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

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

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

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

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

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

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

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

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

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

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

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

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