method development for related substances of atorvastatin & clopidogrel...
TRANSCRIPT
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CHAPTER 2
Method Development for Related substances of Atorvastatin
& Clopidogrel in combination by UPLC
Introduction
Atorvastatin Calcium
Formula : C33H34FN2O5 · 0.5 Ca
CAS Number : 134523-03-8
Molecular Weight : 604.69
Synonyms : (-)-Monocalcium bis[(3R, 5R)-7-[2-(4-fluorophenyl)-5-
isopropyl- 3-phenyl- 4-phenylcarbamoyl-1H- pyrrol-1-yl]-
3,5- dihydroxyheptanoate] Trihydrate; [R-(R',R')]-
2-(4-fluorophenyl)- beta,delta- dihydroxy-5-( 1-
methylethyl)-3-phenyl-4 [(phenylamino)
carbonyl]- lH-pyrrole-1-heptanoic acid, calcium salt (2:1)
Trihydrate;
Melting point : 159.2-160.7 °C, 176 to 178 for a different polymorph
Atorvastatin is a drug belonging to the category known as ‗statins‘. Action wise
these are also called as ‗antihyperlipidemics‘. It is used for lowering blood lipids. It
also stabilizes plaque and prevents strokes through anti-inflammatory and other
mechanisms. Like all statins, Atorvastatin works by inhibiting HMG-CoA reductase,
an enzyme found in liver tissue that plays a key role in production of cholesterol in
the body. It is an efficient drug widely used for cardiac patients. It is also effective in
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secondary prevention in people with coronary heart disease and multiple risk factors
for myocardial infarction, stroke, unstable angina1 and revascularization
2,3
Clopidogrel Hydrogensulfate
Formula : C16H16ClNO2S · H2SO4
CAS Number : 135046-48-9
Molecular Weight : 419.90
Synonyms : (S)-(+)-Methyl (2-chlorophenyl)(6,7-dihydro-4H-thieno[3,2-
c]pyridin-5-yl)acetate hydrogen sulfate, Clopidogrel
hydrogen sulfate; sulfate, Clopidogrel bisulfate.
Melting point : 184°C
Clopidogrel bisulfate is a thienopyridine class anti-platelet agent, a drug that inhibits
the ability of platelets to clump together to form blood clot. Clopidogrel prevents
blood clots by irreversibly binding to the P2Y12 receptor on platelets cell membranes,
preventing adenosine diphosphate (ADP) from activating platelets and eventual cross-
linking by the protein fibrin4. It is administered orally to inhibit blood clots in
coronary artery disease, peripheral vascular disease, and cerebrovascular disease.
Clopidogrel is a pro-drug activated in the liver by cytochrome P450 enzymes,
including CYP2C19. Due to opening of the thiophene ring, the chemical structure of
the active moiety has three sites that are stereochemically relevant, making a total of
eight possible isomers. These are: a stereocentre at C4 (attached to the —SH thiol
group), a double bond at C3—C16, and the original stereocentre at C7. It is also used,
along with aspirin, for the prevention of thrombosis after placement of intracoronary
stent5 or as an alternative antiplatelet drug for patients who are intolerant to aspirin.
6
Combination therapy of Atorvastatin and Clopidogrel
Patients who have undergone heart treatment such as bypass surgery or have been
placed a stent in their arteries need to undergo treatment comprising both of
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antihyperlipidemics such as well as anti clotting agents. Atorvastatin and Clopidogrel
are one of the most preferred and prescribed drugs in this class. A wide research has
been carried out to ascertain if there is any incompatibility between these two drugs
and found that there is absolutely no interference or lowering of the activity of any of
them. Literature survey also states that the activity of Clopidogrel is even enhanced in
presence of Atorvaststin7,
Many articles are available for simultaneous assay determinations of both these drugs
but have not come across any simultaneous determination method for determination
of all the impurities. This experiment aims to achieve very short run times which have
not yet been reported.
