown in Table 1(B)
Development and Validation of a LCMSMS Method for the Determination 909
Table 2 Absolute matrix effect relative recovery and process efficiency for Mirtazapine
Analyte
ISTD
Aa
(CV)
Bb
(CV)
Cc
(CV)
Absolute
matrix
effect
(ME) d
Relative
recovery
(RE) e
Process
efficiency
(PE) f
STD 1
Mirtazapine
Zolpidem
179
(831)
42068
(049)
188
(391)
37907
(135)
109
(432)
40711
(132)
9423
9041
8540
8434
8047
7625
STD 3
Mirtazapine
Zolpidem
567
(323)
52367
(131)
591
(413)
54113
(156)
460
(122)
40951
(180)
10438
10333
7777
7568
8118
7620
STD 5
Mirtazapine
Zolpidem
23157
(174)
50420
(194)
22868
(169)
49366
(165)
18300
(096)
41307
(068)
9875
9791
8002
8367
7902
8193
STD 6
Mirtazapine
Zolpidem
43730
(109)
47700
(153)
43102
(124)
47385
(156)
34517
(101)
38045
(107)
9857
9934
8008
8029
7893
7976
STD 8
Mirtazapine
Zolpidem
77701
(146)
42800
(161)
78734
(194)
43777
(223)
63689
(080)
38217
(533)
10133
10228
8089
8730
8197
8929
a Mean area response of six replicate samples prepared in Mobile phase (neat samples) b Mean area
response of six replicate samples prepared by spiking in post extracted blank c Mean area response of
six replicate samples prepared by spiking in plasma before extraction d Matrix effect Post extracted
mean responseAqueous (Neat) mean response x 100 e Recovery Extracted mean response Post
extracted mean response x 100 f Process efficiency Extracted mean response Aqueous Mean
response x 100
Ruggedness and Dilution Integrity
The results of ruggedness study for Duloxetine was well within the acceptance limit of 15
in Precision and 850-1150 in mean accuracy The precision and accuracy values for
both experiment at LLOQ LQC MQC and HQC levels for Duloxetine ranged from 34 to
76 and 971 to 1085 respectively
The dilution integrity experiment was performed with an aim to validate the dilution test
to be carried out on higher analyte concentration above the upper limit of quantification
(ULOQ) which maybe encountered during real subject sample analysis The precision and
accuracy values for 15th
and 110th
dilution ranged from 326 to 334 and 982 to 1097 for
Duloxetine
Application of the Method in Healthy Human Subjects
The validated method was successfully applied for the assay of Duloxetine in healthy male
Indian volunteers in the age group of 18-45 years Figure 4 shows the plasma concentration
vs time profile of Duloxetine human subjects under fating condition The method was
sensitive enough to monitor the Duloxetine plasma concentration up to 72 h Approximately
520 samples including the calibration and QC samples were within the acceptable limits
D C REDDY et al 910
The pharmacokinetic parameters viz Cmax Tmax AUC0t and AUC0 were
calculated for Duloxetine in test formulations (Figure 4) Shows the data of Mean plasma
concentration-time profile of Duloxetine Hydrochloride 60 mg Delayed Release Capsules
formulation to 12 healthy volunteers
0
5
10
15
20
25
30
35
40
45
0 6 12 18 24 30 36 42 48 54 60 66 72
Time (hr)
Linear mean plot of plasma Duloxetine Concentration Vs Time under Fasting conditions
Parameter Duloxetine
Cmax ngmL 4459418599
Tmax (h) 7251581
AUC0t 984702526502
AUC0 1027147572790
Figure 4 Mean plasma concentration-time profile of Duloxetine Hydrochloride 60 mg
Delayed Release Capsules formulation to 8 healthy volunteers
Conclusion
The proposed method successfully demonstrates chromatographic separation of duloxetine
from human plasma with high resolution The bioanalytical methodology for their
simultaneous determination is highly specific rugged and rapid for therapeutic drug
monitoring The method involved a simple and specific sample preparation by liquid-liquid
extraction followed by isocratic separation in 30 min The overall analysis time is proving
compared to other reported procedures for duloxetine
Acknowledgment
The authors gratefully acknowledge Dr AT Bapuji and Aurobindo Pharma Ltd Hyderabad
India for providing necessary facilities for carrying out this study
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Development and Validation of a LCMSMS Method for the Determination 911
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Evaluation and Research (CDER) and Centre for Veterinary Medicine (CVM) May
2001
16 Viswanathan C T Bansal S Booth B DeStefano A J Rose M J Sailstad J Shah V R
Skelly J P Swann P G and Weiner R APPS J 2007 9 E30
17 Matuszewski B K J Chromatogr B Analyt Technol Biomed Life Sci 2006 830(2)
293-300 Epub 2005 Nov 23
18 FDA Guidance for industry Bioavailability Studies for Orally Administered Drug-
Products-General Considerations US Department of Health and Human Services
Food and Drug Administration Center for drug evaluation and Research (CDER)
2000
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D C REDDY et al 900
Introduction
Duloxetine HCl (DLX) (Figure 1) is chemically 2(+)-(S)-N-methyl-(gamma)-(1-naphthyloxy)-2
thiophenepropylamine hydrochloride1 Duloxetine hydrochloride is a newer selective
serotonin and norepinephrine reuptake inhibitor (SSNRI) used for major depressive
disorders2-3
The empirical formula is C18H19NOSHCl and having a molecular weight of
33388 It is used for the treatment of naturopathic pain associated with peripheral
neuropathy especially diabetic polyneuropathy for which it is first-line and as an add-on
treatment in stress urinary incontinence instead of surgery4-5
also indicated for the
management of fibromyalgia6-7
It restores the balance of neurotransmitters in the brain like
serotonin and norepinehrine8 Moreover it is also being used in the treatment of peripheral
neuropathy caused by certain anti cancer drugs9
Figure 1 Chemical structure for duloxetine HCl
In the references few bioanalytical methods are reported for the determination of
duloxetine in human plasma by LC-MSMS10
HPLC analysis of duloxetine in human
plasma with SPE11
capillary electrophories with laser-induced fluorescence detection12
in
blood using HPLC with spectrometric detection and column switching13
and LC-MS (SIM
mode)14
The following are the advantages of the proposed method over those reported earlier (1)
Greater sensitivity is achieved (0100 ngmL) even with low plasma volumes and method is
well suited for pharmacokinetic analysis (2) Employing a single-step liquid-liquid
extraction procedure minimizes the chances of errors saves considerable time and simplifies
the sample preparation procedure (3) Because of the use of less plasma volume (0300 mL)
the volume of the sample to be collected for time point from subjects during the study is
reduced significantly-this allows inclusion of additional points (4) The rapid sample
analysis turnaround time of 300 minutes makes it an attractive procedure in high-throughput
bioanalysis of duloxetine in human plasma The chromatographic conditions were optimized
and the results of validation in terms of Specificity linearity precision accuracy extraction
efficiency dilution integrity and Stabilities were provided The devised method was used in
duloxetine clinical study which was conducted in accord with USFDA guidelines Typical
bioavailability including AUC0t (the area under plasma concentration-time curve) and Cmax
(the maximum plasma concentration) AUC0 (Area under the concentration time-curves
from time zero to infinity) parameters were compared
Experimental
Reference standards of Duloxetine (Potency (ww 995) and Fluoxetine (999) were
procured from Aurobindo pharma Ltd (Hyderabad India) Methanol and Acetonitrile were
of HPLC Grade purchased from JT Baker (Philipsburg USA) Analytical-grade
Ammonium formate was purchased from sdfine chemicals (Mumbai India) tert-butyl
Development and Validation of a LCMSMS Method for the Determination 901
methyl ether and n-Hexane were of HPLC Grade purchased from Merck specialties (Mumbai
India) and Formic acid (AR Grade) was purchased from (RFCL Chemicals New Delhi
India) Polypropylene vials (Torsens products Pvt Ltd Kolkata India) Water used for the
LC-MSMS analysis was prepared using a Milli Q water purification system procured from
Millipore (Bangalore India) Human plasma was procured from Cauvery Diagnostics and
blood bank Hyderabad India)and was stored at -20degC until use
Liquid Chromatographic Conditions
A waters Acquity UPLC system (Milford MA USA) consisting of binary solvent manager
sample manager and column manager was used for setting the reverse-phase liquid
chromatographic conditions The separation of Duloxetine and Fluoxetine (IS) was
performed on X-terra RP8 (50mmtimes46mm (length inner diameter) with 5 microm particle size)
and was maintained at 35degC in column oven The mobile phase consists of 30mM
Ammonium formate (pH 500005) and acetonitrile in 1090 (vv) ratio For isocratic
elution the flow rate of the mobile phase was kept at 040 mLmin The total
chromatographic run time was 30 min The sample manager temperature was maintained at
10degC and the pressure of the system was 1200 psi
Mass Spectrometric Conditions
Ionization and detection of analyte and IS was carried out on a triple quadrupole mass
spectrometer WATERS Quattro Micro (Milford MA USA) equipped with electro spray
ionization and operating in positive ion mode Quantization was performed using multiple reaction
monitoring (MRM) mode to monitor Parent rarr Product ion (mz) transitions for Duloxetine
29808 rarr 1540 and 31002 rarr 14807 for IS respectively (Figure 2 shows Figure 2 (A) Product
ion mass spectra of Duloxetine (mz 29808rarr1540 scan range 80-320 amu) and Figure 2 (B)
Product ion mass spectra of Fluoxetine (mz 31012rarr14807 amu) scan range 50-350 amu)
Figure 2 (A) Product ion mass spectra of Duloxetine (mz 29808rarr1540 scan range
80-320 amu)
D C REDDY et al 902
Figure 2 (B) Product ion mass spectra of Fluoxetine (mz 31012rarr14807 amu) scan
range 50-350 amu
The source dependent parameters maintained for Duloxetine and Fluoxetine were
capillary 350 kV extractor 00V RF lens 02V source temperature 100degC desolvation
temperature 400degC cone gas flow 80plusmn10 Lh desolvation gas flow 800plusmn10 Lh The
optimum values for compound dependent parameters (MRM file parameters) like cone
voltage and collision energy set were 10 V and 60 eV for the analyte and 150 V and
180 eV for IS respectively The dwell time easy set at 500 ms Mass Lynx software version
41 was used to control all parameters of UPLC and MS
Standard Stock Calibration Standards and Quality Control Sample Preparation
The standard stock solution of 1 mgmL of duloxetine and fluoxetine was prepared by
dissolving requisite amount in methanol Calibration standards and quality control (QC)
samples were prepared by spiking (2 total volume of blank plasma) blank plasma with stock
solution Calibration curve standards were made at 0100 0200 0500 10002 and 25004
50009 80014 and 100017 ngmL respectively while quality control samples were prepared
at four levels viz 77752 ngmL (HQC high quality control) 42064 (MQC middle quality
control) 0300 ngmL (LQC low quality control) 0100 ngmL (LLOQQC lowest level quality
control) Stock solution (1 mgmL) of the internal standard was prepared by dissolving 10 mg
of Fluoxetine in 10 mL of methanol An aliquot of 500 microL of this solution was further diluted
to 100 mL in the same diluent to obtain solution of 5000 microgmL All the solutions (standard
stock calibration standards and quality control samples) were store at 2-8degC until use
Protocol for Sample Preparation
Prior to analysis all frozen subjects samples calibration standards and quality control samples
were thawed and allowed to equilibrate at room temperature To an aliquot of 300 microL of spiked
plasma sample 50 microL internal standard was added and vortexed for 20 s Further 100 microL of
50 mM Ammonium formate was added and vortexed 20 s To these samples 25 mL of
Development and Validation of a LCMSMS Method for the Determination 903
extraction solvent (MTBE n Hexane 2080 vv) was added and samples were extracted on
extractor at 32 times g for 10 10 min centrifugation of the samples was done at 3200 times g for
10 min at 10degC 20 mL supernant was separated and evaporated to dryness under nitrogen at
50degC plusmn 05degC for 15 psi and 15 min The dried samples were reconstituted with 300 microL of
mobile phase and 20 microL was injection in the chromatographic system
Method Validation
The method validation was performed as per USFDA guidelines15
System suitability
experiment was performed by injecting six consecutive injections using aqueous standard
mixture of Duloxetine and internal standard at the start of each batch during the method
validation The carryover effect of the auto sampler was evaluated by injecting a sequence of
injections solutions of aqueous standard Mobile phase standard blank extracted standard
equivalent to highest standard in the calibration range As per the acceptance criteria the
response in blank should not be greater than 20 of LLOQ response16
The linearity of the method was determined by analysis of five linear curves containing
eight non-zero concentration The ratio of area response for drug and IS was used for
regression analysis Each calibration curve was analyzed individually by using least square
weighed (1x2)
linear regression The lowest standard on the calibration curve was accepted
as the lower limit of quantitation (LLOQ) if the analyte response was at least five times
more than that of the drug free (blank) extracted plasma The deviation of than that of drug
free (blank) extracted plasma The deviation of standards other than LLOQ from nominal
concentration should not to be more than plusmn150
The selectivity of the method towards endogenous plasma matrix components was
assessed in twelve batches (7 normal of K2 EDTA plasma 2 haemolysed 2 lipidemic and
and 1 heparinised) of blank human plasma This was done to estimate the extent to which
endogenous plasma components contribute towards interference at the retention time of
analytes and IS The cross talk of MRM for analytes and IS was checked using highest
standard on calibration curve and working solution of IS
For determining the intra-day accuracy and precision replicate analysis of plasma
samples of Duloxetine was performed on the same day The run consisted of a calibration
curve and six replicates of LLOQ LQC MQC and HQC samples The inter-day accuracy
and precision were assessed by analysis of three precision and accuracy batches on three
consecutive validation days The precision of the method was determined by calculating the
percent coefficient of variation (CV) for each level The deviation at each concentration
level from the nominal concentration was expected to be within plusmn150 except LLOQ for
which it should be within plusmn200
The relative recovery matrix effect and process efficiency were assessed as
recommended by Matuszewski et al17
All three parameters were evaluated at Std-1 Std-3
Std-5 Std-6 and Std-8 levels in six replicates Relative recovery (RE) was calculated by
comparing the mean area response of extracted samples (spiked before extraction) to that of
unextracted samples (spiked after extraction) at each CC level The recovery of IS was
similarly estimated Absolute matrix effect (ME) was assessed by comparing the mean area
response of unextracted samples (spiked after extraction) with mean area of neat standard
solutions The overall lsquoprocess efficiencyrsquo (PE) was calculated by comparing the mean
area response of extracted samples (spiked before extraction) to that with mean area of neat
standard solutions at each CC level The assessment of relative matrix effect was based on
direct comparison of the MSMS responses (peak areas) of the analytes spiked into extracts
originating from different lots of plasma The variability in these responses expressed as
CV was considered as the measure of relative matrix effect
D C REDDY et al 904
Stability experiments were carried out to examine the analyte stability in stock solutions
and in plasma samples under different conditions Short term stability at room temperature
and long term stability of spiked solution stored at ndash 70ordmC was assessed by comparing the
area response of stability sample of analyte and IS with the area response of sample prepared
from fresh stock solutions The solutions were considered stable if the deviation from
nominal value was within plusmn10 Autosampler wet extract stability bench top stability dry
extract stability and freeze- thaw stability were performed at LQC and HQC using six
replicates at each level The samples were considered stable if the deviation from the mean
calculated concentration of freshly thawed quality control samples was within plusmn15
To authenticate the ruggedness of the proposed method it was done on two precision
and accuracy batches The first batch was analysed by different analysts while the second
batch was analysed on different column and different LC-MSMS Dilution integrity
experiment was conducted by diluting the stock solution prepared as spiked standard at
concentration of 200034 ngmL for Duloxetine The precision and accuracy for dilution
integrity standards at 15th
and 110th
determined by analyzing the samples against
calibration curve standards
Study Design
A pharmacokinetic study was conducted on 12 healthy adult male human subjects under
fating conditions (n = 12) following oral administration of 60 mg delayed release capsules
Each volunteer was judged to be in good health through medical history physical
examination and routine laboratory tests Written consent was taken from all the volunteers
after informing them about the objectives and possible risks involved in the study An
independent ethics committee constituted as per Indian council of Medical Research (ICMR)
approved the study protocol The study was conducted strictly in accordance with guidelines
laid down by international conference on Harmonization and USFDA18
A single oral dose
of 60 mg drug was given to the volunteers with 240 mL of water Blood samples were
collected at 00 (pre-dose) 100 200 300 400 450 500 550 600 650 700 750
800 1000 1200 1600 2000 2400 3600 4800 and 7200 h after oral administration of
the dose for test formulation in labeled K2 EDTA- vaccuettes Plasma was separated by
centrifugation(3200 times g 10ordmC 10 min) and kept frozen at ndash 70ordmC until analysis During
study volunteers had a standard diet while water intake was free
Results and Discussion
Method Development
Chromatographic resolution of Duloxetine and IS was initiated under isocratic conditions to
obtain adequate response sharp peak shape and a short analysis time Thus separation was
tried using various combinations of methanolacetonitrile acidic buffers and additives like
formic acid on different reversed-phase columns with 5microm particle size viz Chromolith
Hypersil X-terra Kromasil Intertsil and Grace ACE Cyano (150 mm and 250 mm times 46 mm)
Chromolith RP-18 (50 mm times 46 mm) Kromasil (50 mm and 100 mm times 46 mm) and
Gemini C-18 (50 mm times 46 mm) to find the optimal column that produced the best
sensitivity efficiency and peak shape The analytes showed poor separation and
reproducibility for proposed linear range except for x-terra RP-8 column that offered superior
peak shape baseline separation desired linearity and reproducibility The mobile phase
consisting of 30 mM ammonium formate adjusted the pH 500005 with formic acid and
methanol (3070 vv) ratio and having 30 mM ammonium formate pH around 50-55 were
found most suitable for eluting Duloxetine and IS at 150 and 148 min respectively Also
Development and Validation of a LCMSMS Method for the Determination 905
the reproducibility of retention times for the analytes expressed as CV was le2 for 100
injections on the same column
The inherent selectivity of MSMS detection was also expected to be beneficial in
developing a selective and sensitive method The present study was conducted using ESI as
the ionization source as it gave high intensity for drug and IS as they have similar sites for
protonation Initially the extraction of Duloxetine and IS was carried out via protein
precipitation with common solvents like acetonitrile methanol and acetone but the
sensitivity and reproducibility were poor in all the solvents with frequent clogging of the
column which required online flushing of the column Liquid-liquid extraction technique
was also tested to isolate the drugs from plasma using diethyl ether dichromethane ethyl
acetate methyl tert butyl ether and isopropyl alcohol (alone and in combination) as
extracting solvents However the recovery was inconsistent with some ion suppression
(greater than 15 CV) in most of these solvent systems Further use of 100 microL of 50mM
ammonium formate to extraction in methyl tert butyl ether n Hexane (8020 vv) gave
consistent recoveries for the analytes especially at the LLOQ level with minimum matrix
interference A general internal standard was used to minimize any analytical variation due
to solvent evaporation integrity of the column and ionization efficiency of analytes
Fluoxetine was used as an internal standard (IS) in the present study which had similar
chromatographic behavior and was quantitatively extracted with the proposed extraction
procedure Also there was no effect of IS on analyte recovery sensitivity or ion
suppression
System Suitability and Auto Sampler Carryover
Throughout the method validation the CV of system suitability was observed below 40
at the retention time of duloxetine and the IS Carryover evaluation was performed in each
analytical run so as to ensure that it does not affect the accuracy and the precision of the
proposed method There was negligible carryover (le4 of the LLOQ response) observed
during auto sampler carryover experiment No enhancement in the response was observed in
double blank after subsequent injection of highest calibration standard (aqueous and
extracted) at the retention time of analytes and IS
Linearity and Lower Limit of Quantification (LLOQ)