A few methods have been reported for Atorvastatin -Sıdıka Ertürk et al reported ―An
HPLC method for the determination of Atorvastatin and its impurities in bulk drug
and tablets‖8
. BG Chaudhari et al reported ―Stability indicating RP-HPLC method for
simultaneous determination of Atorvastatin and amlodipine from their combination
drug products‖9. A.A. Kadav et al reported ‗Stability indicating UPLC method for
simultaneous determination of Atorvastatin, fenofibrate and their degradation
products in tablets‖10
. Raja Kumar Seshadri et al reported ―Simultaneous Quantitative
Determination of Metoprolol, Atorvastatin and Ramipril in Capsules by a Validated
Stability-Indicating RP-UPLC Method‖11
. D. N. Vora et al reported ―Validated Ultra
HPLC Method for the Simultaneous Determination of Atorvastatin, Aspirin, and their
Degradation Products in Capsules‖12
.
A few methods have also been reported for Clopidogrel- A Mitakos, et al reported ―A
validated LC method for the determination of Clopidogrel in pharmaceutical
preparations‖13
, SS Singh et al reported ―Estimation of carboxylic acid metabolite of
Clopidogrel in Wistar rat plasma by HPLC and its application to a pharmacokinetic
study‖14
, JM Pereillo et al reported Structure and stereochemistry of the active
metabolite of Clopidogrel‖15
.Ramakrishna et al reported ―Quantification of
Clopidogrel in human plasma by sensitive liquid chromatography/tandem mass
spectrometry‖16
. K Anandakumar et al reported ―RP-HPLC analysis of aspirin and
Clopidogrel bisulphate in combination‖17
. H.O. Kaila reported ―A Simple and Rapid
Ultra-Performance Liquid Chromatographic Assay Method for the Simultaneous
determination of Aspirin, Clopidogrel Bisulphate and Atorvastatin Calcium in
Capsule Dosage Form‖18
. Xiuli Yang et al reported ―UPLC for the Determination of
Clopidogrel in Dog Plasma by Tandem Quadrupole MS: Application to a
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Pharmacokinetic Study‖19
. Muhammad. K. Javed et al reported ―Development and
validation of HPLC-UV method for the determination of clopidogrel in
pharmaceutical dosage form and human plasma‖20
.
To the best of the author‘s knowledge no method is available in the literature for
simultaneous determination of Atorvastatin, Clopidogrel and their impurities by
UPLC. The current chapter thus describes a unique and novel method for
simultaneous determination of these drugs along with all their degradation products
using UPLC.
A usual HPLC method for separating 13 peaks along with several unknown
degradation product peaks would require run times of about 60 – 70 minutes. We
succeeded in reducing the run time to less than 12 minutes.
The drug substances, standards and impurities required for this work were obtained
from Dr Reddy‘s laboratories ltd. The drug product used for this exercise was
obtained commercially from the market. The brand called Storclop which contains
Atorvastatin 10mg and Clopidogrel 75mg.
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Structure Confirmation of Atorvastatin Calcium
The following physicochemical techniques were used to confirm the structure of
Atorvastatin calcium. These are given below
Thermal study
UV study
FTIR
NMR spectrophotometry
Mass spectrophotometry
1. Thermal Analysis
2.49 mg of Atorvastatin Calcium was weighed into an aluminum crucible of 25µL
and placed in the DSC. The thermogram was recorded from 30ºC to 300ºC which is
carried out under nitrogen atmosphere at 50mL/min, at 10ºC /min. The thermogram
exhibited broad endotherm at 54.3 ºC which can be attributed to loss of water
molecules. A small endotherm was observed at 161 ºC
2. UV Study
The Ultraviolet spectrum was recorded from 200 nm to 400 nm, with API
concentration of 0.0015% in methanol. The spectrum showed two λmax at 203 and
246 nm.