The calibration curves were linear over the concentration range of 0100100017 ngmL
with correlation coefficient r2 ge 09963 for duloxetine respectively The equations for means
(n=5) of five calibration curves for duloxetine The standard deviation value for slope
intercept observed were 09921 09952 09951 09997 09996 and 000008 000007
00005 00006 00005 and 00005 for duloxetine respectively The accuracy and precision
(CV) observed for the calibration curve standards ranged from 9114 to 10459 and 082
to 1154 respectively The lowest concentration (LLOQ) in the standard curve for both the
isomers was measured at a signal-to-noise ratio (SN) of ge 100 Figure 3 shows the
representative LC-MSMS chromatograms of (A) calibration curve of duloxetine (B)
Double blank plasma with out IS (C) Blank plasma with IS and (D) Duloxetine and
fluoxetine at LLOQ
D C REDDY et al 906
Figure 3 (a) Calibration curve of Duloxetine
Figure 3 (b) Double blank plasma (without IS)
Figure 3 (c) Blank plasma with IS
Res
ponse
Res
idual
Development and Validation of a LCMSMS Method for the Determination 907
Figure 3 (d) Duloxetine and Fluoxetine at LLOQ (mz) 298081540
Selectivity Accuracy and Precision
To establish the selectivity of the method for interference due to endogenous plasma
components from haemolysed lipidemic heparinised and K2 EDTA blank plasmas the
change in the area ratio (analyteIS) at LLOQ level was within 4-8 while the precision
(CV) in their measurement varied from 21 to 56 representative MRM ion
chromatograms extracted (A) blank human plasma (double blank) (B) blank plasma
fortified with IS (mz 31002rarr14807) duloxetine at LLOQ (mz 29808rarr1540) the
selectivity of the method The extraction procedure together with mass detection gave very
good selectivity for the analysis of both the drug and IS in the blank plasma No endogenous
interferences were found at the retention times of analytes and IS
The intra- and inter batch precision and accuracy were established from validation runs
performed at HQC MQC LQC and LLOQ QC levels The intra- and inter batch precision
ranged from 074 to 976 for duloxetineThe accuracy values were within 9714-10449
for both the analytes in intra- and inter batches The precision and accuracy values for intra-
and inter day experiments in plasma are shown in Table 1(A)
Table 1 (A) Comparison of intra- and inter-batch precision and accuracy for Duloxetine
QC ID Nominal
concentration ngmL
Intrabatch
n
Mean
concentration
observed ngmL
CV Accuracy
LLOQQC
LQC
MQC
HQC
0100
0300
42060
77750
6
0104
0307
41255
81240
770
556
379
503
10350
10229
9808
10449
QC ID Nominal
concentration ngmL
Intrabatch
n
Mean
concentration
observed ngmL
CV Accuracy
LLOQQC
LQC
MQC
HQC
0100
0300
42060
77750
24
0103
0308
40863
79684
685
702
521
552
10325
10259
9714
10248
D C REDDY et al 908
(B) Stability of Duloxetine under various conditions (n=6)
Storage conditions Nominal
Concentration ngmL
Mean calculated
conc (ngmL) plusmn SD
Mean
accuracy
Bench top stability
(After 567 h at ~ at 25degC
LQC
HQC
0300
77750
0298 plusmn 00090
75382 plusmn 26005
9928
9695
Freeze thaw stability
(3 Cycles)
LQC
HQC
0300
77750
0298 plusmn 00060
79717 plusmn 49832
9939
10253
Dry extract stability
(2582 h below 10degC)
LQC
HQC
0300
77750
0299 plusmn 00087
83442 plusmn 52259
9967
10732
Wet extract stability
(2643 h below 10degC)
LQC
HQC
0300
77750
0309 plusmn 00136
76943 plusmn 34201
10294
9896
Auto sampler stability
(2188 h 10degC)
LQC
HQC
0300
77750
0297 plusmn 00112
73886 plusmn 43496
9889
9500
Long term stability in plasma
at -70degC
(2366 days at -70degC)
LQC
HQC
0300
77750
0298 plusmn 00060
71398 plusmn 54829
9939
9183
Recovery and Stability Results
The relative recovery absolute matrix effect and process efficiency data at LQC MQC and
HQC levels is presented The recovery for drug and IS in human plasma was 8031 and
8109 Further the extent of matrix effect in different lots of plasma (spiked after
extraction) was within the acceptable limit as evident from the precision (CV) values in
Table 2
Stock solutions for short term stability of Duloxetine and IS were stable at room
temperature for minimum period of about 6 h and between 2 and 8degC for about 7 days
Duloxetine in control human plasma (bench top) at room temperature was stable at least for
567 h at ambient temperature and for minimum of three freeze and thaw cycles Auto
sampler stability of the spiked quality control samples maintained at 10degC was maintained
up to 2188 h Long-term stability of the spiked quality control samples stored at -70degC was
determined up to 2366 days The accuracy values for different stability experiment in
plasma are shown in Table 1(B)
Development and Validation of a LCMSMS Method for the Determination 909
Table 2 Absolute matrix effect relative recovery and process efficiency for Mirtazapine
Analyte
ISTD
Aa
(CV)
Bb
(CV)
Cc
(CV)
Absolute
matrix
effect
(ME) d
Relative
recovery
(RE) e
Process
efficiency
(PE) f
STD 1
Mirtazapine
Zolpidem
179
(831)
42068
(049)
188
(391)
37907
(135)
109
(432)
40711
(132)
9423
9041
8540
8434
8047
7625
STD 3
Mirtazapine
Zolpidem
567
(323)
52367
(131)
591
(413)
54113
(156)
460
(122)
40951
(180)
10438
10333
7777
7568
8118
7620
STD 5
Mirtazapine
Zolpidem
23157
(174)
50420
(194)
22868
(169)
49366
(165)
18300
(096)
41307
(068)
9875
9791
8002
8367
7902
8193
STD 6
Mirtazapine
Zolpidem
43730
(109)
47700
(153)
43102
(124)
47385
(156)
34517
(101)
38045
(107)
9857
9934
8008
8029
7893
7976
STD 8
Mirtazapine
Zolpidem
77701
(146)
42800
(161)
78734
(194)
43777
(223)
63689
(080)
38217
(533)
10133
10228
8089
8730
8197
8929
a Mean area response of six replicate samples prepared in Mobile phase (neat samples) b Mean area
response of six replicate samples prepared by spiking in post extracted blank c Mean area response of
six replicate samples prepared by spiking in plasma before extraction d Matrix effect Post extracted
mean responseAqueous (Neat) mean response x 100 e Recovery Extracted mean response Post
extracted mean response x 100 f Process efficiency Extracted mean response Aqueous Mean
response x 100
Ruggedness and Dilution Integrity
The results of ruggedness study for Duloxetine was well within the acceptance limit of 15
in Precision and 850-1150 in mean accuracy The precision and accuracy values for
both experiment at LLOQ LQC MQC and HQC levels for Duloxetine ranged from 34 to
76 and 971 to 1085 respectively
The dilution integrity experiment was performed with an aim to validate the dilution test
to be carried out on higher analyte concentration above the upper limit of quantification
(ULOQ) which maybe encountered during real subject sample analysis The precision and
accuracy values for 15th
and 110th
dilution ranged from 326 to 334 and 982 to 1097 for
Duloxetine
Application of the Method in Healthy Human Subjects
The validated method was successfully applied for the assay of Duloxetine in healthy male
Indian volunteers in the age group of 18-45 years Figure 4 shows the plasma concentration
vs time profile of Duloxetine human subjects under fating condition The method was
sensitive enough to monitor the Duloxetine plasma concentration up to 72 h Approximately
520 samples including the calibration and QC samples were within the acceptable limits
D C REDDY et al 910
The pharmacokinetic parameters viz Cmax Tmax AUC0t and AUC0 were
calculated for Duloxetine in test formulations (Figure 4) Shows the data of Mean plasma
concentration-time profile of Duloxetine Hydrochloride 60 mg Delayed Release Capsules
formulation to 12 healthy volunteers
0
5
10
15
20
25
30
35
40
45
0 6 12 18 24 30 36 42 48 54 60 66 72
Time (hr)
Linear mean plot of plasma Duloxetine Concentration Vs Time under Fasting conditions
Parameter Duloxetine
Cmax ngmL 4459418599
Tmax (h) 7251581
AUC0t 984702526502
AUC0 1027147572790
Figure 4 Mean plasma concentration-time profile of Duloxetine Hydrochloride 60 mg
Delayed Release Capsules formulation to 8 healthy volunteers
Conclusion
The proposed method successfully demonstrates chromatographic separation of duloxetine
from human plasma with high resolution The bioanalytical methodology for their
simultaneous determination is highly specific rugged and rapid for therapeutic drug
monitoring The method involved a simple and specific sample preparation by liquid-liquid
extraction followed by isocratic separation in 30 min The overall analysis time is proving
compared to other reported procedures for duloxetine
Acknowledgment
The authors gratefully acknowledge Dr AT Bapuji and Aurobindo Pharma Ltd Hyderabad
India for providing necessary facilities for carrying out this study
References
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Ed Whitehouse Station NJ Merck
and Co Inc 2006 p 3465
2 Mishra L Drugs today Vol 1 Delhi Lorina Publications 2006 p 489
3 Stephan A C Luc-Andre G Francois R V Peter R B Frank P B Melissa J J et al J
Neuropsychopharmacol 2003 28 1685ndash93
Mea
n c
once
ntr
atio
n
ngm
L
Time h
Development and Validation of a LCMSMS Method for the Determination 911
4 National Institute for Health and Clinical Excellence Clinical guideline 40 Urinary
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5 National Institute for Health and Clinical Excellence Clinical guideline 96
Neuropathic pain - pharmacological management London 2010
6 httpwwwdrugbankcadrugsDB00476
7 Wolfe F Smythe H A Yunus M B et al The American College of Rheumatology
Criteria for the Classification of Fibromyalgia Report of the Multicenter Criteri
Committee Arthritis Rheum 1990 33(2) 160ndash72 doi101002art1780330203
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8 Douglas R Dolnak Treating Patients for Comorbid Depression Anxiety Disorders
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9 httpwwwcancergovdictionaryCdrID=589399
10 Senthamil Selvan P Gowda K V Mandal U Sam Solomon W D Pal T K J
Chromatogr B Analyt Technol Biomed Life Sci 2007 858(1-2) 269-75 Epub 2007
Sep 14
11 Mercolini L Mandrioli R Cazzolla R Amore M and Raggi M A J Chromatogr B
Analyt Technol Biomed Life Sci 2007 856(1-2) 81-7 Epub 2007 Jun 2
12 Musenga A Amore M Mandrioli R Kenndler E de Martino L and Raggi M A J
Chromatogr B Analyt Technol Biomed Life Sci 2009 877(11-12) 1126-32 Epub
2009 Mar 3
13 Waldschmitt C Vogel F Maurer C and Hiemke C Ther Drug Monit 2007
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14 Ning Ma Bi Kui Zhang Huan De Li Ben Mei Chen Ping Xu Feng Wang Rong
Hua Zhu Sheng Feng Da Xiong Xiang and Yun Gui Zhu Clinica Chimica Acta
2007 380(1-2) 100-105
15 FDA Guidance for industry Bioanalytical Method Validation US Department of
and Health Human Services Food and Drug Administration Centre for Drug
Evaluation and Research (CDER) and Centre for Veterinary Medicine (CVM) May
2001
16 Viswanathan C T Bansal S Booth B DeStefano A J Rose M J Sailstad J Shah V R
Skelly J P Swann P G and Weiner R APPS J 2007 9 E30
17 Matuszewski B K J Chromatogr B Analyt Technol Biomed Life Sci 2006 830(2)
293-300 Epub 2005 Nov 23
18 FDA Guidance for industry Bioavailability Studies for Orally Administered Drug-
Products-General Considerations US Department of Health and Human Services
Food and Drug Administration Center for drug evaluation and Research (CDER)
2000
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Development and Validation of a LCMSMS Method for the Determination 901
methyl ether and n-Hexane were of HPLC Grade purchased from Merck specialties (Mumbai
India) and Formic acid (AR Grade) was purchased from (RFCL Chemicals New Delhi
India) Polypropylene vials (Torsens products Pvt Ltd Kolkata India) Water used for the
LC-MSMS analysis was prepared using a Milli Q water purification system procured from
Millipore (Bangalore India) Human plasma was procured from Cauvery Diagnostics and
blood bank Hyderabad India)and was stored at -20degC until use
Liquid Chromatographic Conditions
A waters Acquity UPLC system (Milford MA USA) consisting of binary solvent manager
sample manager and column manager was used for setting the reverse-phase liquid
chromatographic conditions The separation of Duloxetine and Fluoxetine (IS) was
performed on X-terra RP8 (50mmtimes46mm (length inner diameter) with 5 microm particle size)
and was maintained at 35degC in column oven The mobile phase consists of 30mM
Ammonium formate (pH 500005) and acetonitrile in 1090 (vv) ratio For isocratic
elution the flow rate of the mobile phase was kept at 040 mLmin The total
chromatographic run time was 30 min The sample manager temperature was maintained at
10degC and the pressure of the system was 1200 psi
Mass Spectrometric Conditions
Ionization and detection of analyte and IS was carried out on a triple quadrupole mass
spectrometer WATERS Quattro Micro (Milford MA USA) equipped with electro spray
ionization and operating in positive ion mode Quantization was performed using multiple reaction
monitoring (MRM) mode to monitor Parent rarr Product ion (mz) transitions for Duloxetine
29808 rarr 1540 and 31002 rarr 14807 for IS respectively (Figure 2 shows Figure 2 (A) Product
ion mass spectra of Duloxetine (mz 29808rarr1540 scan range 80-320 amu) and Figure 2 (B)
Product ion mass spectra of Fluoxetine (mz 31012rarr14807 amu) scan range 50-350 amu)
Figure 2 (A) Product ion mass spectra of Duloxetine (mz 29808rarr1540 scan range
80-320 amu)
D C REDDY et al 902
Figure 2 (B) Product ion mass spectra of Fluoxetine (mz 31012rarr14807 amu) scan
range 50-350 amu
The source dependent parameters maintained for Duloxetine and Fluoxetine were
capillary 350 kV extractor 00V RF lens 02V source temperature 100degC desolvation
temperature 400degC cone gas flow 80plusmn10 Lh desolvation gas flow 800plusmn10 Lh The
optimum values for compound dependent parameters (MRM file parameters) like cone
voltage and collision energy set were 10 V and 60 eV for the analyte and 150 V and
180 eV for IS respectively The dwell time easy set at 500 ms Mass Lynx software version
41 was used to control all parameters of UPLC and MS
Standard Stock Calibration Standards and Quality Control Sample Preparation
The standard stock solution of 1 mgmL of duloxetine and fluoxetine was prepared by
dissolving requisite amount in methanol Calibration standards and quality control (QC)
samples were prepared by spiking (2 total volume of blank plasma) blank plasma with stock
solution Calibration curve standards were made at 0100 0200 0500 10002 and 25004
50009 80014 and 100017 ngmL respectively while quality control samples were prepared
at four levels viz 77752 ngmL (HQC high quality control) 42064 (MQC middle quality
control) 0300 ngmL (LQC low quality control) 0100 ngmL (LLOQQC lowest level quality
control) Stock solution (1 mgmL) of the internal standard was prepared by dissolving 10 mg
of Fluoxetine in 10 mL of methanol An aliquot of 500 microL of this solution was further diluted
to 100 mL in the same diluent to obtain solution of 5000 microgmL All the solutions (standard
stock calibration standards and quality control samples) were store at 2-8degC until use
Protocol for Sample Preparation
Prior to analysis all frozen subjects samples calibration standards and quality control samples
were thawed and allowed to equilibrate at room temperature To an aliquot of 300 microL of spiked
plasma sample 50 microL internal standard was added and vortexed for 20 s Further 100 microL of
50 mM Ammonium formate was added and vortexed 20 s To these samples 25 mL of
Development and Validation of a LCMSMS Method for the Determination 903
extraction solvent (MTBE n Hexane 2080 vv) was added and samples were extracted on
extractor at 32 times g for 10 10 min centrifugation of the samples was done at 3200 times g for
10 min at 10degC 20 mL supernant was separated and evaporated to dryness under nitrogen at
50degC plusmn 05degC for 15 psi and 15 min The dried samples were reconstituted with 300 microL of
mobile phase and 20 microL was injection in the chromatographic system
Method Validation
The method validation was performed as per USFDA guidelines15
System suitability
experiment was performed by injecting six consecutive injections using aqueous standard
mixture of Duloxetine and internal standard at the start of each batch during the method
validation The carryover effect of the auto sampler was evaluated by injecting a sequence of
injections solutions of aqueous standard Mobile phase standard blank extracted standard
equivalent to highest standard in the calibration range As per the acceptance criteria the
response in blank should not be greater than 20 of LLOQ response16
The linearity of the method was determined by analysis of five linear curves containing
eight non-zero concentration The ratio of area response for drug and IS was used for
regression analysis Each calibration curve was analyzed individually by using least square
weighed (1x2)
linear regression The lowest standard on the calibration curve was accepted
as the lower limit of quantitation (LLOQ) if the analyte response was at least five times
more than that of the drug free (blank) extracted plasma The deviation of than that of drug
free (blank) extracted plasma The deviation of standards other than LLOQ from nominal
concentration should not to be more than plusmn150
The selectivity of the method towards endogenous plasma matrix components was
assessed in twelve batches (7 normal of K2 EDTA plasma 2 haemolysed 2 lipidemic and
and 1 heparinised) of blank human plasma This was done to estimate the extent to which
endogenous plasma components contribute towards interference at the retention time of
analytes and IS The cross talk of MRM for analytes and IS was checked using highest
standard on calibration curve and working solution of IS
For determining the intra-day accuracy and precision replicate analysis of plasma
samples of Duloxetine was performed on the same day The run consisted of a calibration
curve and six replicates of LLOQ LQC MQC and HQC samples The inter-day accuracy
and precision were assessed by analysis of three precision and accuracy batches on three
consecutive validation days The precision of the method was determined by calculating the
percent coefficient of variation (CV) for each level The deviation at each concentration
level from the nominal concentration was expected to be within plusmn150 except LLOQ for
which it should be within plusmn200
The relative recovery matrix effect and process efficiency were assessed as
recommended by Matuszewski et al17
All three parameters were evaluated at Std-1 Std-3
Std-5 Std-6 and Std-8 levels in six replicates Relative recovery (RE) was calculated by
comparing the mean area response of extracted samples (spiked before extraction) to that of
unextracted samples (spiked after extraction) at each CC level The recovery of IS was
similarly estimated Absolute matrix effect (ME) was assessed by comparing the mean area
response of unextracted samples (spiked after extraction) with mean area of neat standard
solutions The overall lsquoprocess efficiencyrsquo (PE) was calculated by comparing the mean
area response of extracted samples (spiked before extraction) to that with mean area of neat
standard solutions at each CC level The assessment of relative matrix effect was based on
direct comparison of the MSMS responses (peak areas) of the analytes spiked into extracts
originating from different lots of plasma The variability in these responses expressed as
CV was considered as the measure of relative matrix effect
D C REDDY et al 904
Stability experiments were carried out to examine the analyte stability in stock solutions
and in plasma samples under different conditions Short term stability at room temperature
and long term stability of spiked solution stored at ndash 70ordmC was assessed by comparing the
area response of stability sample of analyte and IS with the area response of sample prepared
from fresh stock solutions The solutions were considered stable if the deviation from
nominal value was within plusmn10 Autosampler wet extract stability bench top stability dry
extract stability and freeze- thaw stability were performed at LQC and HQC using six
replicates at each level The samples were considered stable if the deviation from the mean
calculated concentration of freshly thawed quality control samples was within plusmn15
To authenticate the ruggedness of the proposed method it was done on two precision
and accuracy batches The first batch was analysed by different analysts while the second
batch was analysed on different column and different LC-MSMS Dilution integrity
experiment was conducted by diluting the stock solution prepared as spiked standard at
concentration of 200034 ngmL for Duloxetine The precision and accuracy for dilution
integrity standards at 15th
and 110th
determined by analyzing the samples against
calibration curve standards
Study Design
A pharmacokinetic study was conducted on 12 healthy adult male human subjects under
fating conditions (n = 12) following oral administration of 60 mg delayed release capsules
Each volunteer was judged to be in good health through medical history physical
examination and routine laboratory tests Written consent was taken from all the volunteers
after informing them about the objectives and possible risks involved in the study An
independent ethics committee constituted as per Indian council of Medical Research (ICMR)
approved the study protocol The study was conducted strictly in accordance with guidelines
laid down by international conference on Harmonization and USFDA18
A single oral dose
of 60 mg drug was given to the volunteers with 240 mL of water Blood samples were
collected at 00 (pre-dose) 100 200 300 400 450 500 550 600 650 700 750
800 1000 1200 1600 2000 2400 3600 4800 and 7200 h after oral administration of
the dose for test formulation in labeled K2 EDTA- vaccuettes Plasma was separated by
centrifugation(3200 times g 10ordmC 10 min) and kept frozen at ndash 70ordmC until analysis During
study volunteers had a standard diet while water intake was free
Results and Discussion
Method Development
Chromatographic resolution of Duloxetine and IS was initiated under isocratic conditions to
obtain adequate response sharp peak shape and a short analysis time Thus separation was
tried using various combinations of methanolacetonitrile acidic buffers and additives like
formic acid on different reversed-phase columns with 5microm particle size viz Chromolith
Hypersil X-terra Kromasil Intertsil and Grace ACE Cyano (150 mm and 250 mm times 46 mm)
Chromolith RP-18 (50 mm times 46 mm) Kromasil (50 mm and 100 mm times 46 mm) and
Gemini C-18 (50 mm times 46 mm) to find the optimal column that produced the best
sensitivity efficiency and peak shape The analytes showed poor separation and
reproducibility for proposed linear range except for x-terra RP-8 column that offered superior
peak shape baseline separation desired linearity and reproducibility The mobile phase
consisting of 30 mM ammonium formate adjusted the pH 500005 with formic acid and
methanol (3070 vv) ratio and having 30 mM ammonium formate pH around 50-55 were
found most suitable for eluting Duloxetine and IS at 150 and 148 min respectively Also
Development and Validation of a LCMSMS Method for the Determination 905
the reproducibility of retention times for the analytes expressed as CV was le2 for 100
injections on the same column
The inherent selectivity of MSMS detection was also expected to be beneficial in
developing a selective and sensitive method The present study was conducted using ESI as
the ionization source as it gave high intensity for drug and IS as they have similar sites for
protonation Initially the extraction of Duloxetine and IS was carried out via protein
precipitation with common solvents like acetonitrile methanol and acetone but the
sensitivity and reproducibility were poor in all the solvents with frequent clogging of the
column which required online flushing of the column Liquid-liquid extraction technique
was also tested to isolate the drugs from plasma using diethyl ether dichromethane ethyl
acetate methyl tert butyl ether and isopropyl alcohol (alone and in combination) as
extracting solvents However the recovery was inconsistent with some ion suppression
(greater than 15 CV) in most of these solvent systems Further use of 100 microL of 50mM
ammonium formate to extraction in methyl tert butyl ether n Hexane (8020 vv) gave
consistent recoveries for the analytes especially at the LLOQ level with minimum matrix
interference A general internal standard was used to minimize any analytical variation due
to solvent evaporation integrity of the column and ionization efficiency of analytes
Fluoxetine was used as an internal standard (IS) in the present study which had similar
chromatographic behavior and was quantitatively extracted with the proposed extraction
procedure Also there was no effect of IS on analyte recovery sensitivity or ion
suppression
System Suitability and Auto Sampler Carryover
Throughout the method validation the CV of system suitability was observed below 40
at the retention time of duloxetine and the IS Carryover evaluation was performed in each
analytical run so as to ensure that it does not affect the accuracy and the precision of the
proposed method There was negligible carryover (le4 of the LLOQ response) observed
during auto sampler carryover experiment No enhancement in the response was observed in
double blank after subsequent injection of highest calibration standard (aqueous and
extracted) at the retention time of analytes and IS
Linearity and Lower Limit of Quantification (LLOQ)
The calibration curves were linear over the concentration range of 0100100017 ngmL
with correlation coefficient r2 ge 09963 for duloxetine respectively The equations for means
(n=5) of five calibration curves for duloxetine The standard deviation value for slope
intercept observed were 09921 09952 09951 09997 09996 and 000008 000007
00005 00006 00005 and 00005 for duloxetine respectively The accuracy and precision
(CV) observed for the calibration curve standards ranged from 9114 to 10459 and 082
to 1154 respectively The lowest concentration (LLOQ) in the standard curve for both the
isomers was measured at a signal-to-noise ratio (SN) of ge 100 Figure 3 shows the
representative LC-MSMS chromatograms of (A) calibration curve of duloxetine (B)
Double blank plasma with out IS (C) Blank plasma with IS and (D) Duloxetine and
fluoxetine at LLOQ
D C REDDY et al 906
Figure 3 (a) Calibration curve of Duloxetine
Figure 3 (b) Double blank plasma (without IS)
Figure 3 (c) Blank plasma with IS
Res
ponse
Res
idual
Development and Validation of a LCMSMS Method for the Determination 907
Figure 3 (d) Duloxetine and Fluoxetine at LLOQ (mz) 298081540
Selectivity Accuracy and Precision
To establish the selectivity of the method for interference due to endogenous plasma
components from haemolysed lipidemic heparinised and K2 EDTA blank plasmas the
change in the area ratio (analyteIS) at LLOQ level was within 4-8 while the precision
(CV) in their measurement varied from 21 to 56 representative MRM ion
chromatograms extracted (A) blank human plasma (double blank) (B) blank plasma
fortified with IS (mz 31002rarr14807) duloxetine at LLOQ (mz 29808rarr1540) the
selectivity of the method The extraction procedure together with mass detection gave very
good selectivity for the analysis of both the drug and IS in the blank plasma No endogenous
interferences were found at the retention times of analytes and IS
The intra- and inter batch precision and accuracy were established from validation runs
performed at HQC MQC LQC and LLOQ QC levels The intra- and inter batch precision
ranged from 074 to 976 for duloxetineThe accuracy values were within 9714-10449
for both the analytes in intra- and inter batches The precision and accuracy values for intra-
and inter day experiments in plasma are shown in Table 1(A)
Table 1 (A) Comparison of intra- and inter-batch precision and accuracy for Duloxetine
QC ID Nominal
concentration ngmL
Intrabatch
n
Mean
concentration
observed ngmL
CV Accuracy
LLOQQC
LQC
MQC
HQC
0100
0300
42060
77750
6
0104
0307
41255
81240
770
556
379
503
10350
10229
9808
10449
QC ID Nominal
concentration ngmL
Intrabatch
n
Mean
concentration
observed ngmL
CV Accuracy
LLOQQC
LQC
MQC
HQC
0100
0300
42060
77750
24
0103
0308
40863
79684
685
702
521
552
10325
10259
9714
10248
D C REDDY et al 908
(B) Stability of Duloxetine under various conditions (n=6)
Storage conditions Nominal
Concentration ngmL
Mean calculated
conc (ngmL) plusmn SD
Mean
accuracy
Bench top stability
(After 567 h at ~ at 25degC
LQC
HQC
0300
77750
0298 plusmn 00090
75382 plusmn 26005
9928
9695
Freeze thaw stability
(3 Cycles)
LQC
HQC
0300
77750
0298 plusmn 00060
79717 plusmn 49832
9939
10253
Dry extract stability
(2582 h below 10degC)
LQC
HQC
0300
77750
0299 plusmn 00087
83442 plusmn 52259
9967
10732
Wet extract stability
(2643 h below 10degC)
LQC
HQC
0300
77750
0309 plusmn 00136
76943 plusmn 34201
10294
9896
Auto sampler stability
(2188 h 10degC)
LQC
HQC
0300
77750
0297 plusmn 00112
73886 plusmn 43496
9889
9500
Long term stability in plasma
at -70degC
(2366 days at -70degC)
LQC
HQC
0300
77750
0298 plusmn 00060
71398 plusmn 54829
9939
9183
Recovery and Stability Results
The relative recovery absolute matrix effect and process efficiency data at LQC MQC and
HQC levels is presented The recovery for drug and IS in human plasma was 8031 and
8109 Further the extent of matrix effect in different lots of plasma (spiked after
extraction) was within the acceptable limit as evident from the precision (CV) values in
Table 2
Stock solutions for short term stability of Duloxetine and IS were stable at room
temperature for minimum period of about 6 h and between 2 and 8degC for about 7 days
Duloxetine in control human plasma (bench top) at room temperature was stable at least for
567 h at ambient temperature and for minimum of three freeze and thaw cycles Auto
sampler stability of the spiked quality control samples maintained at 10degC was maintained
up to 2188 h Long-term stability of the spiked quality control samples stored at -70degC was
determined up to 2366 days The accuracy values for different stability experiment in
plasma are shown in Table 1(B)
Development and Validation of a LCMSMS Method for the Determination 909
Table 2 Absolute matrix effect relative recovery and process efficiency for Mirtazapine
Analyte
ISTD
Aa
(CV)
Bb
(CV)
Cc
(CV)
Absolute
matrix
effect
(ME) d
Relative
recovery
(RE) e
Process
efficiency
(PE) f
STD 1
Mirtazapine
Zolpidem
179
(831)
42068
(049)
188
(391)
37907
(135)
109
(432)
40711
(132)
9423
9041
8540
8434
8047
7625
STD 3
Mirtazapine
Zolpidem
567
(323)
52367
(131)
591
(413)
54113
(156)
460
(122)
40951
(180)
10438
10333
7777
7568
8118
7620
STD 5
Mirtazapine
Zolpidem
23157
(174)
50420
(194)
22868
(169)
49366
(165)
18300
(096)
41307
(068)
9875
9791
8002
8367
7902
8193
STD 6
Mirtazapine
Zolpidem
43730
(109)
47700
(153)
43102
(124)
47385
(156)
34517
(101)
38045
(107)
9857
9934
8008
8029
7893
7976
STD 8
Mirtazapine
Zolpidem
77701
(146)
42800
(161)
78734
(194)
43777
(223)
63689
(080)
38217
(533)
10133
10228
8089
8730
8197
8929
a Mean area response of six replicate samples prepared in Mobile phase (neat samples) b Mean area
response of six replicate samples prepared by spiking in post extracted blank c Mean area response of
six replicate samples prepared by spiking in plasma before extraction d Matrix effect Post extracted
mean responseAqueous (Neat) mean response x 100 e Recovery Extracted mean response Post
extracted mean response x 100 f Process efficiency Extracted mean response Aqueous Mean
response x 100
Ruggedness and Dilution Integrity
The results of ruggedness study for Duloxetine was well within the acceptance limit of 15
in Precision and 850-1150 in mean accuracy The precision and accuracy values for
both experiment at LLOQ LQC MQC and HQC levels for Duloxetine ranged from 34 to
76 and 971 to 1085 respectively
The dilution integrity experiment was performed with an aim to validate the dilution test
to be carried out on higher analyte concentration above the upper limit of quantification
(ULOQ) which maybe encountered during real subject sample analysis The precision and
accuracy values for 15th
and 110th
dilution ranged from 326 to 334 and 982 to 1097 for
Duloxetine
Application of the Method in Healthy Human Subjects
The validated method was successfully applied for the assay of Duloxetine in healthy male
Indian volunteers in the age group of 18-45 years Figure 4 shows the plasma concentration
vs time profile of Duloxetine human subjects under fating condition The method was
sensitive enough to monitor the Duloxetine plasma concentration up to 72 h Approximately
520 samples including the calibration and QC samples were within the acceptable limits
D C REDDY et al 910
The pharmacokinetic parameters viz Cmax Tmax AUC0t and AUC0 were
calculated for Duloxetine in test formulations (Figure 4) Shows the data of Mean plasma
concentration-time profile of Duloxetine Hydrochloride 60 mg Delayed Release Capsules
formulation to 12 healthy volunteers
0
5
10
15
20
25
30
35
40
45
0 6 12 18 24 30 36 42 48 54 60 66 72
Time (hr)
Linear mean plot of plasma Duloxetine Concentration Vs Time under Fasting conditions
Parameter Duloxetine
Cmax ngmL 4459418599
Tmax (h) 7251581
AUC0t 984702526502
AUC0 1027147572790
Figure 4 Mean plasma concentration-time profile of Duloxetine Hydrochloride 60 mg
Delayed Release Capsules formulation to 8 healthy volunteers
Conclusion
The proposed method successfully demonstrates chromatographic separation of duloxetine
from human plasma with high resolution The bioanalytical methodology for their
simultaneous determination is highly specific rugged and rapid for therapeutic drug
monitoring The method involved a simple and specific sample preparation by liquid-liquid
extraction followed by isocratic separation in 30 min The overall analysis time is proving
compared to other reported procedures for duloxetine
Acknowledgment
The authors gratefully acknowledge Dr AT Bapuji and Aurobindo Pharma Ltd Hyderabad
India for providing necessary facilities for carrying out this study
References
1 Maryadele J O editor The Merck Index 14th
Ed Whitehouse Station NJ Merck
and Co Inc 2006 p 3465
2 Mishra L Drugs today Vol 1 Delhi Lorina Publications 2006 p 489
3 Stephan A C Luc-Andre G Francois R V Peter R B Frank P B Melissa J J et al J
Neuropsychopharmacol 2003 28 1685ndash93
Mea
n c
once
ntr
atio
n
ngm
L
Time h
Development and Validation of a LCMSMS Method for the Determination 911
4 National Institute for Health and Clinical Excellence Clinical guideline 40 Urinary
incontinence London 2006
5 National Institute for Health and Clinical Excellence Clinical guideline 96
Neuropathic pain - pharmacological management London 2010
6 httpwwwdrugbankcadrugsDB00476
7 Wolfe F Smythe H A Yunus M B et al The American College of Rheumatology
Criteria for the Classification of Fibromyalgia Report of the Multicenter Criteri
Committee Arthritis Rheum 1990 33(2) 160ndash72 doi101002art1780330203
PMID 2306288
8 Douglas R Dolnak Treating Patients for Comorbid Depression Anxiety Disorders
and Somatic Illnesses 2006 JAOA 106 8
9 httpwwwcancergovdictionaryCdrID=589399
10 Senthamil Selvan P Gowda K V Mandal U Sam Solomon W D Pal T K J
Chromatogr B Analyt Technol Biomed Life Sci 2007 858(1-2) 269-75 Epub 2007
Sep 14
11 Mercolini L Mandrioli R Cazzolla R Amore M and Raggi M A J Chromatogr B
Analyt Technol Biomed Life Sci 2007 856(1-2) 81-7 Epub 2007 Jun 2
12 Musenga A Amore M Mandrioli R Kenndler E de Martino L and Raggi M A J
Chromatogr B Analyt Technol Biomed Life Sci 2009 877(11-12) 1126-32 Epub
2009 Mar 3
13 Waldschmitt C Vogel F Maurer C and Hiemke C Ther Drug Monit 2007
29(6) 767-72
14 Ning Ma Bi Kui Zhang Huan De Li Ben Mei Chen Ping Xu Feng Wang Rong
Hua Zhu Sheng Feng Da Xiong Xiang and Yun Gui Zhu Clinica Chimica Acta
2007 380(1-2) 100-105
15 FDA Guidance for industry Bioanalytical Method Validation US Department of
and Health Human Services Food and Drug Administration Centre for Drug
Evaluation and Research (CDER) and Centre for Veterinary Medicine (CVM) May
2001
16 Viswanathan C T Bansal S Booth B DeStefano A J Rose M J Sailstad J Shah V R
Skelly J P Swann P G and Weiner R APPS J 2007 9 E30
17 Matuszewski B K J Chromatogr B Analyt Technol Biomed Life Sci 2006 830(2)
293-300 Epub 2005 Nov 23
18 FDA Guidance for industry Bioavailability Studies for Orally Administered Drug-
Products-General Considerations US Department of Health and Human Services
Food and Drug Administration Center for drug evaluation and Research (CDER)
2000
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
D C REDDY et al 902
Figure 2 (B) Product ion mass spectra of Fluoxetine (mz 31012rarr14807 amu) scan
range 50-350 amu
The source dependent parameters maintained for Duloxetine and Fluoxetine were
capillary 350 kV extractor 00V RF lens 02V source temperature 100degC desolvation
temperature 400degC cone gas flow 80plusmn10 Lh desolvation gas flow 800plusmn10 Lh The
optimum values for compound dependent parameters (MRM file parameters) like cone
voltage and collision energy set were 10 V and 60 eV for the analyte and 150 V and
180 eV for IS respectively The dwell time easy set at 500 ms Mass Lynx software version
41 was used to control all parameters of UPLC and MS
Standard Stock Calibration Standards and Quality Control Sample Preparation
The standard stock solution of 1 mgmL of duloxetine and fluoxetine was prepared by
dissolving requisite amount in methanol Calibration standards and quality control (QC)
samples were prepared by spiking (2 total volume of blank plasma) blank plasma with stock
solution Calibration curve standards were made at 0100 0200 0500 10002 and 25004
50009 80014 and 100017 ngmL respectively while quality control samples were prepared
at four levels viz 77752 ngmL (HQC high quality control) 42064 (MQC middle quality
control) 0300 ngmL (LQC low quality control) 0100 ngmL (LLOQQC lowest level quality
control) Stock solution (1 mgmL) of the internal standard was prepared by dissolving 10 mg
of Fluoxetine in 10 mL of methanol An aliquot of 500 microL of this solution was further diluted
to 100 mL in the same diluent to obtain solution of 5000 microgmL All the solutions (standard
stock calibration standards and quality control samples) were store at 2-8degC until use
Protocol for Sample Preparation
Prior to analysis all frozen subjects samples calibration standards and quality control samples
were thawed and allowed to equilibrate at room temperature To an aliquot of 300 microL of spiked
plasma sample 50 microL internal standard was added and vortexed for 20 s Further 100 microL of
50 mM Ammonium formate was added and vortexed 20 s To these samples 25 mL of
Development and Validation of a LCMSMS Method for the Determination 903
extraction solvent (MTBE n Hexane 2080 vv) was added and samples were extracted on
extractor at 32 times g for 10 10 min centrifugation of the samples was done at 3200 times g for
10 min at 10degC 20 mL supernant was separated and evaporated to dryness under nitrogen at
50degC plusmn 05degC for 15 psi and 15 min The dried samples were reconstituted with 300 microL of
mobile phase and 20 microL was injection in the chromatographic system
Method Validation
The method validation was performed as per USFDA guidelines15
System suitability
experiment was performed by injecting six consecutive injections using aqueous standard
mixture of Duloxetine and internal standard at the start of each batch during the method
validation The carryover effect of the auto sampler was evaluated by injecting a sequence of
injections solutions of aqueous standard Mobile phase standard blank extracted standard
equivalent to highest standard in the calibration range As per the acceptance criteria the
response in blank should not be greater than 20 of LLOQ response16
The linearity of the method was determined by analysis of five linear curves containing
eight non-zero concentration The ratio of area response for drug and IS was used for
regression analysis Each calibration curve was analyzed individually by using least square
weighed (1x2)
linear regression The lowest standard on the calibration curve was accepted
as the lower limit of quantitation (LLOQ) if the analyte response was at least five times
more than that of the drug free (blank) extracted plasma The deviation of than that of drug
free (blank) extracted plasma The deviation of standards other than LLOQ from nominal
concentration should not to be more than plusmn150
The selectivity of the method towards endogenous plasma matrix components was
assessed in twelve batches (7 normal of K2 EDTA plasma 2 haemolysed 2 lipidemic and
and 1 heparinised) of blank human plasma This was done to estimate the extent to which
endogenous plasma components contribute towards interference at the retention time of
analytes and IS The cross talk of MRM for analytes and IS was checked using highest
standard on calibration curve and working solution of IS
For determining the intra-day accuracy and precision replicate analysis of plasma
samples of Duloxetine was performed on the same day The run consisted of a calibration
curve and six replicates of LLOQ LQC MQC and HQC samples The inter-day accuracy
and precision were assessed by analysis of three precision and accuracy batches on three
consecutive validation days The precision of the method was determined by calculating the
percent coefficient of variation (CV) for each level The deviation at each concentration
level from the nominal concentration was expected to be within plusmn150 except LLOQ for
which it should be within plusmn200
The relative recovery matrix effect and process efficiency were assessed as
recommended by Matuszewski et al17
All three parameters were evaluated at Std-1 Std-3
Std-5 Std-6 and Std-8 levels in six replicates Relative recovery (RE) was calculated by
comparing the mean area response of extracted samples (spiked before extraction) to that of
unextracted samples (spiked after extraction) at each CC level The recovery of IS was
similarly estimated Absolute matrix effect (ME) was assessed by comparing the mean area
response of unextracted samples (spiked after extraction) with mean area of neat standard
solutions The overall lsquoprocess efficiencyrsquo (PE) was calculated by comparing the mean
area response of extracted samples (spiked before extraction) to that with mean area of neat
standard solutions at each CC level The assessment of relative matrix effect was based on
direct comparison of the MSMS responses (peak areas) of the analytes spiked into extracts
originating from different lots of plasma The variability in these responses expressed as
CV was considered as the measure of relative matrix effect
D C REDDY et al 904
Stability experiments were carried out to examine the analyte stability in stock solutions
and in plasma samples under different conditions Short term stability at room temperature
and long term stability of spiked solution stored at ndash 70ordmC was assessed by comparing the
area response of stability sample of analyte and IS with the area response of sample prepared
from fresh stock solutions The solutions were considered stable if the deviation from
nominal value was within plusmn10 Autosampler wet extract stability bench top stability dry
extract stability and freeze- thaw stability were performed at LQC and HQC using six
replicates at each level The samples were considered stable if the deviation from the mean
calculated concentration of freshly thawed quality control samples was within plusmn15
To authenticate the ruggedness of the proposed method it was done on two precision
and accuracy batches The first batch was analysed by different analysts while the second