3. FTIR Study
The FTIR of spectrum of Atorvastatin calcium was recorded by preparation of pellet
with KBr. The assignments are given in table No 2.1.
Table 2.1 FTIR assignments for Atorvastatin.
Wave number (cm-1
) Assignment Mode of vibration
3402 -Q-H/-N-H Stretching
2962 Aliphatic -C-H Stretching
1651 -C=O Stretching
1595,1531 Aromatic -C=C Stretching
1437, 1314 Aliphatic –C-H Bending
1224 -C-F Stretching
1157 -C-N Stretching
843, 753 Aromatic -C-H Stretching
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4. NMR Study:
The 1H and
13C NMR (Fig 6&7) data of Atorvastatin Calcium were recorded In
DMSO-d6 at 400 MHz and 100MHz respectively on a 400MHz spectrometer. The
chemical shift values are reported on 3 scale in ppm with respect to TMS (δ 0.00ppm)
and DMSO-d6 (δ 39.5ppm) as internal standard respectively. The exchangeable
proton was observed from M exchange spectrum The NMR assignment are given in
the Table No 2.2.
NMR assignments of Atorvastatin Calcium (BHA premix)
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Table 2.2 NMR assignments for Atorvastatin.
Position1 1
H δ (ppm) J (Hz)2 13
C
2 - - - 134.9
3 - - - 123.0
4 - - - 117.5
5 - - - 127.6
6 - - - 125.3
7 1H 6.90-7.60 m 133.3 (8.4*)
8 1H 6.90-7.60 m 115.3 (21.4*)
9 - - - 161.6 (d,243.6)
10 1H 6.90-7.60 m 115.3 (21.4*)
11 1H 6.90-7.60 m 133.2 (8.4*)
12 - - - 136.0
13 1H 6.90-7.60 m 128.7
14 1H 6.90-7.60 m 129.1
15 1H 6.90-7.60 m 127.3
16 1H 6.90-7.60 m 129.1
17 1H 6.90-7.90 m 128.7
18 - - - 166.1
19 NH* 9.76 s -
20 - - - 139.4
21 1H 6.90-7.60 m 119.4
22 1H 6.90-7.60 m 128.4
23 1H 6.90-7.60 m 120.6
24 1H 6.90-7.60 m 128.4
25 1H 6.90-7.60 m 119.4
26 1H 3.37 m 25.6
27 3H 1.37 d,6.4 22.3
28 3H 1.37 d,6.4 22.3
29 Ha 2.01 m 44.0
Hb 2.10 d,15.6 -
30 Ha 3.96 m 40.9
Hb 3.80 m
31 1H 3.54 m 66.3
32 Ha 1.23 m 43.5
Hb 1.62 m
33 1H 3.96 m 66.3
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34 2H 1.37 m 39.0
35 - - - 178.4
36 OH 4.74 br -
37 OH 5.70 br -
5. Mass spectral study
The ESI mass spectrum of Atorvastatin calcium was studied on 400Q trap LCMSMS
system. The sample is introduced through HPLC system by bypassing the column.
The ESI +ve mass spectrum of Atorvastatin displayed the protonated molecular ion at
m/z =559 which corresponds to the molecular formula C33H35FN2O5. The possible
fragmentation pattern is shown below.
Figure 2.1 Mass fragmentation pattern of Atorvastatin
m+= 558
m/z= 440
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Impurities of Atorvastatin
1.Atorvastatin Desfluoro Impurity:
Chemical Name: [R"(R '" ,R "')]-2,3-diphenyl-~,a-dihydroxy-5-(1-
methylethyl)-4.
[(phenylamino )carbonyl]-l H -pyrrole~ l .. heptanoicacid,
hemicalciurn Salt
(or)
[(3R,5R)-7 -[3-(phenylcarbamoyl)-2-isopropyl4 ,5-diphenyJ-l
H
pyrrol-l-ylJ-3,5-dihydroxybeptanoic acid, calcium salt]
(or)
(3R,SR)-7-[2,3..diphenyl-4-(phenylcarbamoyl)-S-(propan-2-
yl) – lHpyrrol-l-yl]-3,5-dibydroxyheptanoic acid.