batch was analysed on different column and different LC-MSMS Dilution integrity
experiment was conducted by diluting the stock solution prepared as spiked standard at
concentration of 200034 ngmL for Duloxetine The precision and accuracy for dilution
integrity standards at 15th
and 110th
determined by analyzing the samples against
calibration curve standards
Study Design
A pharmacokinetic study was conducted on 12 healthy adult male human subjects under
fating conditions (n = 12) following oral administration of 60 mg delayed release capsules
Each volunteer was judged to be in good health through medical history physical
examination and routine laboratory tests Written consent was taken from all the volunteers
after informing them about the objectives and possible risks involved in the study An
independent ethics committee constituted as per Indian council of Medical Research (ICMR)
approved the study protocol The study was conducted strictly in accordance with guidelines
laid down by international conference on Harmonization and USFDA18
A single oral dose
of 60 mg drug was given to the volunteers with 240 mL of water Blood samples were
collected at 00 (pre-dose) 100 200 300 400 450 500 550 600 650 700 750
800 1000 1200 1600 2000 2400 3600 4800 and 7200 h after oral administration of
the dose for test formulation in labeled K2 EDTA- vaccuettes Plasma was separated by
centrifugation(3200 times g 10ordmC 10 min) and kept frozen at ndash 70ordmC until analysis During
study volunteers had a standard diet while water intake was free
Results and Discussion
Method Development
Chromatographic resolution of Duloxetine and IS was initiated under isocratic conditions to
obtain adequate response sharp peak shape and a short analysis time Thus separation was
tried using various combinations of methanolacetonitrile acidic buffers and additives like
formic acid on different reversed-phase columns with 5microm particle size viz Chromolith
Hypersil X-terra Kromasil Intertsil and Grace ACE Cyano (150 mm and 250 mm times 46 mm)
Chromolith RP-18 (50 mm times 46 mm) Kromasil (50 mm and 100 mm times 46 mm) and
Gemini C-18 (50 mm times 46 mm) to find the optimal column that produced the best
sensitivity efficiency and peak shape The analytes showed poor separation and
reproducibility for proposed linear range except for x-terra RP-8 column that offered superior
peak shape baseline separation desired linearity and reproducibility The mobile phase
consisting of 30 mM ammonium formate adjusted the pH 500005 with formic acid and
methanol (3070 vv) ratio and having 30 mM ammonium formate pH around 50-55 were
found most suitable for eluting Duloxetine and IS at 150 and 148 min respectively Also
Development and Validation of a LCMSMS Method for the Determination 905
the reproducibility of retention times for the analytes expressed as CV was le2 for 100
injections on the same column
The inherent selectivity of MSMS detection was also expected to be beneficial in
developing a selective and sensitive method The present study was conducted using ESI as
the ionization source as it gave high intensity for drug and IS as they have similar sites for
protonation Initially the extraction of Duloxetine and IS was carried out via protein
precipitation with common solvents like acetonitrile methanol and acetone but the
sensitivity and reproducibility were poor in all the solvents with frequent clogging of the
column which required online flushing of the column Liquid-liquid extraction technique
was also tested to isolate the drugs from plasma using diethyl ether dichromethane ethyl
acetate methyl tert butyl ether and isopropyl alcohol (alone and in combination) as
extracting solvents However the recovery was inconsistent with some ion suppression
(greater than 15 CV) in most of these solvent systems Further use of 100 microL of 50mM
ammonium formate to extraction in methyl tert butyl ether n Hexane (8020 vv) gave
consistent recoveries for the analytes especially at the LLOQ level with minimum matrix
interference A general internal standard was used to minimize any analytical variation due
to solvent evaporation integrity of the column and ionization efficiency of analytes
Fluoxetine was used as an internal standard (IS) in the present study which had similar
chromatographic behavior and was quantitatively extracted with the proposed extraction
procedure Also there was no effect of IS on analyte recovery sensitivity or ion
suppression
System Suitability and Auto Sampler Carryover
Throughout the method validation the CV of system suitability was observed below 40
at the retention time of duloxetine and the IS Carryover evaluation was performed in each
analytical run so as to ensure that it does not affect the accuracy and the precision of the
proposed method There was negligible carryover (le4 of the LLOQ response) observed
during auto sampler carryover experiment No enhancement in the response was observed in
double blank after subsequent injection of highest calibration standard (aqueous and
extracted) at the retention time of analytes and IS
Linearity and Lower Limit of Quantification (LLOQ)
The calibration curves were linear over the concentration range of 0100100017 ngmL
with correlation coefficient r2 ge 09963 for duloxetine respectively The equations for means
(n=5) of five calibration curves for duloxetine The standard deviation value for slope
intercept observed were 09921 09952 09951 09997 09996 and 000008 000007
00005 00006 00005 and 00005 for duloxetine respectively The accuracy and precision
(CV) observed for the calibration curve standards ranged from 9114 to 10459 and 082
to 1154 respectively The lowest concentration (LLOQ) in the standard curve for both the
isomers was measured at a signal-to-noise ratio (SN) of ge 100 Figure 3 shows the
representative LC-MSMS chromatograms of (A) calibration curve of duloxetine (B)
Double blank plasma with out IS (C) Blank plasma with IS and (D) Duloxetine and
fluoxetine at LLOQ
D C REDDY et al 906
Figure 3 (a) Calibration curve of Duloxetine
Figure 3 (b) Double blank plasma (without IS)
Figure 3 (c) Blank plasma with IS
Res
ponse
Res
idual
Development and Validation of a LCMSMS Method for the Determination 907
Figure 3 (d) Duloxetine and Fluoxetine at LLOQ (mz) 298081540
Selectivity Accuracy and Precision
To establish the selectivity of the method for interference due to endogenous plasma
components from haemolysed lipidemic heparinised and K2 EDTA blank plasmas the
change in the area ratio (analyteIS) at LLOQ level was within 4-8 while the precision
(CV) in their measurement varied from 21 to 56 representative MRM ion
chromatograms extracted (A) blank human plasma (double blank) (B) blank plasma
fortified with IS (mz 31002rarr14807) duloxetine at LLOQ (mz 29808rarr1540) the
selectivity of the method The extraction procedure together with mass detection gave very
good selectivity for the analysis of both the drug and IS in the blank plasma No endogenous
interferences were found at the retention times of analytes and IS
The intra- and inter batch precision and accuracy were established from validation runs
performed at HQC MQC LQC and LLOQ QC levels The intra- and inter batch precision
ranged from 074 to 976 for duloxetineThe accuracy values were within 9714-10449
for both the analytes in intra- and inter batches The precision and accuracy values for intra-
and inter day experiments in plasma are shown in Table 1(A)
Table 1 (A) Comparison of intra- and inter-batch precision and accuracy for Duloxetine
QC ID Nominal
concentration ngmL
Intrabatch
n
Mean
concentration
observed ngmL
CV Accuracy
LLOQQC
LQC
MQC
HQC
0100
0300
42060
77750
6
0104
0307
41255
81240
770
556
379
503
10350
10229
9808
10449
QC ID Nominal
concentration ngmL
Intrabatch
n
Mean
concentration
observed ngmL
CV Accuracy
LLOQQC
LQC
MQC
HQC
0100
0300
42060
77750
24
0103
0308
40863
79684
685
702
521
552
10325
10259
9714
10248
D C REDDY et al 908
(B) Stability of Duloxetine under various conditions (n=6)
Storage conditions Nominal
Concentration ngmL
Mean calculated
conc (ngmL) plusmn SD
Mean
accuracy
Bench top stability
(After 567 h at ~ at 25degC
LQC
HQC
0300
77750
0298 plusmn 00090
75382 plusmn 26005
9928
9695
Freeze thaw stability
(3 Cycles)
LQC
HQC
0300
77750
0298 plusmn 00060
79717 plusmn 49832
9939
10253
Dry extract stability
(2582 h below 10degC)
LQC
HQC
0300
77750
0299 plusmn 00087
83442 plusmn 52259
9967
10732
Wet extract stability
(2643 h below 10degC)
LQC
HQC
0300
77750
0309 plusmn 00136
76943 plusmn 34201
10294
9896
Auto sampler stability
(2188 h 10degC)
LQC
HQC
0300
77750
0297 plusmn 00112
73886 plusmn 43496
9889
9500
Long term stability in plasma
at -70degC
(2366 days at -70degC)
LQC
HQC
0300
77750
0298 plusmn 00060
71398 plusmn 54829
9939
9183
Recovery and Stability Results
The relative recovery absolute matrix effect and process efficiency data at LQC MQC and
HQC levels is presented The recovery for drug and IS in human plasma was 8031 and
8109 Further the extent of matrix effect in different lots of plasma (spiked after
extraction) was within the acceptable limit as evident from the precision (CV) values in
Table 2
Stock solutions for short term stability of Duloxetine and IS were stable at room
temperature for minimum period of about 6 h and between 2 and 8degC for about 7 days
Duloxetine in control human plasma (bench top) at room temperature was stable at least for
567 h at ambient temperature and for minimum of three freeze and thaw cycles Auto
sampler stability of the spiked quality control samples maintained at 10degC was maintained
up to 2188 h Long-term stability of the spiked quality control samples stored at -70degC was
determined up to 2366 days The accuracy values for different stability experiment in
plasma are shown in Table 1(B)
Development and Validation of a LCMSMS Method for the Determination 909
Table 2 Absolute matrix effect relative recovery and process efficiency for Mirtazapine
Analyte
ISTD
Aa
(CV)
Bb
(CV)
Cc
(CV)
Absolute
matrix
effect
(ME) d
Relative
recovery
(RE) e
Process
efficiency
(PE) f
STD 1
Mirtazapine
Zolpidem
179
(831)
42068
(049)
188
(391)
37907
(135)
109
(432)
40711
(132)
9423
9041
8540
8434
8047
7625
STD 3
Mirtazapine
Zolpidem
567
(323)
52367
(131)
591
(413)
54113
(156)
460
(122)
40951
(180)
10438
10333
7777
7568
8118
7620
STD 5
Mirtazapine
Zolpidem
23157
(174)
50420
(194)
22868
(169)
49366
(165)
18300
(096)
41307
(068)
9875
9791
8002
8367
7902
8193
STD 6
Mirtazapine
Zolpidem
43730
(109)
47700
(153)
43102
(124)
47385
(156)
34517
(101)
38045
(107)
9857
9934
8008
8029
7893
7976
STD 8
Mirtazapine
Zolpidem
77701
(146)
42800
(161)
78734
(194)
43777
(223)
63689
(080)
38217
(533)
10133
10228
8089
8730
8197
8929
a Mean area response of six replicate samples prepared in Mobile phase (neat samples) b Mean area
response of six replicate samples prepared by spiking in post extracted blank c Mean area response of
six replicate samples prepared by spiking in plasma before extraction d Matrix effect Post extracted
mean responseAqueous (Neat) mean response x 100 e Recovery Extracted mean response Post
extracted mean response x 100 f Process efficiency Extracted mean response Aqueous Mean
response x 100
Ruggedness and Dilution Integrity
The results of ruggedness study for Duloxetine was well within the acceptance limit of 15
in Precision and 850-1150 in mean accuracy The precision and accuracy values for
both experiment at LLOQ LQC MQC and HQC levels for Duloxetine ranged from 34 to
76 and 971 to 1085 respectively
The dilution integrity experiment was performed with an aim to validate the dilution test
to be carried out on higher analyte concentration above the upper limit of quantification
(ULOQ) which maybe encountered during real subject sample analysis The precision and
accuracy values for 15th
and 110th
dilution ranged from 326 to 334 and 982 to 1097 for
Duloxetine
Application of the Method in Healthy Human Subjects
The validated method was successfully applied for the assay of Duloxetine in healthy male
Indian volunteers in the age group of 18-45 years Figure 4 shows the plasma concentration
vs time profile of Duloxetine human subjects under fating condition The method was
sensitive enough to monitor the Duloxetine plasma concentration up to 72 h Approximately
520 samples including the calibration and QC samples were within the acceptable limits
D C REDDY et al 910
The pharmacokinetic parameters viz Cmax Tmax AUC0t and AUC0 were
calculated for Duloxetine in test formulations (Figure 4) Shows the data of Mean plasma
concentration-time profile of Duloxetine Hydrochloride 60 mg Delayed Release Capsules
formulation to 12 healthy volunteers
0
5
10
15
20
25
30
35
40
45
0 6 12 18 24 30 36 42 48 54 60 66 72
Time (hr)
Linear mean plot of plasma Duloxetine Concentration Vs Time under Fasting conditions
Parameter Duloxetine
Cmax ngmL 4459418599
Tmax (h) 7251581
AUC0t 984702526502
AUC0 1027147572790
Figure 4 Mean plasma concentration-time profile of Duloxetine Hydrochloride 60 mg
Delayed Release Capsules formulation to 8 healthy volunteers
Conclusion
The proposed method successfully demonstrates chromatographic separation of duloxetine
from human plasma with high resolution The bioanalytical methodology for their
simultaneous determination is highly specific rugged and rapid for therapeutic drug
monitoring The method involved a simple and specific sample preparation by liquid-liquid
extraction followed by isocratic separation in 30 min The overall analysis time is proving
compared to other reported procedures for duloxetine
Acknowledgment
The authors gratefully acknowledge Dr AT Bapuji and Aurobindo Pharma Ltd Hyderabad
India for providing necessary facilities for carrying out this study
References
1 Maryadele J O editor The Merck Index 14th
Ed Whitehouse Station NJ Merck
and Co Inc 2006 p 3465
2 Mishra L Drugs today Vol 1 Delhi Lorina Publications 2006 p 489
3 Stephan A C Luc-Andre G Francois R V Peter R B Frank P B Melissa J J et al J
Neuropsychopharmacol 2003 28 1685ndash93
Mea
n c
once
ntr
atio
n
ngm
L
Time h
Development and Validation of a LCMSMS Method for the Determination 911
4 National Institute for Health and Clinical Excellence Clinical guideline 40 Urinary
incontinence London 2006
5 National Institute for Health and Clinical Excellence Clinical guideline 96
Neuropathic pain - pharmacological management London 2010
6 httpwwwdrugbankcadrugsDB00476
7 Wolfe F Smythe H A Yunus M B et al The American College of Rheumatology
Criteria for the Classification of Fibromyalgia Report of the Multicenter Criteri
Committee Arthritis Rheum 1990 33(2) 160ndash72 doi101002art1780330203
PMID 2306288
8 Douglas R Dolnak Treating Patients for Comorbid Depression Anxiety Disorders
and Somatic Illnesses 2006 JAOA 106 8
9 httpwwwcancergovdictionaryCdrID=589399
10 Senthamil Selvan P Gowda K V Mandal U Sam Solomon W D Pal T K J
Chromatogr B Analyt Technol Biomed Life Sci 2007 858(1-2) 269-75 Epub 2007
Sep 14
11 Mercolini L Mandrioli R Cazzolla R Amore M and Raggi M A J Chromatogr B
Analyt Technol Biomed Life Sci 2007 856(1-2) 81-7 Epub 2007 Jun 2
12 Musenga A Amore M Mandrioli R Kenndler E de Martino L and Raggi M A J
Chromatogr B Analyt Technol Biomed Life Sci 2009 877(11-12) 1126-32 Epub
2009 Mar 3
13 Waldschmitt C Vogel F Maurer C and Hiemke C Ther Drug Monit 2007
29(6) 767-72
14 Ning Ma Bi Kui Zhang Huan De Li Ben Mei Chen Ping Xu Feng Wang Rong
Hua Zhu Sheng Feng Da Xiong Xiang and Yun Gui Zhu Clinica Chimica Acta
2007 380(1-2) 100-105
15 FDA Guidance for industry Bioanalytical Method Validation US Department of
and Health Human Services Food and Drug Administration Centre for Drug
Evaluation and Research (CDER) and Centre for Veterinary Medicine (CVM) May
2001
16 Viswanathan C T Bansal S Booth B DeStefano A J Rose M J Sailstad J Shah V R
Skelly J P Swann P G and Weiner R APPS J 2007 9 E30
17 Matuszewski B K J Chromatogr B Analyt Technol Biomed Life Sci 2006 830(2)
293-300 Epub 2005 Nov 23
18 FDA Guidance for industry Bioavailability Studies for Orally Administered Drug-
Products-General Considerations US Department of Health and Human Services
Food and Drug Administration Center for drug evaluation and Research (CDER)
2000
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Development and Validation of a LCMSMS Method for the Determination 903
extraction solvent (MTBE n Hexane 2080 vv) was added and samples were extracted on
extractor at 32 times g for 10 10 min centrifugation of the samples was done at 3200 times g for
10 min at 10degC 20 mL supernant was separated and evaporated to dryness under nitrogen at
50degC plusmn 05degC for 15 psi and 15 min The dried samples were reconstituted with 300 microL of
mobile phase and 20 microL was injection in the chromatographic system
Method Validation
The method validation was performed as per USFDA guidelines15
System suitability
experiment was performed by injecting six consecutive injections using aqueous standard
mixture of Duloxetine and internal standard at the start of each batch during the method
validation The carryover effect of the auto sampler was evaluated by injecting a sequence of
injections solutions of aqueous standard Mobile phase standard blank extracted standard
equivalent to highest standard in the calibration range As per the acceptance criteria the
response in blank should not be greater than 20 of LLOQ response16
The linearity of the method was determined by analysis of five linear curves containing
eight non-zero concentration The ratio of area response for drug and IS was used for
regression analysis Each calibration curve was analyzed individually by using least square
weighed (1x2)
linear regression The lowest standard on the calibration curve was accepted
as the lower limit of quantitation (LLOQ) if the analyte response was at least five times
more than that of the drug free (blank) extracted plasma The deviation of than that of drug
free (blank) extracted plasma The deviation of standards other than LLOQ from nominal
concentration should not to be more than plusmn150
The selectivity of the method towards endogenous plasma matrix components was
assessed in twelve batches (7 normal of K2 EDTA plasma 2 haemolysed 2 lipidemic and
and 1 heparinised) of blank human plasma This was done to estimate the extent to which
endogenous plasma components contribute towards interference at the retention time of
analytes and IS The cross talk of MRM for analytes and IS was checked using highest
standard on calibration curve and working solution of IS
For determining the intra-day accuracy and precision replicate analysis of plasma
samples of Duloxetine was performed on the same day The run consisted of a calibration
curve and six replicates of LLOQ LQC MQC and HQC samples The inter-day accuracy
and precision were assessed by analysis of three precision and accuracy batches on three
consecutive validation days The precision of the method was determined by calculating the
percent coefficient of variation (CV) for each level The deviation at each concentration
level from the nominal concentration was expected to be within plusmn150 except LLOQ for
which it should be within plusmn200
The relative recovery matrix effect and process efficiency were assessed as
recommended by Matuszewski et al17
All three parameters were evaluated at Std-1 Std-3
Std-5 Std-6 and Std-8 levels in six replicates Relative recovery (RE) was calculated by
comparing the mean area response of extracted samples (spiked before extraction) to that of
unextracted samples (spiked after extraction) at each CC level The recovery of IS was
similarly estimated Absolute matrix effect (ME) was assessed by comparing the mean area
response of unextracted samples (spiked after extraction) with mean area of neat standard
solutions The overall lsquoprocess efficiencyrsquo (PE) was calculated by comparing the mean
area response of extracted samples (spiked before extraction) to that with mean area of neat
standard solutions at each CC level The assessment of relative matrix effect was based on
direct comparison of the MSMS responses (peak areas) of the analytes spiked into extracts
originating from different lots of plasma The variability in these responses expressed as
CV was considered as the measure of relative matrix effect
D C REDDY et al 904
Stability experiments were carried out to examine the analyte stability in stock solutions
and in plasma samples under different conditions Short term stability at room temperature
and long term stability of spiked solution stored at ndash 70ordmC was assessed by comparing the
area response of stability sample of analyte and IS with the area response of sample prepared
from fresh stock solutions The solutions were considered stable if the deviation from
nominal value was within plusmn10 Autosampler wet extract stability bench top stability dry
extract stability and freeze- thaw stability were performed at LQC and HQC using six
replicates at each level The samples were considered stable if the deviation from the mean
calculated concentration of freshly thawed quality control samples was within plusmn15
To authenticate the ruggedness of the proposed method it was done on two precision
and accuracy batches The first batch was analysed by different analysts while the second
batch was analysed on different column and different LC-MSMS Dilution integrity
experiment was conducted by diluting the stock solution prepared as spiked standard at
concentration of 200034 ngmL for Duloxetine The precision and accuracy for dilution
integrity standards at 15th
and 110th
determined by analyzing the samples against
calibration curve standards
Study Design
A pharmacokinetic study was conducted on 12 healthy adult male human subjects under
fating conditions (n = 12) following oral administration of 60 mg delayed release capsules
Each volunteer was judged to be in good health through medical history physical