Molecular Formula: C66 H70 N4 O10 Ca
Structure:
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3. Atorvastatin tertiary butyl Ester impurity
Chemical Name: [R-(R*, R*)]~2-(4.fluorophenyl)-p,&-dihydroxy .. 5-[(1-
methylethyl) -3- phenyl-4-(phenylamino )-carbonyl]-l H-
pyrrole- l-heptanoic acid, tertbutyl ester
(or)
(3R,5R)-tert-butyl 7 -[2 -(4-fluorophenyl)-5-isopropyl~ 3-
phenyl-4-(phenylcarbamoyl)-l H-pyrrol-l-yl]-3,5-
dihydroxyheptanoate
Structure:
Molecular Formula: C37 H43 FN2 O5
Molecular Weight: 614.75
4. Atorvastatin Lactone:
Chemical name: (2R trans)-5-( 4-fluoro phenyl)-Z-(1-methylethyl)-N,4-
dipbenyI- l-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2·yl)ethyl]-lH-
pyrrole-3- carboxamide
(or)
(2R,4R)-2-[2-[2-( 4-fluorophenyl)-J -phenyl-4-
(phenylcarbamoyI)-5-
(propan-2-yl)-IH-pyrrol-l-yl]ethyl]-4-hydroxytetrahydro-2H-
pyran-6-one.
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Structure:
Molecular Formula: C33H33FN2O4
Molecular Weight: 540.62
5. Difluoro Impurity:
Chemical name: (3R,5R)-7-[3-(phenylcarbamoyl)4,5-bis(4·fluorophenyl)-2-
isopropyl IH-pyrrol·l-yl]-3,5-dihydroxyheptanoic acid,
calcium salt
(or)
(3R,SR)-7 .[2,3-bis{ 4-fluorophenyl)-4-(phenylcarbamoyl)-5-
propan-2- yl)-lH-pyrrol-l-yl].3,5-dihydroxyheptanoic
acid
Structure:
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6. Oxirane impurity
Chemical Name: 3-(4-Fluorobenzoyl)-2-isobutyryl-3-phenyloxirane-2-
carboxylic Acid Phenylamide; 3-(4-Fluorobenzoyl)-2-(2-methylpropyl)-
N,3-diphenyl- 2-oxiranecarboxamide;
Structure:
Mol. Formula: C26H24FNO3
Mol. Weight: 417.47
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Structure Confirmation of Clopidogrel Bisulphate
The following physicochemical techniques were used to confirm the structure of
Clopidogrel Bisulphate. These are given below
Thermal study
UV study
FTIR
NMR spectrophotometry
Mass spectrophotometry
1. Thermal Analysis
2.49 mg of Clopidogrel Bisulphate was weighed into an aluminum crucible of 25µL
and placed in to a DSC. The thermogram was recorded from 30ºC to 300ºC which is
carried out under nitrogen atmosphere at 50mL/min, at 10ºC /min. The thermogram
exhibited broad endotherm at 54.3 ºC which can be attributed to loss of water
molecules. A small endotherm was observed at 161 ºC
2. UV Study
The Ultraviolet spectrum was recorded from 200 nm to 400 nm, with API
concentration of 0.0015% in methanol. The spectrum showed two λmax at 203 and
246 nm.
3. FTIR Study
The FTIR of spectrum of Clopidogrel Bisulphate was recorded by preparation of
pellet with KBr. The assignments are given in table No 2.3.
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Table No 2.3- FTIR assignments for Clopidogrel Bisulphate.
Wave number (cm-1
) Assignment Mode of vibration
2721, 2625 -N+-H
- Stretching
3108 Aromatic -C-H Stretching
2987 Aliphatic –C-H Stretching
1754 -C=O Stretching
1434, 1299 Aliphatic –C-H Bending
1175 -C-N Stretching
1220 -C-O Stretching
840, 715 Aromatic -C-H Bending
4. NMR Study
The 1H and
13C NMR (Fig 6&7) data of Clopidogrel Bisulphate were recorded In
DMSO-d6 at 200 MHz and 50MHz respectively on 200MHz spectrometer. The
chemical shift values are reported on 3 scale in ppm with respect to TMS (δ 0.00ppm)
and DMSO-d6 (δ 39.5ppm) as internal standard respectively. The NMR assignment is
given in Table No 2.4
NMR assignments of Clopidogrel Bisulphate
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Table 2.4- NMR assignments for Clopidogrel Bisulphate
Position1 1
H δ (ppm) J (Hz)2 13
C
1 - - - 127.7
2 - - - 134.7
3 1H 7.50-7.80 m 128.9
4 1H 7.50-7.80 m 130.9
5 1H 7.50-7.80 m 131.0
6 1H 7.50-7.80 m 132.9
7 1H 5.63 s 65.1
8 1H 4.22 br, s 50.7
9 1H 3.76 br, s 22.3
10 - - - 131.8
11 1H 7.45 d, 5.2 125.5
12 1H 6.90 d, 5.2 125.8
13 - - - 128.2
14 1H 3.47 d, 5.2 49.5
15 - - - 167.4
16 1H 3.76 s 54.2
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5. Mass spectral study
The ESI mass spectrum of Clopidogrel Bisulphate was studied on 400Q trap
LCMSMS system. The sample is introduced through HPLC system by bypassing the
column. The ESI +ve mass spectrum of Clopidogrel Bisulphate displayed the
protonated molecular ion at m/z =322 which corresponds to the molecular formula
C16H16ClN2S. The possible fragmentation pattern is shown in Figure 2.2.
Figure 2.2- Mass fragmentation pattern for Clopidogrel bisulphate
M
+=321 M
++1 =322
m/z =262
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Impurities of Clopidogrel Bisulphate
1. Impurity A
Chemical name: (+) - (S)-(o-chIorophenyI)-6~7-djhydrothieno-[3;2-c]pyridine-
5(4B:)-acctic acid,
Structure:
2. Impurity B:
Chemical name: Methyi (±)-(o-chlofopheny1)-4,5-dihydrothieno [2,3-
c]pyridine-6 (7H) Acetate hydrogen sulfate
Structure:
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3. Impurity C
Chemical Name : Methyle (-) –(R)- (o-chlorophenyl)-6,7-dihydrothieno[3,2-c]
pyridine-5 (AH) acetate, hydrogen sulphate.
Structure:
4. Impurity D
Chemical Name: Thieno[3,2-c]-4,5,6,7 tetrahydopyridine hydrochloride
Structure:
5. Impurity 3:
Chemical Name: 2-chlororo-α-[2-(2-theinyl)ethyl]amino]benzene acetic acid
methyl ester bisulphate
Structure:
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Method development by UPLC
Aim:
To develop an analytical method for related substances of Atorvastatin and
Clopidogrel in a combination product.
Scope:
This method can be used for routine analysis in Quality control laboratories for
routine analysis and Stability testing for determination of related substance in a
combination drug product of Atorvastatin and Clopidogrel.
Chemicals and reagents:
All the chemicals, solvents and reagents used for the following experiments e.g. Ortho
phosphoric acid, Acetonitrile, methanol, Potassium dihydrogen Orthophosphate
(KH2PO4), water etc. of HPLC grade
Glassware:
All the glassware used for the following experimentation is of class A grade to obtain
maximum precision.