examination and routine laboratory tests Written consent was taken from all the volunteers
after informing them about the objectives and possible risks involved in the study An
independent ethics committee constituted as per Indian council of Medical Research (ICMR)
approved the study protocol The study was conducted strictly in accordance with guidelines
laid down by international conference on Harmonization and USFDA18
A single oral dose
of 60 mg drug was given to the volunteers with 240 mL of water Blood samples were
collected at 00 (pre-dose) 100 200 300 400 450 500 550 600 650 700 750
800 1000 1200 1600 2000 2400 3600 4800 and 7200 h after oral administration of
the dose for test formulation in labeled K2 EDTA- vaccuettes Plasma was separated by
centrifugation(3200 times g 10ordmC 10 min) and kept frozen at ndash 70ordmC until analysis During
study volunteers had a standard diet while water intake was free
Results and Discussion
Method Development
Chromatographic resolution of Duloxetine and IS was initiated under isocratic conditions to
obtain adequate response sharp peak shape and a short analysis time Thus separation was
tried using various combinations of methanolacetonitrile acidic buffers and additives like
formic acid on different reversed-phase columns with 5microm particle size viz Chromolith
Hypersil X-terra Kromasil Intertsil and Grace ACE Cyano (150 mm and 250 mm times 46 mm)
Chromolith RP-18 (50 mm times 46 mm) Kromasil (50 mm and 100 mm times 46 mm) and
Gemini C-18 (50 mm times 46 mm) to find the optimal column that produced the best
sensitivity efficiency and peak shape The analytes showed poor separation and
reproducibility for proposed linear range except for x-terra RP-8 column that offered superior
peak shape baseline separation desired linearity and reproducibility The mobile phase
consisting of 30 mM ammonium formate adjusted the pH 500005 with formic acid and
methanol (3070 vv) ratio and having 30 mM ammonium formate pH around 50-55 were
found most suitable for eluting Duloxetine and IS at 150 and 148 min respectively Also
Development and Validation of a LCMSMS Method for the Determination 905
the reproducibility of retention times for the analytes expressed as CV was le2 for 100
injections on the same column
The inherent selectivity of MSMS detection was also expected to be beneficial in
developing a selective and sensitive method The present study was conducted using ESI as
the ionization source as it gave high intensity for drug and IS as they have similar sites for
protonation Initially the extraction of Duloxetine and IS was carried out via protein
precipitation with common solvents like acetonitrile methanol and acetone but the
sensitivity and reproducibility were poor in all the solvents with frequent clogging of the
column which required online flushing of the column Liquid-liquid extraction technique
was also tested to isolate the drugs from plasma using diethyl ether dichromethane ethyl
acetate methyl tert butyl ether and isopropyl alcohol (alone and in combination) as
extracting solvents However the recovery was inconsistent with some ion suppression
(greater than 15 CV) in most of these solvent systems Further use of 100 microL of 50mM
ammonium formate to extraction in methyl tert butyl ether n Hexane (8020 vv) gave
consistent recoveries for the analytes especially at the LLOQ level with minimum matrix
interference A general internal standard was used to minimize any analytical variation due
to solvent evaporation integrity of the column and ionization efficiency of analytes
Fluoxetine was used as an internal standard (IS) in the present study which had similar
chromatographic behavior and was quantitatively extracted with the proposed extraction
procedure Also there was no effect of IS on analyte recovery sensitivity or ion
suppression
System Suitability and Auto Sampler Carryover
Throughout the method validation the CV of system suitability was observed below 40
at the retention time of duloxetine and the IS Carryover evaluation was performed in each
analytical run so as to ensure that it does not affect the accuracy and the precision of the
proposed method There was negligible carryover (le4 of the LLOQ response) observed
during auto sampler carryover experiment No enhancement in the response was observed in
double blank after subsequent injection of highest calibration standard (aqueous and
extracted) at the retention time of analytes and IS
Linearity and Lower Limit of Quantification (LLOQ)
The calibration curves were linear over the concentration range of 0100100017 ngmL
with correlation coefficient r2 ge 09963 for duloxetine respectively The equations for means
(n=5) of five calibration curves for duloxetine The standard deviation value for slope
intercept observed were 09921 09952 09951 09997 09996 and 000008 000007
00005 00006 00005 and 00005 for duloxetine respectively The accuracy and precision
(CV) observed for the calibration curve standards ranged from 9114 to 10459 and 082
to 1154 respectively The lowest concentration (LLOQ) in the standard curve for both the
isomers was measured at a signal-to-noise ratio (SN) of ge 100 Figure 3 shows the
representative LC-MSMS chromatograms of (A) calibration curve of duloxetine (B)
Double blank plasma with out IS (C) Blank plasma with IS and (D) Duloxetine and
fluoxetine at LLOQ
D C REDDY et al 906
Figure 3 (a) Calibration curve of Duloxetine
Figure 3 (b) Double blank plasma (without IS)
Figure 3 (c) Blank plasma with IS
Res
ponse
Res
idual
Development and Validation of a LCMSMS Method for the Determination 907
Figure 3 (d) Duloxetine and Fluoxetine at LLOQ (mz) 298081540
Selectivity Accuracy and Precision
To establish the selectivity of the method for interference due to endogenous plasma
components from haemolysed lipidemic heparinised and K2 EDTA blank plasmas the
change in the area ratio (analyteIS) at LLOQ level was within 4-8 while the precision
(CV) in their measurement varied from 21 to 56 representative MRM ion
chromatograms extracted (A) blank human plasma (double blank) (B) blank plasma
fortified with IS (mz 31002rarr14807) duloxetine at LLOQ (mz 29808rarr1540) the
selectivity of the method The extraction procedure together with mass detection gave very
good selectivity for the analysis of both the drug and IS in the blank plasma No endogenous
interferences were found at the retention times of analytes and IS
The intra- and inter batch precision and accuracy were established from validation runs
performed at HQC MQC LQC and LLOQ QC levels The intra- and inter batch precision
ranged from 074 to 976 for duloxetineThe accuracy values were within 9714-10449
for both the analytes in intra- and inter batches The precision and accuracy values for intra-
and inter day experiments in plasma are shown in Table 1(A)
Table 1 (A) Comparison of intra- and inter-batch precision and accuracy for Duloxetine
QC ID Nominal
concentration ngmL
Intrabatch
n
Mean
concentration
observed ngmL
CV Accuracy
LLOQQC
LQC
MQC
HQC
0100
0300
42060
77750
6
0104
0307
41255
81240
770
556
379
503
10350
10229
9808
10449
QC ID Nominal
concentration ngmL
Intrabatch
n
Mean
concentration
observed ngmL
CV Accuracy
LLOQQC
LQC
MQC
HQC
0100
0300
42060
77750
24
0103
0308
40863
79684
685
702
521
552
10325
10259
9714
10248
D C REDDY et al 908
(B) Stability of Duloxetine under various conditions (n=6)
Storage conditions Nominal
Concentration ngmL
Mean calculated
conc (ngmL) plusmn SD
Mean
accuracy
Bench top stability
(After 567 h at ~ at 25degC
LQC
HQC
0300
77750
0298 plusmn 00090
75382 plusmn 26005
9928
9695
Freeze thaw stability
(3 Cycles)
LQC
HQC
0300
77750
0298 plusmn 00060
79717 plusmn 49832
9939
10253
Dry extract stability
(2582 h below 10degC)
LQC
HQC
0300
77750
0299 plusmn 00087
83442 plusmn 52259
9967
10732
Wet extract stability
(2643 h below 10degC)
LQC
HQC
0300
77750
0309 plusmn 00136
76943 plusmn 34201
10294
9896
Auto sampler stability
(2188 h 10degC)
LQC
HQC
0300
77750
0297 plusmn 00112
73886 plusmn 43496
9889
9500
Long term stability in plasma
at -70degC
(2366 days at -70degC)
LQC
HQC
0300
77750
0298 plusmn 00060
71398 plusmn 54829
9939
9183
Recovery and Stability Results
The relative recovery absolute matrix effect and process efficiency data at LQC MQC and
HQC levels is presented The recovery for drug and IS in human plasma was 8031 and
8109 Further the extent of matrix effect in different lots of plasma (spiked after
extraction) was within the acceptable limit as evident from the precision (CV) values in
Table 2
Stock solutions for short term stability of Duloxetine and IS were stable at room
temperature for minimum period of about 6 h and between 2 and 8degC for about 7 days
Duloxetine in control human plasma (bench top) at room temperature was stable at least for
567 h at ambient temperature and for minimum of three freeze and thaw cycles Auto
sampler stability of the spiked quality control samples maintained at 10degC was maintained
up to 2188 h Long-term stability of the spiked quality control samples stored at -70degC was
determined up to 2366 days The accuracy values for different stability experiment in
plasma are shown in Table 1(B)
Development and Validation of a LCMSMS Method for the Determination 909
Table 2 Absolute matrix effect relative recovery and process efficiency for Mirtazapine
Analyte
ISTD
Aa
(CV)
Bb
(CV)
Cc
(CV)
Absolute
matrix
effect
(ME) d
Relative
recovery
(RE) e
Process
efficiency
(PE) f
STD 1
Mirtazapine
Zolpidem
179
(831)
42068
(049)
188
(391)
37907
(135)
109
(432)
40711
(132)
9423
9041
8540
8434
8047
7625
STD 3
Mirtazapine
Zolpidem
567
(323)
52367
(131)
591
(413)
54113
(156)
460
(122)
40951
(180)
10438
10333
7777
7568
8118
7620
STD 5
Mirtazapine
Zolpidem
23157
(174)
50420
(194)
22868
(169)
49366
(165)
18300
(096)
41307
(068)
9875
9791
8002
8367
7902
8193
STD 6
Mirtazapine
Zolpidem
43730
(109)
47700
(153)
43102
(124)
47385
(156)
34517
(101)
38045
(107)
9857
9934
8008
8029
7893
7976
STD 8
Mirtazapine
Zolpidem
77701
(146)
42800
(161)
78734
(194)
43777
(223)
63689
(080)
38217
(533)
10133
10228
8089
8730
8197
8929
a Mean area response of six replicate samples prepared in Mobile phase (neat samples) b Mean area
response of six replicate samples prepared by spiking in post extracted blank c Mean area response of
six replicate samples prepared by spiking in plasma before extraction d Matrix effect Post extracted
mean responseAqueous (Neat) mean response x 100 e Recovery Extracted mean response Post
extracted mean response x 100 f Process efficiency Extracted mean response Aqueous Mean
response x 100
Ruggedness and Dilution Integrity
The results of ruggedness study for Duloxetine was well within the acceptance limit of 15
in Precision and 850-1150 in mean accuracy The precision and accuracy values for
both experiment at LLOQ LQC MQC and HQC levels for Duloxetine ranged from 34 to
76 and 971 to 1085 respectively
The dilution integrity experiment was performed with an aim to validate the dilution test
to be carried out on higher analyte concentration above the upper limit of quantification
(ULOQ) which maybe encountered during real subject sample analysis The precision and
accuracy values for 15th
and 110th
dilution ranged from 326 to 334 and 982 to 1097 for
Duloxetine
Application of the Method in Healthy Human Subjects
The validated method was successfully applied for the assay of Duloxetine in healthy male
Indian volunteers in the age group of 18-45 years Figure 4 shows the plasma concentration
vs time profile of Duloxetine human subjects under fating condition The method was
sensitive enough to monitor the Duloxetine plasma concentration up to 72 h Approximately
520 samples including the calibration and QC samples were within the acceptable limits
D C REDDY et al 910
The pharmacokinetic parameters viz Cmax Tmax AUC0t and AUC0 were
calculated for Duloxetine in test formulations (Figure 4) Shows the data of Mean plasma
concentration-time profile of Duloxetine Hydrochloride 60 mg Delayed Release Capsules
formulation to 12 healthy volunteers
0
5
10
15
20
25
30
35
40
45
0 6 12 18 24 30 36 42 48 54 60 66 72
Time (hr)
Linear mean plot of plasma Duloxetine Concentration Vs Time under Fasting conditions
Parameter Duloxetine
Cmax ngmL 4459418599
Tmax (h) 7251581
AUC0t 984702526502
AUC0 1027147572790
Figure 4 Mean plasma concentration-time profile of Duloxetine Hydrochloride 60 mg
Delayed Release Capsules formulation to 8 healthy volunteers
Conclusion
The proposed method successfully demonstrates chromatographic separation of duloxetine
from human plasma with high resolution The bioanalytical methodology for their
simultaneous determination is highly specific rugged and rapid for therapeutic drug
monitoring The method involved a simple and specific sample preparation by liquid-liquid
extraction followed by isocratic separation in 30 min The overall analysis time is proving
compared to other reported procedures for duloxetine
Acknowledgment
The authors gratefully acknowledge Dr AT Bapuji and Aurobindo Pharma Ltd Hyderabad
India for providing necessary facilities for carrying out this study
References
1 Maryadele J O editor The Merck Index 14th
Ed Whitehouse Station NJ Merck
and Co Inc 2006 p 3465
2 Mishra L Drugs today Vol 1 Delhi Lorina Publications 2006 p 489
3 Stephan A C Luc-Andre G Francois R V Peter R B Frank P B Melissa J J et al J
Neuropsychopharmacol 2003 28 1685ndash93
Mea
n c
once
ntr
atio
n
ngm
L
Time h
Development and Validation of a LCMSMS Method for the Determination 911
4 National Institute for Health and Clinical Excellence Clinical guideline 40 Urinary
incontinence London 2006
5 National Institute for Health and Clinical Excellence Clinical guideline 96
Neuropathic pain - pharmacological management London 2010
6 httpwwwdrugbankcadrugsDB00476
7 Wolfe F Smythe H A Yunus M B et al The American College of Rheumatology
Criteria for the Classification of Fibromyalgia Report of the Multicenter Criteri
Committee Arthritis Rheum 1990 33(2) 160ndash72 doi101002art1780330203
PMID 2306288
8 Douglas R Dolnak Treating Patients for Comorbid Depression Anxiety Disorders
and Somatic Illnesses 2006 JAOA 106 8
9 httpwwwcancergovdictionaryCdrID=589399
10 Senthamil Selvan P Gowda K V Mandal U Sam Solomon W D Pal T K J
Chromatogr B Analyt Technol Biomed Life Sci 2007 858(1-2) 269-75 Epub 2007
Sep 14
11 Mercolini L Mandrioli R Cazzolla R Amore M and Raggi M A J Chromatogr B
Analyt Technol Biomed Life Sci 2007 856(1-2) 81-7 Epub 2007 Jun 2
12 Musenga A Amore M Mandrioli R Kenndler E de Martino L and Raggi M A J
Chromatogr B Analyt Technol Biomed Life Sci 2009 877(11-12) 1126-32 Epub
2009 Mar 3
13 Waldschmitt C Vogel F Maurer C and Hiemke C Ther Drug Monit 2007
29(6) 767-72
14 Ning Ma Bi Kui Zhang Huan De Li Ben Mei Chen Ping Xu Feng Wang Rong
Hua Zhu Sheng Feng Da Xiong Xiang and Yun Gui Zhu Clinica Chimica Acta
2007 380(1-2) 100-105
15 FDA Guidance for industry Bioanalytical Method Validation US Department of
and Health Human Services Food and Drug Administration Centre for Drug
Evaluation and Research (CDER) and Centre for Veterinary Medicine (CVM) May
2001
16 Viswanathan C T Bansal S Booth B DeStefano A J Rose M J Sailstad J Shah V R
Skelly J P Swann P G and Weiner R APPS J 2007 9 E30
17 Matuszewski B K J Chromatogr B Analyt Technol Biomed Life Sci 2006 830(2)
293-300 Epub 2005 Nov 23
18 FDA Guidance for industry Bioavailability Studies for Orally Administered Drug-
Products-General Considerations US Department of Health and Human Services
Food and Drug Administration Center for drug evaluation and Research (CDER)
2000
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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D C REDDY et al 904
Stability experiments were carried out to examine the analyte stability in stock solutions
and in plasma samples under different conditions Short term stability at room temperature
and long term stability of spiked solution stored at ndash 70ordmC was assessed by comparing the
area response of stability sample of analyte and IS with the area response of sample prepared
from fresh stock solutions The solutions were considered stable if the deviation from
nominal value was within plusmn10 Autosampler wet extract stability bench top stability dry
extract stability and freeze- thaw stability were performed at LQC and HQC using six
replicates at each level The samples were considered stable if the deviation from the mean
calculated concentration of freshly thawed quality control samples was within plusmn15
To authenticate the ruggedness of the proposed method it was done on two precision
and accuracy batches The first batch was analysed by different analysts while the second
batch was analysed on different column and different LC-MSMS Dilution integrity
experiment was conducted by diluting the stock solution prepared as spiked standard at
concentration of 200034 ngmL for Duloxetine The precision and accuracy for dilution
integrity standards at 15th
and 110th
determined by analyzing the samples against
calibration curve standards
Study Design
A pharmacokinetic study was conducted on 12 healthy adult male human subjects under
fating conditions (n = 12) following oral administration of 60 mg delayed release capsules
Each volunteer was judged to be in good health through medical history physical
examination and routine laboratory tests Written consent was taken from all the volunteers
after informing them about the objectives and possible risks involved in the study An
independent ethics committee constituted as per Indian council of Medical Research (ICMR)
approved the study protocol The study was conducted strictly in accordance with guidelines
laid down by international conference on Harmonization and USFDA18
A single oral dose
of 60 mg drug was given to the volunteers with 240 mL of water Blood samples were
collected at 00 (pre-dose) 100 200 300 400 450 500 550 600 650 700 750
800 1000 1200 1600 2000 2400 3600 4800 and 7200 h after oral administration of
the dose for test formulation in labeled K2 EDTA- vaccuettes Plasma was separated by
centrifugation(3200 times g 10ordmC 10 min) and kept frozen at ndash 70ordmC until analysis During
study volunteers had a standard diet while water intake was free
Results and Discussion
Method Development
Chromatographic resolution of Duloxetine and IS was initiated under isocratic conditions to
obtain adequate response sharp peak shape and a short analysis time Thus separation was
tried using various combinations of methanolacetonitrile acidic buffers and additives like
formic acid on different reversed-phase columns with 5microm particle size viz Chromolith
Hypersil X-terra Kromasil Intertsil and Grace ACE Cyano (150 mm and 250 mm times 46 mm)
Chromolith RP-18 (50 mm times 46 mm) Kromasil (50 mm and 100 mm times 46 mm) and
Gemini C-18 (50 mm times 46 mm) to find the optimal column that produced the best
sensitivity efficiency and peak shape The analytes showed poor separation and
reproducibility for proposed linear range except for x-terra RP-8 column that offered superior
peak shape baseline separation desired linearity and reproducibility The mobile phase
consisting of 30 mM ammonium formate adjusted the pH 500005 with formic acid and
methanol (3070 vv) ratio and having 30 mM ammonium formate pH around 50-55 were
found most suitable for eluting Duloxetine and IS at 150 and 148 min respectively Also
Development and Validation of a LCMSMS Method for the Determination 905
the reproducibility of retention times for the analytes expressed as CV was le2 for 100
injections on the same column
The inherent selectivity of MSMS detection was also expected to be beneficial in
developing a selective and sensitive method The present study was conducted using ESI as
the ionization source as it gave high intensity for drug and IS as they have similar sites for
protonation Initially the extraction of Duloxetine and IS was carried out via protein
precipitation with common solvents like acetonitrile methanol and acetone but the
sensitivity and reproducibility were poor in all the solvents with frequent clogging of the
column which required online flushing of the column Liquid-liquid extraction technique
was also tested to isolate the drugs from plasma using diethyl ether dichromethane ethyl
acetate methyl tert butyl ether and isopropyl alcohol (alone and in combination) as
extracting solvents However the recovery was inconsistent with some ion suppression
(greater than 15 CV) in most of these solvent systems Further use of 100 microL of 50mM
ammonium formate to extraction in methyl tert butyl ether n Hexane (8020 vv) gave
consistent recoveries for the analytes especially at the LLOQ level with minimum matrix
interference A general internal standard was used to minimize any analytical variation due
to solvent evaporation integrity of the column and ionization efficiency of analytes
Fluoxetine was used as an internal standard (IS) in the present study which had similar
chromatographic behavior and was quantitatively extracted with the proposed extraction
procedure Also there was no effect of IS on analyte recovery sensitivity or ion
suppression
System Suitability and Auto Sampler Carryover
Throughout the method validation the CV of system suitability was observed below 40
at the retention time of duloxetine and the IS Carryover evaluation was performed in each
analytical run so as to ensure that it does not affect the accuracy and the precision of the
proposed method There was negligible carryover (le4 of the LLOQ response) observed
during auto sampler carryover experiment No enhancement in the response was observed in
double blank after subsequent injection of highest calibration standard (aqueous and
extracted) at the retention time of analytes and IS
Linearity and Lower Limit of Quantification (LLOQ)
The calibration curves were linear over the concentration range of 0100100017 ngmL
with correlation coefficient r2 ge 09963 for duloxetine respectively The equations for means
(n=5) of five calibration curves for duloxetine The standard deviation value for slope
intercept observed were 09921 09952 09951 09997 09996 and 000008 000007
00005 00006 00005 and 00005 for duloxetine respectively The accuracy and precision
(CV) observed for the calibration curve standards ranged from 9114 to 10459 and 082
to 1154 respectively The lowest concentration (LLOQ) in the standard curve for both the
isomers was measured at a signal-to-noise ratio (SN) of ge 100 Figure 3 shows the
representative LC-MSMS chromatograms of (A) calibration curve of duloxetine (B)
Double blank plasma with out IS (C) Blank plasma with IS and (D) Duloxetine and
fluoxetine at LLOQ
D C REDDY et al 906
Figure 3 (a) Calibration curve of Duloxetine
Figure 3 (b) Double blank plasma (without IS)
Figure 3 (c) Blank plasma with IS
Res
ponse
Res
idual
Development and Validation of a LCMSMS Method for the Determination 907
Figure 3 (d) Duloxetine and Fluoxetine at LLOQ (mz) 298081540
Selectivity Accuracy and Precision
To establish the selectivity of the method for interference due to endogenous plasma
components from haemolysed lipidemic heparinised and K2 EDTA blank plasmas the
change in the area ratio (analyteIS) at LLOQ level was within 4-8 while the precision
(CV) in their measurement varied from 21 to 56 representative MRM ion
chromatograms extracted (A) blank human plasma (double blank) (B) blank plasma
fortified with IS (mz 31002rarr14807) duloxetine at LLOQ (mz 29808rarr1540) the
selectivity of the method The extraction procedure together with mass detection gave very
good selectivity for the analysis of both the drug and IS in the blank plasma No endogenous
interferences were found at the retention times of analytes and IS
The intra- and inter batch precision and accuracy were established from validation runs
performed at HQC MQC LQC and LLOQ QC levels The intra- and inter batch precision
ranged from 074 to 976 for duloxetineThe accuracy values were within 9714-10449
for both the analytes in intra- and inter batches The precision and accuracy values for intra-
and inter day experiments in plasma are shown in Table 1(A)
Table 1 (A) Comparison of intra- and inter-batch precision and accuracy for Duloxetine
QC ID Nominal
concentration ngmL
Intrabatch
n
Mean
concentration
observed ngmL
CV Accuracy
LLOQQC
LQC
MQC
HQC
0100
0300
42060
77750
6
0104
0307
41255
81240
770
556
379
503
10350
10229
9808
10449
QC ID Nominal
concentration ngmL
Intrabatch
n
Mean
concentration
observed ngmL
CV Accuracy
LLOQQC
LQC
MQC
HQC
0100
0300
42060
77750
24
0103
0308
40863
79684
685
702
521
552
10325
10259
9714
10248
D C REDDY et al 908
(B) Stability of Duloxetine under various conditions (n=6)
Storage conditions Nominal
Concentration ngmL
Mean calculated
conc (ngmL) plusmn SD
Mean
accuracy
Bench top stability
(After 567 h at ~ at 25degC
LQC
HQC
0300
77750
0298 plusmn 00090
75382 plusmn 26005
9928
9695
Freeze thaw stability
(3 Cycles)
LQC
HQC
0300
77750
0298 plusmn 00060
79717 plusmn 49832
9939
10253
Dry extract stability
(2582 h below 10degC)
LQC
HQC
0300
77750
0299 plusmn 00087
83442 plusmn 52259
9967
10732
Wet extract stability
(2643 h below 10degC)
LQC
HQC
0300
77750
0309 plusmn 00136
76943 plusmn 34201
10294
9896
Auto sampler stability
(2188 h 10degC)
LQC
HQC
0300
77750
0297 plusmn 00112
73886 plusmn 43496
9889
9500
Long term stability in plasma
at -70degC
(2366 days at -70degC)
LQC
HQC
0300
77750
0298 plusmn 00060
71398 plusmn 54829
9939
9183
Recovery and Stability Results
The relative recovery absolute matrix effect and process efficiency data at LQC MQC and
HQC levels is presented The recovery for drug and IS in human plasma was 8031 and
8109 Further the extent of matrix effect in different lots of plasma (spiked after
extraction) was within the acceptable limit as evident from the precision (CV) values in
Table 2
Stock solutions for short term stability of Duloxetine and IS were stable at room
temperature for minimum period of about 6 h and between 2 and 8degC for about 7 days
Duloxetine in control human plasma (bench top) at room temperature was stable at least for
567 h at ambient temperature and for minimum of three freeze and thaw cycles Auto
sampler stability of the spiked quality control samples maintained at 10degC was maintained
up to 2188 h Long-term stability of the spiked quality control samples stored at -70degC was
determined up to 2366 days The accuracy values for different stability experiment in
plasma are shown in Table 1(B)
Development and Validation of a LCMSMS Method for the Determination 909
Table 2 Absolute matrix effect relative recovery and process efficiency for Mirtazapine
Analyte
ISTD
Aa
(CV)
Bb
(CV)
Cc
(CV)
Absolute
matrix
effect
(ME) d
Relative
recovery
(RE) e
Process
efficiency
(PE) f
STD 1
Mirtazapine
Zolpidem
179
(831)
42068
(049)
188
(391)
37907
(135)
109
(432)
40711
(132)
9423
9041
8540
8434
8047
7625
STD 3
Mirtazapine
Zolpidem
567
(323)
52367
(131)
591
(413)
54113
(156)
460
(122)
40951
(180)
10438
10333
7777
7568
8118
7620
STD 5
Mirtazapine
Zolpidem
23157
(174)
50420
(194)
22868
(169)
49366
(165)
18300
(096)
41307
(068)
9875
9791
8002
8367
7902
8193
STD 6
Mirtazapine
Zolpidem
43730
(109)
47700
(153)
43102
(124)
47385
(156)
34517
(101)
38045
(107)
9857
9934
8008
8029
7893
7976
STD 8
Mirtazapine
Zolpidem
77701
(146)
42800
(161)
78734
(194)
43777
(223)
63689
(080)
38217
(533)
10133
10228
8089
8730
8197
8929
a Mean area response of six replicate samples prepared in Mobile phase (neat samples) b Mean area
response of six replicate samples prepared by spiking in post extracted blank c Mean area response of
six replicate samples prepared by spiking in plasma before extraction d Matrix effect Post extracted
mean responseAqueous (Neat) mean response x 100 e Recovery Extracted mean response Post
extracted mean response x 100 f Process efficiency Extracted mean response Aqueous Mean
response x 100
Ruggedness and Dilution Integrity
The results of ruggedness study for Duloxetine was well within the acceptance limit of 15
in Precision and 850-1150 in mean accuracy The precision and accuracy values for
both experiment at LLOQ LQC MQC and HQC levels for Duloxetine ranged from 34 to
76 and 971 to 1085 respectively
The dilution integrity experiment was performed with an aim to validate the dilution test
to be carried out on higher analyte concentration above the upper limit of quantification
(ULOQ) which maybe encountered during real subject sample analysis The precision and
accuracy values for 15th
and 110th
dilution ranged from 326 to 334 and 982 to 1097 for
Duloxetine
Application of the Method in Healthy Human Subjects
The validated method was successfully applied for the assay of Duloxetine in healthy male
Indian volunteers in the age group of 18-45 years Figure 4 shows the plasma concentration
vs time profile of Duloxetine human subjects under fating condition The method was
sensitive enough to monitor the Duloxetine plasma concentration up to 72 h Approximately
520 samples including the calibration and QC samples were within the acceptable limits
D C REDDY et al 910
The pharmacokinetic parameters viz Cmax Tmax AUC0t and AUC0 were
calculated for Duloxetine in test formulations (Figure 4) Shows the data of Mean plasma
concentration-time profile of Duloxetine Hydrochloride 60 mg Delayed Release Capsules
formulation to 12 healthy volunteers
0
5
10
15
20
25
30
35
40
45
0 6 12 18 24 30 36 42 48 54 60 66 72
Time (hr)
Linear mean plot of plasma Duloxetine Concentration Vs Time under Fasting conditions
Parameter Duloxetine
Cmax ngmL 4459418599
Tmax (h) 7251581
AUC0t 984702526502
AUC0 1027147572790
Figure 4 Mean plasma concentration-time profile of Duloxetine Hydrochloride 60 mg
Delayed Release Capsules formulation to 8 healthy volunteers
Conclusion
The proposed method successfully demonstrates chromatographic separation of duloxetine
from human plasma with high resolution The bioanalytical methodology for their
simultaneous determination is highly specific rugged and rapid for therapeutic drug
monitoring The method involved a simple and specific sample preparation by liquid-liquid
extraction followed by isocratic separation in 30 min The overall analysis time is proving
compared to other reported procedures for duloxetine
Acknowledgment
The authors gratefully acknowledge Dr AT Bapuji and Aurobindo Pharma Ltd Hyderabad
India for providing necessary facilities for carrying out this study
References
1 Maryadele J O editor The Merck Index 14th
Ed Whitehouse Station NJ Merck
and Co Inc 2006 p 3465
2 Mishra L Drugs today Vol 1 Delhi Lorina Publications 2006 p 489
3 Stephan A C Luc-Andre G Francois R V Peter R B Frank P B Melissa J J et al J
Neuropsychopharmacol 2003 28 1685ndash93
Mea
n c
once
ntr
atio
n
ngm
L
Time h
Development and Validation of a LCMSMS Method for the Determination 911
4 National Institute for Health and Clinical Excellence Clinical guideline 40 Urinary
incontinence London 2006
5 National Institute for Health and Clinical Excellence Clinical guideline 96
Neuropathic pain - pharmacological management London 2010
6 httpwwwdrugbankcadrugsDB00476
7 Wolfe F Smythe H A Yunus M B et al The American College of Rheumatology
Criteria for the Classification of Fibromyalgia Report of the Multicenter Criteri
Committee Arthritis Rheum 1990 33(2) 160ndash72 doi101002art1780330203
PMID 2306288
8 Douglas R Dolnak Treating Patients for Comorbid Depression Anxiety Disorders
and Somatic Illnesses 2006 JAOA 106 8
9 httpwwwcancergovdictionaryCdrID=589399
10 Senthamil Selvan P Gowda K V Mandal U Sam Solomon W D Pal T K J
Chromatogr B Analyt Technol Biomed Life Sci 2007 858(1-2) 269-75 Epub 2007
Sep 14
11 Mercolini L Mandrioli R Cazzolla R Amore M and Raggi M A J Chromatogr B
Analyt Technol Biomed Life Sci 2007 856(1-2) 81-7 Epub 2007 Jun 2
12 Musenga A Amore M Mandrioli R Kenndler E de Martino L and Raggi M A J
Chromatogr B Analyt Technol Biomed Life Sci 2009 877(11-12) 1126-32 Epub
2009 Mar 3
13 Waldschmitt C Vogel F Maurer C and Hiemke C Ther Drug Monit 2007
29(6) 767-72
14 Ning Ma Bi Kui Zhang Huan De Li Ben Mei Chen Ping Xu Feng Wang Rong
Hua Zhu Sheng Feng Da Xiong Xiang and Yun Gui Zhu Clinica Chimica Acta
2007 380(1-2) 100-105
15 FDA Guidance for industry Bioanalytical Method Validation US Department of
and Health Human Services Food and Drug Administration Centre for Drug
Evaluation and Research (CDER) and Centre for Veterinary Medicine (CVM) May
2001
16 Viswanathan C T Bansal S Booth B DeStefano A J Rose M J Sailstad J Shah V R
Skelly J P Swann P G and Weiner R APPS J 2007 9 E30
17 Matuszewski B K J Chromatogr B Analyt Technol Biomed Life Sci 2006 830(2)
293-300 Epub 2005 Nov 23
18 FDA Guidance for industry Bioavailability Studies for Orally Administered Drug-
Products-General Considerations US Department of Health and Human Services
Food and Drug Administration Center for drug evaluation and Research (CDER)
2000
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Development and Validation of a LCMSMS Method for the Determination 905
the reproducibility of retention times for the analytes expressed as CV was le2 for 100
injections on the same column
The inherent selectivity of MSMS detection was also expected to be beneficial in
developing a selective and sensitive method The present study was conducted using ESI as
the ionization source as it gave high intensity for drug and IS as they have similar sites for
protonation Initially the extraction of Duloxetine and IS was carried out via protein
precipitation with common solvents like acetonitrile methanol and acetone but the
sensitivity and reproducibility were poor in all the solvents with frequent clogging of the
column which required online flushing of the column Liquid-liquid extraction technique
was also tested to isolate the drugs from plasma using diethyl ether dichromethane ethyl
acetate methyl tert butyl ether and isopropyl alcohol (alone and in combination) as
extracting solvents However the recovery was inconsistent with some ion suppression
(greater than 15 CV) in most of these solvent systems Further use of 100 microL of 50mM
ammonium formate to extraction in methyl tert butyl ether n Hexane (8020 vv) gave
consistent recoveries for the analytes especially at the LLOQ level with minimum matrix
interference A general internal standard was used to minimize any analytical variation due
to solvent evaporation integrity of the column and ionization efficiency of analytes
Fluoxetine was used as an internal standard (IS) in the present study which had similar
chromatographic behavior and was quantitatively extracted with the proposed extraction
procedure Also there was no effect of IS on analyte recovery sensitivity or ion
suppression
System Suitability and Auto Sampler Carryover
Throughout the method validation the CV of system suitability was observed below 40
at the retention time of duloxetine and the IS Carryover evaluation was performed in each
analytical run so as to ensure that it does not affect the accuracy and the precision of the
proposed method There was negligible carryover (le4 of the LLOQ response) observed
during auto sampler carryover experiment No enhancement in the response was observed in
double blank after subsequent injection of highest calibration standard (aqueous and
extracted) at the retention time of analytes and IS
Linearity and Lower Limit of Quantification (LLOQ)
The calibration curves were linear over the concentration range of 0100100017 ngmL
with correlation coefficient r2 ge 09963 for duloxetine respectively The equations for means
(n=5) of five calibration curves for duloxetine The standard deviation value for slope
intercept observed were 09921 09952 09951 09997 09996 and 000008 000007
00005 00006 00005 and 00005 for duloxetine respectively The accuracy and precision
(CV) observed for the calibration curve standards ranged from 9114 to 10459 and 082
to 1154 respectively The lowest concentration (LLOQ) in the standard curve for both the
isomers was measured at a signal-to-noise ratio (SN) of ge 100 Figure 3 shows the
representative LC-MSMS chromatograms of (A) calibration curve of duloxetine (B)
Double blank plasma with out IS (C) Blank plasma with IS and (D) Duloxetine and
fluoxetine at LLOQ
D C REDDY et al 906
Figure 3 (a) Calibration curve of Duloxetine
Figure 3 (b) Double blank plasma (without IS)
Figure 3 (c) Blank plasma with IS
Res
ponse
Res
idual
Development and Validation of a LCMSMS Method for the Determination 907
Figure 3 (d) Duloxetine and Fluoxetine at LLOQ (mz) 298081540
Selectivity Accuracy and Precision
To establish the selectivity of the method for interference due to endogenous plasma
components from haemolysed lipidemic heparinised and K2 EDTA blank plasmas the
change in the area ratio (analyteIS) at LLOQ level was within 4-8 while the precision
(CV) in their measurement varied from 21 to 56 representative MRM ion
chromatograms extracted (A) blank human plasma (double blank) (B) blank plasma
fortified with IS (mz 31002rarr14807) duloxetine at LLOQ (mz 29808rarr1540) the
selectivity of the method The extraction procedure together with mass detection gave very
good selectivity for the analysis of both the drug and IS in the blank plasma No endogenous
interferences were found at the retention times of analytes and IS
The intra- and inter batch precision and accuracy were established from validation runs
performed at HQC MQC LQC and LLOQ QC levels The intra- and inter batch precision
ranged from 074 to 976 for duloxetineThe accuracy values were within 9714-10449
for both the analytes in intra- and inter batches The precision and accuracy values for intra-
and inter day experiments in plasma are shown in Table 1(A)
Table 1 (A) Comparison of intra- and inter-batch precision and accuracy for Duloxetine
QC ID Nominal
concentration ngmL
Intrabatch
n
Mean
concentration
observed ngmL
CV Accuracy
LLOQQC
LQC
MQC
HQC
0100
0300
42060
77750
6
0104
0307
41255
81240
770
556
379
503
10350
10229
9808
10449
QC ID Nominal
concentration ngmL
Intrabatch
n
Mean
concentration
observed ngmL
CV Accuracy
LLOQQC
LQC
MQC
HQC
0100
0300
42060
77750
24
0103
0308
40863
79684
685
702
521
552
10325
10259
9714
10248
D C REDDY et al 908
(B) Stability of Duloxetine under various conditions (n=6)
Storage conditions Nominal
Concentration ngmL
Mean calculated
conc (ngmL) plusmn SD
Mean
accuracy
Bench top stability
(After 567 h at ~ at 25degC
LQC
HQC
0300
77750
0298 plusmn 00090
75382 plusmn 26005
9928
9695
Freeze thaw stability
(3 Cycles)
LQC
HQC
0300
77750
0298 plusmn 00060
79717 plusmn 49832
9939
10253
Dry extract stability
(2582 h below 10degC)
LQC
HQC
0300
77750
0299 plusmn 00087
83442 plusmn 52259
9967
10732
Wet extract stability
(2643 h below 10degC)
LQC
HQC
0300
77750
0309 plusmn 00136
76943 plusmn 34201
10294
9896
Auto sampler stability
(2188 h 10degC)
LQC
HQC
0300
77750
0297 plusmn 00112
73886 plusmn 43496
9889
9500
Long term stability in plasma
at -70degC
(2366 days at -70degC)
LQC
HQC
0300
77750
0298 plusmn 00060
71398 plusmn 54829
9939
9183
Recovery and Stability Results
The relative recovery absolute matrix effect and process efficiency data at LQC MQC and
HQC levels is presented The recovery for drug and IS in human plasma was 8031 and
8109 Further the extent of matrix effect in different lots of plasma (spiked after
extraction) was within the acceptable limit as evident from the precision (CV) values in
Table 2
Stock solutions for short term stability of Duloxetine and IS were stable at room
temperature for minimum period of about 6 h and between 2 and 8degC for about 7 days
Duloxetine in control human plasma (bench top) at room temperature was stable at least for
567 h at ambient temperature and for minimum of three freeze and thaw cycles Auto
sampler stability of the spiked quality control samples maintained at 10degC was maintained
up to 2188 h Long-term stability of the spiked quality control samples stored at -70degC was
determined up to 2366 days The accuracy values for different stability experiment in
plasma are shown in Table 1(B)
Development and Validation of a LCMSMS Method for the Determination 909
Table 2 Absolute matrix effect relative recovery and process efficiency for Mirtazapine
Analyte
ISTD
Aa
(CV)
Bb
(CV)
Cc
(CV)
Absolute
matrix
effect
(ME) d
Relative
recovery
(RE) e
Process
efficiency
(PE) f
STD 1
Mirtazapine
Zolpidem
179
(831)
42068
(049)
188
(391)
37907
(135)
109
(432)
40711
(132)
9423
9041
8540
8434
8047
7625
STD 3
Mirtazapine
Zolpidem
567
(323)
52367
(131)
591
(413)
54113
(156)
460
(122)
40951
(180)
10438
10333
7777
7568
8118
7620
STD 5
Mirtazapine
Zolpidem
23157
(174)
50420
(194)
22868
(169)
49366
(165)
18300
(096)
41307
(068)
9875
9791
8002
8367
7902
8193
STD 6
Mirtazapine
Zolpidem
43730
(109)
47700
(153)
43102
(124)
47385
(156)
34517
(101)
38045
(107)
9857
9934
8008
8029
7893
7976
STD 8
Mirtazapine
Zolpidem
77701
(146)
42800
(161)
78734
(194)
43777
(223)
63689
(080)
38217
(533)
10133
10228
8089
8730
8197
8929
a Mean area response of six replicate samples prepared in Mobile phase (neat samples) b Mean area
response of six replicate samples prepared by spiking in post extracted blank c Mean area response of
six replicate samples prepared by spiking in plasma before extraction d Matrix effect Post extracted
mean responseAqueous (Neat) mean response x 100 e Recovery Extracted mean response Post
extracted mean response x 100 f Process efficiency Extracted mean response Aqueous Mean
response x 100
Ruggedness and Dilution Integrity
The results of ruggedness study for Duloxetine was well within the acceptance limit of 15
in Precision and 850-1150 in mean accuracy The precision and accuracy values for
both experiment at LLOQ LQC MQC and HQC levels for Duloxetine ranged from 34 to
76 and 971 to 1085 respectively
The dilution integrity experiment was performed with an aim to validate the dilution test
to be carried out on higher analyte concentration above the upper limit of quantification
(ULOQ) which maybe encountered during real subject sample analysis The precision and
accuracy values for 15th
and 110th
dilution ranged from 326 to 334 and 982 to 1097 for
Duloxetine
Application of the Method in Healthy Human Subjects
The validated method was successfully applied for the assay of Duloxetine in healthy male
Indian volunteers in the age group of 18-45 years Figure 4 shows the plasma concentration
vs time profile of Duloxetine human subjects under fating condition The method was
sensitive enough to monitor the Duloxetine plasma concentration up to 72 h Approximately
520 samples including the calibration and QC samples were within the acceptable limits
D C REDDY et al 910
The pharmacokinetic parameters viz Cmax Tmax AUC0t and AUC0 were
calculated for Duloxetine in test formulations (Figure 4) Shows the data of Mean plasma
concentration-time profile of Duloxetine Hydrochloride 60 mg Delayed Release Capsules
formulation to 12 healthy volunteers
0
5
10
15
20
25
30
35
40
45
0 6 12 18 24 30 36 42 48 54 60 66 72
Time (hr)
Linear mean plot of plasma Duloxetine Concentration Vs Time under Fasting conditions
Parameter Duloxetine
Cmax ngmL 4459418599
Tmax (h) 7251581
AUC0t 984702526502
AUC0 1027147572790
Figure 4 Mean plasma concentration-time profile of Duloxetine Hydrochloride 60 mg
Delayed Release Capsules formulation to 8 healthy volunteers
Conclusion
The proposed method successfully demonstrates chromatographic separation of duloxetine
from human plasma with high resolution The bioanalytical methodology for their
simultaneous determination is highly specific rugged and rapid for therapeutic drug
monitoring The method involved a simple and specific sample preparation by liquid-liquid
extraction followed by isocratic separation in 30 min The overall analysis time is proving
compared to other reported procedures for duloxetine
Acknowledgment
The authors gratefully acknowledge Dr AT Bapuji and Aurobindo Pharma Ltd Hyderabad
India for providing necessary facilities for carrying out this study
References
1 Maryadele J O editor The Merck Index 14th
Ed Whitehouse Station NJ Merck
and Co Inc 2006 p 3465
2 Mishra L Drugs today Vol 1 Delhi Lorina Publications 2006 p 489
3 Stephan A C Luc-Andre G Francois R V Peter R B Frank P B Melissa J J et al J
Neuropsychopharmacol 2003 28 1685ndash93
Mea
n c
once
ntr
atio
n
ngm
L
Time h
Development and Validation of a LCMSMS Method for the Determination 911
4 National Institute for Health and Clinical Excellence Clinical guideline 40 Urinary
incontinence London 2006
5 National Institute for Health and Clinical Excellence Clinical guideline 96
Neuropathic pain - pharmacological management London 2010
6 httpwwwdrugbankcadrugsDB00476
7 Wolfe F Smythe H A Yunus M B et al The American College of Rheumatology
Criteria for the Classification of Fibromyalgia Report of the Multicenter Criteri
Committee Arthritis Rheum 1990 33(2) 160ndash72 doi101002art1780330203
PMID 2306288
8 Douglas R Dolnak Treating Patients for Comorbid Depression Anxiety Disorders
and Somatic Illnesses 2006 JAOA 106 8
9 httpwwwcancergovdictionaryCdrID=589399
10 Senthamil Selvan P Gowda K V Mandal U Sam Solomon W D Pal T K J
Chromatogr B Analyt Technol Biomed Life Sci 2007 858(1-2) 269-75 Epub 2007
Sep 14
11 Mercolini L Mandrioli R Cazzolla R Amore M and Raggi M A J Chromatogr B
Analyt Technol Biomed Life Sci 2007 856(1-2) 81-7 Epub 2007 Jun 2
12 Musenga A Amore M Mandrioli R Kenndler E de Martino L and Raggi M A J
Chromatogr B Analyt Technol Biomed Life Sci 2009 877(11-12) 1126-32 Epub
2009 Mar 3
13 Waldschmitt C Vogel F Maurer C and Hiemke C Ther Drug Monit 2007
29(6) 767-72
14 Ning Ma Bi Kui Zhang Huan De Li Ben Mei Chen Ping Xu Feng Wang Rong
Hua Zhu Sheng Feng Da Xiong Xiang and Yun Gui Zhu Clinica Chimica Acta
2007 380(1-2) 100-105
15 FDA Guidance for industry Bioanalytical Method Validation US Department of
and Health Human Services Food and Drug Administration Centre for Drug
Evaluation and Research (CDER) and Centre for Veterinary Medicine (CVM) May
2001
16 Viswanathan C T Bansal S Booth B DeStefano A J Rose M J Sailstad J Shah V R
Skelly J P Swann P G and Weiner R APPS J 2007 9 E30
17 Matuszewski B K J Chromatogr B Analyt Technol Biomed Life Sci 2006 830(2)
293-300 Epub 2005 Nov 23
18 FDA Guidance for industry Bioavailability Studies for Orally Administered Drug-
Products-General Considerations US Department of Health and Human Services
Food and Drug Administration Center for drug evaluation and Research (CDER)
2000
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
D C REDDY et al 906
Figure 3 (a) Calibration curve of Duloxetine
Figure 3 (b) Double blank plasma (without IS)
Figure 3 (c) Blank plasma with IS
Res
ponse
Res
idual
Development and Validation of a LCMSMS Method for the Determination 907
Figure 3 (d) Duloxetine and Fluoxetine at LLOQ (mz) 298081540
Selectivity Accuracy and Precision
To establish the selectivity of the method for interference due to endogenous plasma
components from haemolysed lipidemic heparinised and K2 EDTA blank plasmas the
change in the area ratio (analyteIS) at LLOQ level was within 4-8 while the precision
(CV) in their measurement varied from 21 to 56 representative MRM ion
chromatograms extracted (A) blank human plasma (double blank) (B) blank plasma
fortified with IS (mz 31002rarr14807) duloxetine at LLOQ (mz 29808rarr1540) the
selectivity of the method The extraction procedure together with mass detection gave very
good selectivity for the analysis of both the drug and IS in the blank plasma No endogenous
interferences were found at the retention times of analytes and IS
The intra- and inter batch precision and accuracy were established from validation runs
performed at HQC MQC LQC and LLOQ QC levels The intra- and inter batch precision
ranged from 074 to 976 for duloxetineThe accuracy values were within 9714-10449
for both the analytes in intra- and inter batches The precision and accuracy values for intra-
and inter day experiments in plasma are shown in Table 1(A)
Table 1 (A) Comparison of intra- and inter-batch precision and accuracy for Duloxetine
QC ID Nominal
concentration ngmL
Intrabatch
n
Mean
concentration
observed ngmL
CV Accuracy
LLOQQC
LQC
MQC
HQC
0100
0300
42060
77750
6
0104
0307
41255
81240
770
556
379
503
10350
10229
9808
10449
QC ID Nominal
concentration ngmL
Intrabatch
n
Mean
concentration
observed ngmL
CV Accuracy
LLOQQC
LQC
MQC
HQC
0100
0300
42060
77750
24
0103
0308
40863
79684
685
702
521
552
10325
10259
9714
10248
D C REDDY et al 908
(B) Stability of Duloxetine under various conditions (n=6)
Storage conditions Nominal
Concentration ngmL
Mean calculated
conc (ngmL) plusmn SD
Mean
accuracy
Bench top stability
(After 567 h at ~ at 25degC
LQC
HQC
0300
77750
0298 plusmn 00090
75382 plusmn 26005
9928
9695
Freeze thaw stability
(3 Cycles)
LQC
HQC
0300
77750
0298 plusmn 00060
79717 plusmn 49832
9939
10253
Dry extract stability
(2582 h below 10degC)
LQC
HQC
0300
77750
0299 plusmn 00087
83442 plusmn 52259
9967
10732
Wet extract stability
(2643 h below 10degC)
LQC
HQC
0300
77750
0309 plusmn 00136
76943 plusmn 34201
10294
9896
Auto sampler stability
(2188 h 10degC)
LQC
HQC
0300
77750
0297 plusmn 00112
73886 plusmn 43496
9889
9500
Long term stability in plasma
at -70degC
(2366 days at -70degC)
LQC
HQC
0300
77750
0298 plusmn 00060
71398 plusmn 54829
9939
9183
Recovery and Stability Results
The relative recovery absolute matrix effect and process efficiency data at LQC MQC and
HQC levels is presented The recovery for drug and IS in human plasma was 8031 and
8109 Further the extent of matrix effect in different lots of plasma (spiked after
extraction) was within the acceptable limit as evident from the precision (CV) values in
Table 2
Stock solutions for short term stability of Duloxetine and IS were stable at room
temperature for minimum period of about 6 h and between 2 and 8degC for about 7 days
Duloxetine in control human plasma (bench top) at room temperature was stable at least for
567 h at ambient temperature and for minimum of three freeze and thaw cycles Auto
sampler stability of the spiked quality control samples maintained at 10degC was maintained
up to 2188 h Long-term stability of the spiked quality control samples stored at -70degC was
determined up to 2366 days The accuracy values for different stability experiment in
plasma are shown in Table 1(B)
Development and Validation of a LCMSMS Method for the Determination 909
Table 2 Absolute matrix effect relative recovery and process efficiency for Mirtazapine
Analyte
ISTD
Aa
(CV)
Bb
(CV)
Cc
(CV)
Absolute
matrix
effect
(ME) d
Relative
recovery
(RE) e
Process
efficiency
(PE) f
STD 1
Mirtazapine
Zolpidem
179
(831)
42068
(049)
188
(391)
37907
(135)
109
(432)
40711
(132)
9423
9041
8540
8434
8047
7625
STD 3
Mirtazapine
Zolpidem
567
(323)
52367
(131)
591
(413)
54113
(156)
460
(122)
40951
(180)
10438
10333
7777
7568
8118
7620
STD 5
Mirtazapine
Zolpidem
23157
(174)
50420
(194)
22868
(169)
49366
(165)
18300
(096)
41307
(068)
9875
9791
8002
8367
7902
8193
STD 6
Mirtazapine
Zolpidem
43730
(109)
47700
(153)
43102
(124)
47385
(156)
34517
(101)
38045
(107)
9857
9934
8008
8029
7893
7976
STD 8
Mirtazapine
Zolpidem
77701
(146)
42800
(161)
78734
(194)
43777
(223)
63689
(080)
38217
(533)
10133
10228
8089
8730
8197
8929
a Mean area response of six replicate samples prepared in Mobile phase (neat samples) b Mean area
response of six replicate samples prepared by spiking in post extracted blank c Mean area response of
six replicate samples prepared by spiking in plasma before extraction d Matrix effect Post extracted
mean responseAqueous (Neat) mean response x 100 e Recovery Extracted mean response Post
extracted mean response x 100 f Process efficiency Extracted mean response Aqueous Mean
response x 100
Ruggedness and Dilution Integrity
The results of ruggedness study for Duloxetine was well within the acceptance limit of 15
in Precision and 850-1150 in mean accuracy The precision and accuracy values for
both experiment at LLOQ LQC MQC and HQC levels for Duloxetine ranged from 34 to
76 and 971 to 1085 respectively
The dilution integrity experiment was performed with an aim to validate the dilution test
to be carried out on higher analyte concentration above the upper limit of quantification
(ULOQ) which maybe encountered during real subject sample analysis The precision and
accuracy values for 15th
and 110th
dilution ranged from 326 to 334 and 982 to 1097 for
Duloxetine
Application of the Method in Healthy Human Subjects
The validated method was successfully applied for the assay of Duloxetine in healthy male
Indian volunteers in the age group of 18-45 years Figure 4 shows the plasma concentration
vs time profile of Duloxetine human subjects under fating condition The method was
sensitive enough to monitor the Duloxetine plasma concentration up to 72 h Approximately
520 samples including the calibration and QC samples were within the acceptable limits
D C REDDY et al 910
The pharmacokinetic parameters viz Cmax Tmax AUC0t and AUC0 were
calculated for Duloxetine in test formulations (Figure 4) Shows the data of Mean plasma
concentration-time profile of Duloxetine Hydrochloride 60 mg Delayed Release Capsules
formulation to 12 healthy volunteers
0
5
10
15
20
25
30
35
40
45
0 6 12 18 24 30 36 42 48 54 60 66 72
Time (hr)
Linear mean plot of plasma Duloxetine Concentration Vs Time under Fasting conditions
Parameter Duloxetine
Cmax ngmL 4459418599
Tmax (h) 7251581
AUC0t 984702526502
AUC0 1027147572790
Figure 4 Mean plasma concentration-time profile of Duloxetine Hydrochloride 60 mg
Delayed Release Capsules formulation to 8 healthy volunteers
Conclusion
The proposed method successfully demonstrates chromatographic separation of duloxetine
from human plasma with high resolution The bioanalytical methodology for their
simultaneous determination is highly specific rugged and rapid for therapeutic drug
monitoring The method involved a simple and specific sample preparation by liquid-liquid
extraction followed by isocratic separation in 30 min The overall analysis time is proving
compared to other reported procedures for duloxetine
Acknowledgment
The authors gratefully acknowledge Dr AT Bapuji and Aurobindo Pharma Ltd Hyderabad
India for providing necessary facilities for carrying out this study
References
1 Maryadele J O editor The Merck Index 14th
Ed Whitehouse Station NJ Merck
and Co Inc 2006 p 3465
2 Mishra L Drugs today Vol 1 Delhi Lorina Publications 2006 p 489
3 Stephan A C Luc-Andre G Francois R V Peter R B Frank P B Melissa J J et al J
Neuropsychopharmacol 2003 28 1685ndash93
Mea
n c
once
ntr
atio
n
ngm
L
Time h
Development and Validation of a LCMSMS Method for the Determination 911
4 National Institute for Health and Clinical Excellence Clinical guideline 40 Urinary
incontinence London 2006
5 National Institute for Health and Clinical Excellence Clinical guideline 96
Neuropathic pain - pharmacological management London 2010
6 httpwwwdrugbankcadrugsDB00476
7 Wolfe F Smythe H A Yunus M B et al The American College of Rheumatology
Criteria for the Classification of Fibromyalgia Report of the Multicenter Criteri
Committee Arthritis Rheum 1990 33(2) 160ndash72 doi101002art1780330203
PMID 2306288
8 Douglas R Dolnak Treating Patients for Comorbid Depression Anxiety Disorders
and Somatic Illnesses 2006 JAOA 106 8
9 httpwwwcancergovdictionaryCdrID=589399
10 Senthamil Selvan P Gowda K V Mandal U Sam Solomon W D Pal T K J
Chromatogr B Analyt Technol Biomed Life Sci 2007 858(1-2) 269-75 Epub 2007
Sep 14
11 Mercolini L Mandrioli R Cazzolla R Amore M and Raggi M A J Chromatogr B
Analyt Technol Biomed Life Sci 2007 856(1-2) 81-7 Epub 2007 Jun 2
12 Musenga A Amore M Mandrioli R Kenndler E de Martino L and Raggi M A J
Chromatogr B Analyt Technol Biomed Life Sci 2009 877(11-12) 1126-32 Epub
2009 Mar 3
13 Waldschmitt C Vogel F Maurer C and Hiemke C Ther Drug Monit 2007
29(6) 767-72
14 Ning Ma Bi Kui Zhang Huan De Li Ben Mei Chen Ping Xu Feng Wang Rong
Hua Zhu Sheng Feng Da Xiong Xiang and Yun Gui Zhu Clinica Chimica Acta
2007 380(1-2) 100-105
15 FDA Guidance for industry Bioanalytical Method Validation US Department of
and Health Human Services Food and Drug Administration Centre for Drug
Evaluation and Research (CDER) and Centre for Veterinary Medicine (CVM) May
2001
16 Viswanathan C T Bansal S Booth B DeStefano A J Rose M J Sailstad J Shah V R
Skelly J P Swann P G and Weiner R APPS J 2007 9 E30
17 Matuszewski B K J Chromatogr B Analyt Technol Biomed Life Sci 2006 830(2)
293-300 Epub 2005 Nov 23
18 FDA Guidance for industry Bioavailability Studies for Orally Administered Drug-
Products-General Considerations US Department of Health and Human Services
Food and Drug Administration Center for drug evaluation and Research (CDER)
2000
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Development and Validation of a LCMSMS Method for the Determination 907
Figure 3 (d) Duloxetine and Fluoxetine at LLOQ (mz) 298081540
Selectivity Accuracy and Precision
To establish the selectivity of the method for interference due to endogenous plasma
components from haemolysed lipidemic heparinised and K2 EDTA blank plasmas the
change in the area ratio (analyteIS) at LLOQ level was within 4-8 while the precision
(CV) in their measurement varied from 21 to 56 representative MRM ion
chromatograms extracted (A) blank human plasma (double blank) (B) blank plasma
fortified with IS (mz 31002rarr14807) duloxetine at LLOQ (mz 29808rarr1540) the
selectivity of the method The extraction procedure together with mass detection gave very
good selectivity for the analysis of both the drug and IS in the blank plasma No endogenous
interferences were found at the retention times of analytes and IS
The intra- and inter batch precision and accuracy were established from validation runs
performed at HQC MQC LQC and LLOQ QC levels The intra- and inter batch precision
ranged from 074 to 976 for duloxetineThe accuracy values were within 9714-10449
for both the analytes in intra- and inter batches The precision and accuracy values for intra-
and inter day experiments in plasma are shown in Table 1(A)
Table 1 (A) Comparison of intra- and inter-batch precision and accuracy for Duloxetine
QC ID Nominal
concentration ngmL
Intrabatch
n
Mean
concentration
observed ngmL
CV Accuracy
LLOQQC
LQC
MQC
HQC
0100
0300
42060
77750
6
0104
0307
41255
81240
770
556
379
503
10350
10229
9808
10449
QC ID Nominal
concentration ngmL
Intrabatch
n
Mean
concentration
observed ngmL
CV Accuracy
LLOQQC
LQC
MQC
HQC
0100
0300
42060
77750
24
0103
0308
40863
79684
685
702
521
552
10325
10259
9714
10248
D C REDDY et al 908
(B) Stability of Duloxetine under various conditions (n=6)
Storage conditions Nominal
Concentration ngmL
Mean calculated
conc (ngmL) plusmn SD
Mean
accuracy
Bench top stability
(After 567 h at ~ at 25degC
LQC
HQC
0300
77750
0298 plusmn 00090
75382 plusmn 26005
9928
9695
Freeze thaw stability
(3 Cycles)
LQC
HQC
0300
77750
0298 plusmn 00060
79717 plusmn 49832
9939
10253
Dry extract stability
(2582 h below 10degC)
LQC
HQC
0300
77750
0299 plusmn 00087
83442 plusmn 52259
9967
10732
Wet extract stability
(2643 h below 10degC)
LQC
HQC
0300
77750
0309 plusmn 00136
76943 plusmn 34201
10294
9896
Auto sampler stability
(2188 h 10degC)
LQC
HQC
0300
77750
0297 plusmn 00112
73886 plusmn 43496
9889
9500
Long term stability in plasma
at -70degC
(2366 days at -70degC)
LQC
HQC
0300
77750
0298 plusmn 00060
71398 plusmn 54829
9939
9183
Recovery and Stability Results
The relative recovery absolute matrix effect and process efficiency data at LQC MQC and
HQC levels is presented The recovery for drug and IS in human plasma was 8031 and
8109 Further the extent of matrix effect in different lots of plasma (spiked after
extraction) was within the acceptable limit as evident from the precision (CV) values in
Table 2
Stock solutions for short term stability of Duloxetine and IS were stable at room
temperature for minimum period of about 6 h and between 2 and 8degC for about 7 days
Duloxetine in control human plasma (bench top) at room temperature was stable at least for
567 h at ambient temperature and for minimum of three freeze and thaw cycles Auto
sampler stability of the spiked quality control samples maintained at 10degC was maintained
up to 2188 h Long-term stability of the spiked quality control samples stored at -70degC was
determined up to 2366 days The accuracy values for different stability experiment in
plasma are shown in Table 1(B)
Development and Validation of a LCMSMS Method for the Determination 909
Table 2 Absolute matrix effect relative recovery and process efficiency for Mirtazapine
Analyte
ISTD
Aa
(CV)
Bb
(CV)
Cc
(CV)
Absolute
matrix
effect
(ME) d
Relative
recovery
(RE) e
Process
efficiency
(PE) f
STD 1
Mirtazapine
Zolpidem
179
(831)
42068
(049)
188
(391)
37907
(135)
109
(432)
40711
(132)
9423
9041
8540
8434
8047
7625
STD 3
Mirtazapine
Zolpidem
567
(323)
52367
(131)
591
(413)
54113
(156)
460
(122)
40951
(180)
10438
10333
7777
7568
8118
7620
STD 5
Mirtazapine
Zolpidem
23157
(174)
50420
(194)
22868
(169)
49366
(165)
18300
(096)
41307
(068)
9875
9791
8002
8367
7902
8193
STD 6
Mirtazapine
Zolpidem
43730
(109)
47700
(153)
43102
(124)
47385
(156)
34517
(101)
38045
(107)
9857
9934
8008
8029
7893
7976
STD 8
Mirtazapine
Zolpidem
77701
(146)
42800
(161)
78734
(194)
43777
(223)
63689
(080)
38217
(533)
10133
10228
8089
8730
8197
8929
a Mean area response of six replicate samples prepared in Mobile phase (neat samples) b Mean area
response of six replicate samples prepared by spiking in post extracted blank c Mean area response of
six replicate samples prepared by spiking in plasma before extraction d Matrix effect Post extracted
mean responseAqueous (Neat) mean response x 100 e Recovery Extracted mean response Post
extracted mean response x 100 f Process efficiency Extracted mean response Aqueous Mean
response x 100
Ruggedness and Dilution Integrity
The results of ruggedness study for Duloxetine was well within the acceptance limit of 15
in Precision and 850-1150 in mean accuracy The precision and accuracy values for
both experiment at LLOQ LQC MQC and HQC levels for Duloxetine ranged from 34 to
76 and 971 to 1085 respectively
The dilution integrity experiment was performed with an aim to validate the dilution test
to be carried out on higher analyte concentration above the upper limit of quantification
(ULOQ) which maybe encountered during real subject sample analysis The precision and
accuracy values for 15th
and 110th
dilution ranged from 326 to 334 and 982 to 1097 for
Duloxetine
Application of the Method in Healthy Human Subjects
The validated method was successfully applied for the assay of Duloxetine in healthy male
Indian volunteers in the age group of 18-45 years Figure 4 shows the plasma concentration
vs time profile of Duloxetine human subjects under fating condition The method was
sensitive enough to monitor the Duloxetine plasma concentration up to 72 h Approximately
520 samples including the calibration and QC samples were within the acceptable limits
D C REDDY et al 910
The pharmacokinetic parameters viz Cmax Tmax AUC0t and AUC0 were
calculated for Duloxetine in test formulations (Figure 4) Shows the data of Mean plasma
concentration-time profile of Duloxetine Hydrochloride 60 mg Delayed Release Capsules
formulation to 12 healthy volunteers
0
5
10
15
20
25
30
35
40
45
0 6 12 18 24 30 36 42 48 54 60 66 72
Time (hr)
Linear mean plot of plasma Duloxetine Concentration Vs Time under Fasting conditions
Parameter Duloxetine
Cmax ngmL 4459418599
Tmax (h) 7251581
AUC0t 984702526502
AUC0 1027147572790
Figure 4 Mean plasma concentration-time profile of Duloxetine Hydrochloride 60 mg
Delayed Release Capsules formulation to 8 healthy volunteers
Conclusion
The proposed method successfully demonstrates chromatographic separation of duloxetine
from human plasma with high resolution The bioanalytical methodology for their
simultaneous determination is highly specific rugged and rapid for therapeutic drug
monitoring The method involved a simple and specific sample preparation by liquid-liquid
extraction followed by isocratic separation in 30 min The overall analysis time is proving
compared to other reported procedures for duloxetine
Acknowledgment
The authors gratefully acknowledge Dr AT Bapuji and Aurobindo Pharma Ltd Hyderabad
India for providing necessary facilities for carrying out this study
References
1 Maryadele J O editor The Merck Index 14th
Ed Whitehouse Station NJ Merck
and Co Inc 2006 p 3465
2 Mishra L Drugs today Vol 1 Delhi Lorina Publications 2006 p 489
3 Stephan A C Luc-Andre G Francois R V Peter R B Frank P B Melissa J J et al J
Neuropsychopharmacol 2003 28 1685ndash93
Mea
n c
once
ntr
atio
n
ngm
L
Time h
Development and Validation of a LCMSMS Method for the Determination 911
4 National Institute for Health and Clinical Excellence Clinical guideline 40 Urinary
incontinence London 2006
5 National Institute for Health and Clinical Excellence Clinical guideline 96
Neuropathic pain - pharmacological management London 2010
6 httpwwwdrugbankcadrugsDB00476
7 Wolfe F Smythe H A Yunus M B et al The American College of Rheumatology
Criteria for the Classification of Fibromyalgia Report of the Multicenter Criteri
Committee Arthritis Rheum 1990 33(2) 160ndash72 doi101002art1780330203
PMID 2306288
8 Douglas R Dolnak Treating Patients for Comorbid Depression Anxiety Disorders
and Somatic Illnesses 2006 JAOA 106 8
9 httpwwwcancergovdictionaryCdrID=589399
10 Senthamil Selvan P Gowda K V Mandal U Sam Solomon W D Pal T K J
Chromatogr B Analyt Technol Biomed Life Sci 2007 858(1-2) 269-75 Epub 2007
Sep 14
11 Mercolini L Mandrioli R Cazzolla R Amore M and Raggi M A J Chromatogr B
Analyt Technol Biomed Life Sci 2007 856(1-2) 81-7 Epub 2007 Jun 2
12 Musenga A Amore M Mandrioli R Kenndler E de Martino L and Raggi M A J
Chromatogr B Analyt Technol Biomed Life Sci 2009 877(11-12) 1126-32 Epub
2009 Mar 3
13 Waldschmitt C Vogel F Maurer C and Hiemke C Ther Drug Monit 2007
29(6) 767-72
14 Ning Ma Bi Kui Zhang Huan De Li Ben Mei Chen Ping Xu Feng Wang Rong
Hua Zhu Sheng Feng Da Xiong Xiang and Yun Gui Zhu Clinica Chimica Acta
2007 380(1-2) 100-105
15 FDA Guidance for industry Bioanalytical Method Validation US Department of
and Health Human Services Food and Drug Administration Centre for Drug
Evaluation and Research (CDER) and Centre for Veterinary Medicine (CVM) May
2001
16 Viswanathan C T Bansal S Booth B DeStefano A J Rose M J Sailstad J Shah V R
Skelly J P Swann P G and Weiner R APPS J 2007 9 E30
17 Matuszewski B K J Chromatogr B Analyt Technol Biomed Life Sci 2006 830(2)
293-300 Epub 2005 Nov 23
18 FDA Guidance for industry Bioavailability Studies for Orally Administered Drug-
Products-General Considerations US Department of Health and Human Services
Food and Drug Administration Center for drug evaluation and Research (CDER)
2000
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
D C REDDY et al 908
(B) Stability of Duloxetine under various conditions (n=6)
Storage conditions Nominal
Concentration ngmL
Mean calculated
conc (ngmL) plusmn SD
Mean
accuracy
Bench top stability
(After 567 h at ~ at 25degC
LQC
HQC
0300
77750
0298 plusmn 00090
75382 plusmn 26005
9928
9695
Freeze thaw stability
(3 Cycles)
LQC
HQC
0300
77750
0298 plusmn 00060
79717 plusmn 49832
9939
10253
Dry extract stability
(2582 h below 10degC)
LQC
HQC
0300
77750
0299 plusmn 00087
83442 plusmn 52259
9967
10732
Wet extract stability
(2643 h below 10degC)
LQC
HQC
0300
77750
0309 plusmn 00136
76943 plusmn 34201
10294
9896
Auto sampler stability
(2188 h 10degC)
LQC
HQC
0300
77750
0297 plusmn 00112
73886 plusmn 43496
9889
9500
Long term stability in plasma
at -70degC
(2366 days at -70degC)
LQC
HQC
0300
77750
0298 plusmn 00060
71398 plusmn 54829
9939
9183
Recovery and Stability Results
The relative recovery absolute matrix effect and process efficiency data at LQC MQC and
HQC levels is presented The recovery for drug and IS in human plasma was 8031 and
8109 Further the extent of matrix effect in different lots of plasma (spiked after
extraction) was within the acceptable limit as evident from the precision (CV) values in
Table 2
Stock solutions for short term stability of Duloxetine and IS were stable at room
temperature for minimum period of about 6 h and between 2 and 8degC for about 7 days
Duloxetine in control human plasma (bench top) at room temperature was stable at least for
567 h at ambient temperature and for minimum of three freeze and thaw cycles Auto
sampler stability of the spiked quality control samples maintained at 10degC was maintained
up to 2188 h Long-term stability of the spiked quality control samples stored at -70degC was
determined up to 2366 days The accuracy values for different stability experiment in
plasma are shown in Table 1(B)
Development and Validation of a LCMSMS Method for the Determination 909
Table 2 Absolute matrix effect relative recovery and process efficiency for Mirtazapine
Analyte
ISTD
Aa
(CV)
Bb
(CV)
Cc
(CV)
Absolute
matrix
effect
(ME) d
Relative
recovery
(RE) e
Process
efficiency
(PE) f
STD 1
Mirtazapine
Zolpidem
179
(831)
42068
(049)
188
(391)
37907
(135)
109
(432)
40711
(132)
9423
9041
8540
8434
8047
7625
STD 3
Mirtazapine
Zolpidem
567
(323)
52367
(131)
591
(413)
54113
(156)
460
(122)
40951
(180)
10438
10333
7777
7568
8118
7620
STD 5
Mirtazapine
Zolpidem
23157
(174)
50420
(194)
22868
(169)
49366
(165)
18300
(096)
41307
(068)
9875
9791
8002
8367
7902
8193
STD 6
Mirtazapine
Zolpidem
43730
(109)
47700
(153)
43102
(124)
47385
(156)
34517
(101)
38045
(107)
9857
9934
8008
8029
7893
7976
STD 8
Mirtazapine
Zolpidem
77701
(146)
42800
(161)
78734
(194)
43777
(223)
63689
(080)
38217
(533)
10133
10228
8089
8730
8197
8929
a Mean area response of six replicate samples prepared in Mobile phase (neat samples) b Mean area
response of six replicate samples prepared by spiking in post extracted blank c Mean area response of
six replicate samples prepared by spiking in plasma before extraction d Matrix effect Post extracted
mean responseAqueous (Neat) mean response x 100 e Recovery Extracted mean response Post
extracted mean response x 100 f Process efficiency Extracted mean response Aqueous Mean
response x 100
Ruggedness and Dilution Integrity
The results of ruggedness study for Duloxetine was well within the acceptance limit of 15
in Precision and 850-1150 in mean accuracy The precision and accuracy values for
both experiment at LLOQ LQC MQC and HQC levels for Duloxetine ranged from 34 to
76 and 971 to 1085 respectively
The dilution integrity experiment was performed with an aim to validate the dilution test
to be carried out on higher analyte concentration above the upper limit of quantification
(ULOQ) which maybe encountered during real subject sample analysis The precision and
accuracy values for 15th
and 110th
dilution ranged from 326 to 334 and 982 to 1097 for
Duloxetine
Application of the Method in Healthy Human Subjects
The validated method was successfully applied for the assay of Duloxetine in healthy male
Indian volunteers in the age group of 18-45 years Figure 4 shows the plasma concentration
vs time profile of Duloxetine human subjects under fating condition The method was
sensitive enough to monitor the Duloxetine plasma concentration up to 72 h Approximately
520 samples including the calibration and QC samples were within the acceptable limits
D C REDDY et al 910
The pharmacokinetic parameters viz Cmax Tmax AUC0t and AUC0 were
calculated for Duloxetine in test formulations (Figure 4) Shows the data of Mean plasma
concentration-time profile of Duloxetine Hydrochloride 60 mg Delayed Release Capsules
formulation to 12 healthy volunteers
0
5
10
15
20
25
30
35
40
45
0 6 12 18 24 30 36 42 48 54 60 66 72
Time (hr)
Linear mean plot of plasma Duloxetine Concentration Vs Time under Fasting conditions
Parameter Duloxetine
Cmax ngmL 4459418599
Tmax (h) 7251581
AUC0t 984702526502
AUC0 1027147572790
Figure 4 Mean plasma concentration-time profile of Duloxetine Hydrochloride 60 mg
Delayed Release Capsules formulation to 8 healthy volunteers
Conclusion
The proposed method successfully demonstrates chromatographic separation of duloxetine
from human plasma with high resolution The bioanalytical methodology for their
simultaneous determination is highly specific rugged and rapid for therapeutic drug
monitoring The method involved a simple and specific sample preparation by liquid-liquid
extraction followed by isocratic separation in 30 min The overall analysis time is proving
compared to other reported procedures for duloxetine
Acknowledgment
The authors gratefully acknowledge Dr AT Bapuji and Aurobindo Pharma Ltd Hyderabad
India for providing necessary facilities for carrying out this study
References
1 Maryadele J O editor The Merck Index 14th
Ed Whitehouse Station NJ Merck
and Co Inc 2006 p 3465
2 Mishra L Drugs today Vol 1 Delhi Lorina Publications 2006 p 489
3 Stephan A C Luc-Andre G Francois R V Peter R B Frank P B Melissa J J et al J
Neuropsychopharmacol 2003 28 1685ndash93
Mea
n c
once
ntr
atio
n
ngm
L
Time h
Development and Validation of a LCMSMS Method for the Determination 911
4 National Institute for Health and Clinical Excellence Clinical guideline 40 Urinary
incontinence London 2006
5 National Institute for Health and Clinical Excellence Clinical guideline 96
Neuropathic pain - pharmacological management London 2010
6 httpwwwdrugbankcadrugsDB00476
7 Wolfe F Smythe H A Yunus M B et al The American College of Rheumatology
Criteria for the Classification of Fibromyalgia Report of the Multicenter Criteri
Committee Arthritis Rheum 1990 33(2) 160ndash72 doi101002art1780330203
PMID 2306288
8 Douglas R Dolnak Treating Patients for Comorbid Depression Anxiety Disorders
and Somatic Illnesses 2006 JAOA 106 8
9 httpwwwcancergovdictionaryCdrID=589399
10 Senthamil Selvan P Gowda K V Mandal U Sam Solomon W D Pal T K J
Chromatogr B Analyt Technol Biomed Life Sci 2007 858(1-2) 269-75 Epub 2007
Sep 14
11 Mercolini L Mandrioli R Cazzolla R Amore M and Raggi M A J Chromatogr B
Analyt Technol Biomed Life Sci 2007 856(1-2) 81-7 Epub 2007 Jun 2
12 Musenga A Amore M Mandrioli R Kenndler E de Martino L and Raggi M A J
Chromatogr B Analyt Technol Biomed Life Sci 2009 877(11-12) 1126-32 Epub
2009 Mar 3
13 Waldschmitt C Vogel F Maurer C and Hiemke C Ther Drug Monit 2007
29(6) 767-72
14 Ning Ma Bi Kui Zhang Huan De Li Ben Mei Chen Ping Xu Feng Wang Rong
Hua Zhu Sheng Feng Da Xiong Xiang and Yun Gui Zhu Clinica Chimica Acta
2007 380(1-2) 100-105
15 FDA Guidance for industry Bioanalytical Method Validation US Department of
and Health Human Services Food and Drug Administration Centre for Drug
Evaluation and Research (CDER) and Centre for Veterinary Medicine (CVM) May
2001
16 Viswanathan C T Bansal S Booth B DeStefano A J Rose M J Sailstad J Shah V R
Skelly J P Swann P G and Weiner R APPS J 2007 9 E30
17 Matuszewski B K J Chromatogr B Analyt Technol Biomed Life Sci 2006 830(2)
293-300 Epub 2005 Nov 23
18 FDA Guidance for industry Bioavailability Studies for Orally Administered Drug-
Products-General Considerations US Department of Health and Human Services
Food and Drug Administration Center for drug evaluation and Research (CDER)
2000
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Development and Validation of a LCMSMS Method for the Determination 909
Table 2 Absolute matrix effect relative recovery and process efficiency for Mirtazapine
Analyte
ISTD
Aa
(CV)
Bb
(CV)
Cc
(CV)
Absolute
matrix
effect
(ME) d
Relative
recovery
(RE) e
Process
efficiency
(PE) f
STD 1
Mirtazapine
Zolpidem
179
(831)
42068
(049)
188
(391)
37907
(135)
109
(432)
40711
(132)
9423
9041
8540
8434
8047
7625
STD 3
Mirtazapine
Zolpidem
567
(323)
52367
(131)
591
(413)
54113
(156)
460
(122)
40951
(180)
10438
10333
7777
7568
8118
7620
STD 5
Mirtazapine
Zolpidem
23157
(174)
50420
(194)
22868
(169)
49366
(165)
18300
(096)
41307
(068)
9875
9791
8002
8367
7902
8193
STD 6
Mirtazapine
Zolpidem
43730
(109)
47700
(153)
43102
(124)
47385
(156)
34517
(101)
38045
(107)
9857
9934
8008
8029
7893
7976
STD 8
Mirtazapine
Zolpidem
77701
(146)
42800
(161)
78734
(194)
43777
(223)
63689
(080)
38217
(533)
10133
10228
8089
8730
8197
8929
a Mean area response of six replicate samples prepared in Mobile phase (neat samples) b Mean area
response of six replicate samples prepared by spiking in post extracted blank c Mean area response of
six replicate samples prepared by spiking in plasma before extraction d Matrix effect Post extracted
mean responseAqueous (Neat) mean response x 100 e Recovery Extracted mean response Post
extracted mean response x 100 f Process efficiency Extracted mean response Aqueous Mean
response x 100
Ruggedness and Dilution Integrity
The results of ruggedness study for Duloxetine was well within the acceptance limit of 15
in Precision and 850-1150 in mean accuracy The precision and accuracy values for
both experiment at LLOQ LQC MQC and HQC levels for Duloxetine ranged from 34 to
76 and 971 to 1085 respectively
The dilution integrity experiment was performed with an aim to validate the dilution test
to be carried out on higher analyte concentration above the upper limit of quantification
(ULOQ) which maybe encountered during real subject sample analysis The precision and
accuracy values for 15th
and 110th
dilution ranged from 326 to 334 and 982 to 1097 for
Duloxetine
Application of the Method in Healthy Human Subjects
The validated method was successfully applied for the assay of Duloxetine in healthy male
Indian volunteers in the age group of 18-45 years Figure 4 shows the plasma concentration
vs time profile of Duloxetine human subjects under fating condition The method was
sensitive enough to monitor the Duloxetine plasma concentration up to 72 h Approximately
520 samples including the calibration and QC samples were within the acceptable limits
D C REDDY et al 910
The pharmacokinetic parameters viz Cmax Tmax AUC0t and AUC0 were
calculated for Duloxetine in test formulations (Figure 4) Shows the data of Mean plasma
concentration-time profile of Duloxetine Hydrochloride 60 mg Delayed Release Capsules
formulation to 12 healthy volunteers
0
5
10
15
20
25
30
35
40
45
0 6 12 18 24 30 36 42 48 54 60 66 72
Time (hr)
Linear mean plot of plasma Duloxetine Concentration Vs Time under Fasting conditions
Parameter Duloxetine
Cmax ngmL 4459418599
Tmax (h) 7251581
AUC0t 984702526502
AUC0 1027147572790
Figure 4 Mean plasma concentration-time profile of Duloxetine Hydrochloride 60 mg
Delayed Release Capsules formulation to 8 healthy volunteers
Conclusion
The proposed method successfully demonstrates chromatographic separation of duloxetine
from human plasma with high resolution The bioanalytical methodology for their
simultaneous determination is highly specific rugged and rapid for therapeutic drug
monitoring The method involved a simple and specific sample preparation by liquid-liquid
extraction followed by isocratic separation in 30 min The overall analysis time is proving
compared to other reported procedures for duloxetine
Acknowledgment
The authors gratefully acknowledge Dr AT Bapuji and Aurobindo Pharma Ltd Hyderabad
India for providing necessary facilities for carrying out this study
References
1 Maryadele J O editor The Merck Index 14th
Ed Whitehouse Station NJ Merck
and Co Inc 2006 p 3465
2 Mishra L Drugs today Vol 1 Delhi Lorina Publications 2006 p 489
3 Stephan A C Luc-Andre G Francois R V Peter R B Frank P B Melissa J J et al J
Neuropsychopharmacol 2003 28 1685ndash93
Mea
n c
once
ntr
atio
n
ngm
L
Time h
Development and Validation of a LCMSMS Method for the Determination 911
4 National Institute for Health and Clinical Excellence Clinical guideline 40 Urinary
incontinence London 2006
5 National Institute for Health and Clinical Excellence Clinical guideline 96
Neuropathic pain - pharmacological management London 2010
6 httpwwwdrugbankcadrugsDB00476
7 Wolfe F Smythe H A Yunus M B et al The American College of Rheumatology
Criteria for the Classification of Fibromyalgia Report of the Multicenter Criteri
Committee Arthritis Rheum 1990 33(2) 160ndash72 doi101002art1780330203
PMID 2306288
8 Douglas R Dolnak Treating Patients for Comorbid Depression Anxiety Disorders
and Somatic Illnesses 2006 JAOA 106 8
9 httpwwwcancergovdictionaryCdrID=589399
10 Senthamil Selvan P Gowda K V Mandal U Sam Solomon W D Pal T K J
Chromatogr B Analyt Technol Biomed Life Sci 2007 858(1-2) 269-75 Epub 2007
Sep 14
11 Mercolini L Mandrioli R Cazzolla R Amore M and Raggi M A J Chromatogr B
Analyt Technol Biomed Life Sci 2007 856(1-2) 81-7 Epub 2007 Jun 2
12 Musenga A Amore M Mandrioli R Kenndler E de Martino L and Raggi M A J
Chromatogr B Analyt Technol Biomed Life Sci 2009 877(11-12) 1126-32 Epub
2009 Mar 3
13 Waldschmitt C Vogel F Maurer C and Hiemke C Ther Drug Monit 2007
29(6) 767-72
14 Ning Ma Bi Kui Zhang Huan De Li Ben Mei Chen Ping Xu Feng Wang Rong
Hua Zhu Sheng Feng Da Xiong Xiang and Yun Gui Zhu Clinica Chimica Acta
2007 380(1-2) 100-105
15 FDA Guidance for industry Bioanalytical Method Validation US Department of
and Health Human Services Food and Drug Administration Centre for Drug
Evaluation and Research (CDER) and Centre for Veterinary Medicine (CVM) May
2001
16 Viswanathan C T Bansal S Booth B DeStefano A J Rose M J Sailstad J Shah V R
Skelly J P Swann P G and Weiner R APPS J 2007 9 E30
17 Matuszewski B K J Chromatogr B Analyt Technol Biomed Life Sci 2006 830(2)
293-300 Epub 2005 Nov 23
18 FDA Guidance for industry Bioavailability Studies for Orally Administered Drug-
Products-General Considerations US Department of Health and Human Services
Food and Drug Administration Center for drug evaluation and Research (CDER)
2000
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
D C REDDY et al 910
The pharmacokinetic parameters viz Cmax Tmax AUC0t and AUC0 were
calculated for Duloxetine in test formulations (Figure 4) Shows the data of Mean plasma
concentration-time profile of Duloxetine Hydrochloride 60 mg Delayed Release Capsules
formulation to 12 healthy volunteers
0
5
10
15
20
25
30
35
40
45
0 6 12 18 24 30 36 42 48 54 60 66 72
Time (hr)
Linear mean plot of plasma Duloxetine Concentration Vs Time under Fasting conditions
Parameter Duloxetine
Cmax ngmL 4459418599
Tmax (h) 7251581
AUC0t 984702526502
AUC0 1027147572790
Figure 4 Mean plasma concentration-time profile of Duloxetine Hydrochloride 60 mg
Delayed Release Capsules formulation to 8 healthy volunteers
Conclusion
The proposed method successfully demonstrates chromatographic separation of duloxetine
from human plasma with high resolution The bioanalytical methodology for their
simultaneous determination is highly specific rugged and rapid for therapeutic drug
monitoring The method involved a simple and specific sample preparation by liquid-liquid
extraction followed by isocratic separation in 30 min The overall analysis time is proving
compared to other reported procedures for duloxetine
Acknowledgment
The authors gratefully acknowledge Dr AT Bapuji and Aurobindo Pharma Ltd Hyderabad
India for providing necessary facilities for carrying out this study
References
1 Maryadele J O editor The Merck Index 14th
Ed Whitehouse Station NJ Merck
and Co Inc 2006 p 3465
2 Mishra L Drugs today Vol 1 Delhi Lorina Publications 2006 p 489
3 Stephan A C Luc-Andre G Francois R V Peter R B Frank P B Melissa J J et al J
Neuropsychopharmacol 2003 28 1685ndash93
Mea
n c
once
ntr
atio
n
ngm
L
Time h
Development and Validation of a LCMSMS Method for the Determination 911
4 National Institute for Health and Clinical Excellence Clinical guideline 40 Urinary
incontinence London 2006
5 National Institute for Health and Clinical Excellence Clinical guideline 96
Neuropathic pain - pharmacological management London 2010
6 httpwwwdrugbankcadrugsDB00476
7 Wolfe F Smythe H A Yunus M B et al The American College of Rheumatology
Criteria for the Classification of Fibromyalgia Report of the Multicenter Criteri
Committee Arthritis Rheum 1990 33(2) 160ndash72 doi101002art1780330203
PMID 2306288
8 Douglas R Dolnak Treating Patients for Comorbid Depression Anxiety Disorders
and Somatic Illnesses 2006 JAOA 106 8
9 httpwwwcancergovdictionaryCdrID=589399
10 Senthamil Selvan P Gowda K V Mandal U Sam Solomon W D Pal T K J
Chromatogr B Analyt Technol Biomed Life Sci 2007 858(1-2) 269-75 Epub 2007
Sep 14
11 Mercolini L Mandrioli R Cazzolla R Amore M and Raggi M A J Chromatogr B
Analyt Technol Biomed Life Sci 2007 856(1-2) 81-7 Epub 2007 Jun 2
12 Musenga A Amore M Mandrioli R Kenndler E de Martino L and Raggi M A J
Chromatogr B Analyt Technol Biomed Life Sci 2009 877(11-12) 1126-32 Epub
2009 Mar 3
13 Waldschmitt C Vogel F Maurer C and Hiemke C Ther Drug Monit 2007
29(6) 767-72
14 Ning Ma Bi Kui Zhang Huan De Li Ben Mei Chen Ping Xu Feng Wang Rong
Hua Zhu Sheng Feng Da Xiong Xiang and Yun Gui Zhu Clinica Chimica Acta
2007 380(1-2) 100-105
15 FDA Guidance for industry Bioanalytical Method Validation US Department of
and Health Human Services Food and Drug Administration Centre for Drug
Evaluation and Research (CDER) and Centre for Veterinary Medicine (CVM) May
2001
16 Viswanathan C T Bansal S Booth B DeStefano A J Rose M J Sailstad J Shah V R
Skelly J P Swann P G and Weiner R APPS J 2007 9 E30
17 Matuszewski B K J Chromatogr B Analyt Technol Biomed Life Sci 2006 830(2)
293-300 Epub 2005 Nov 23
18 FDA Guidance for industry Bioavailability Studies for Orally Administered Drug-
Products-General Considerations US Department of Health and Human Services
Food and Drug Administration Center for drug evaluation and Research (CDER)
2000
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Development and Validation of a LCMSMS Method for the Determination 911
4 National Institute for Health and Clinical Excellence Clinical guideline 40 Urinary
incontinence London 2006
5 National Institute for Health and Clinical Excellence Clinical guideline 96
Neuropathic pain - pharmacological management London 2010
6 httpwwwdrugbankcadrugsDB00476
7 Wolfe F Smythe H A Yunus M B et al The American College of Rheumatology
Criteria for the Classification of Fibromyalgia Report of the Multicenter Criteri
Committee Arthritis Rheum 1990 33(2) 160ndash72 doi101002art1780330203
PMID 2306288
8 Douglas R Dolnak Treating Patients for Comorbid Depression Anxiety Disorders
and Somatic Illnesses 2006 JAOA 106 8
9 httpwwwcancergovdictionaryCdrID=589399
10 Senthamil Selvan P Gowda K V Mandal U Sam Solomon W D Pal T K J
Chromatogr B Analyt Technol Biomed Life Sci 2007 858(1-2) 269-75 Epub 2007
Sep 14
11 Mercolini L Mandrioli R Cazzolla R Amore M and Raggi M A J Chromatogr B
Analyt Technol Biomed Life Sci 2007 856(1-2) 81-7 Epub 2007 Jun 2
12 Musenga A Amore M Mandrioli R Kenndler E de Martino L and Raggi M A J
Chromatogr B Analyt Technol Biomed Life Sci 2009 877(11-12) 1126-32 Epub
2009 Mar 3
13 Waldschmitt C Vogel F Maurer C and Hiemke C Ther Drug Monit 2007
29(6) 767-72
14 Ning Ma Bi Kui Zhang Huan De Li Ben Mei Chen Ping Xu Feng Wang Rong
Hua Zhu Sheng Feng Da Xiong Xiang and Yun Gui Zhu Clinica Chimica Acta
2007 380(1-2) 100-105
15 FDA Guidance for industry Bioanalytical Method Validation US Department of
and Health Human Services Food and Drug Administration Centre for Drug
Evaluation and Research (CDER) and Centre for Veterinary Medicine (CVM) May
2001
16 Viswanathan C T Bansal S Booth B DeStefano A J Rose M J Sailstad J Shah V R
Skelly J P Swann P G and Weiner R APPS J 2007 9 E30
17 Matuszewski B K J Chromatogr B Analyt Technol Biomed Life Sci 2006 830(2)
293-300 Epub 2005 Nov 23
18 FDA Guidance for industry Bioavailability Studies for Orally Administered Drug-
Products-General Considerations US Department of Health and Human Services
Food and Drug Administration Center for drug evaluation and Research (CDER)
2000
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014