Equipment:
The Ultra Performance liquid chromatograph used for this experiment is Waters
Acquity
Selection of Mobile phase:
Mobile phase was selected on the basis of chemical properties of Clopidogrel and
Atorvastatin. Clopidogrel and its impurity showed different polarity as Imp B,C,A,
were eluting faster and Imp D and Imp E was eluting slower. The elution of
Atorvastatin and its impurities is dependent on high ratio of organic modifiers, 10mM
KH2PO4 buffer with 1 ml of TEA, pH adjusted to 2.5 with OPA and 0.1% of 1-
Octane Sulfonic acid Sodium salt was used . The reason for the use of ion pairing
reagent in buffer preparation was only because the Clopidogrel Imp B and Imp A, as
they were eluting at the same retention time and at dead volume of the column. With
the help of Ion paring reagent separation was achieved and retention time of impurity
was increased. Methanol was introduced in low concentration which helped improve
separation of Atorvastatin impurities however in high concentration Atorvastatin
impurities didn‘t elute at all. An organic phase used in the beginning was a
combination Acetonitrile and 0.2% OPA in the ratio 90:10 and 10:90, but at this
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combination impurities were closely eluting. Hence addition of buffer became
essential.
Selection of Column:
Column study was done extensively on Waters Acquity BEH C18, 100X2.1, 1.7µm
column and Agilent Eclipse plus C18 RRHD 50X2.1 1.8µm, both columns were
good, however Agilent Eclipse plus column showed better separation.
Selection of Diluent:
Methanol was used as diluent for Atorvastatin because of its highly non polar nature
whereas for Clopidogrel , 1: 1 ratio of water and Acetonitrile was used. The final
dilution was performed with 1:1 ratio of Water and Acetonitrile
Selection of wavelength:
USP method for related substance analysis of Clopidogrel uses a wavelength of 220
nm and for Atorvastatin, it is 244 nm. All the impurities of Atorvastatin and
Clopidogrel showed excellent absorption at 220nm moreover at this wavelength, the
method sensitivity is also increased thus it was able to detect low concentration of
impurities. The sensitivity and response was found to decrease with increase in
wavelength
Experiment 1:
Buffer: Dissolve 2mL of 85% Orthophosphoric Acid in 500mL of water, shake well
to dissolve, make it up to 1000mL with water, subject it to ultra sonication, filter it
through 0.45µm membrane filter.
Mobile Phase A: Buffer : Acetonitrile::90:10% v/v
Mobile Phase B: Buffer : Acetonitrile:10:90% v/v
Diluent: Methanol for Atorvastatin ,
Water: Acetonitrile:50:50% v/v for Clopidogrel and Final dilution with Water :
Acetonitrile: 50:50% v/v
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Chromatographic Conditions:
Flow rate 0.5 ml/min
Wavelength 220 nm
Sample temperature Ambient
Column temperature 25°C
Column Waters BEH C18, 100x2.1mm, 1.7µm
Gradient program
Figure 2.3- Chromatogram obtained with experiment No 1
Time %A %B
0.01 85 15
3 80 20
5 65 35
7 60 40
10 50 50
13 50 50
14 85 15
15 85 15
Atorvastatin
Difluoro Clopidogrel
Experiment 1
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Observation:
Atorvastatin and all its Impurities were eluted between 11.3 to 13.5 minutes.
Clopidogrel and all its impurity were eluted within 5 minutes. Almost all impurities of
Atorvastatin merged together showing only 3 impurity peaks. The differences in
polarity of the eluents have caused this impact i.e. all the polar compounds have
eluted very early and the nonpolar ones have been retained on the column and eluted
at the last due to increase in nonpolarity of the mobile phase.
Way forward:
In order to make the elution pattern uniform and bring about separation between the
peaks the non polar solvent increment of organic phase in the gradient and increase in
column oven temperature needs to be implemented. This may cause early elution and
better separations in Atorvastatin and its related substances.
Experiment 2:
Buffer: Dissolve 2mL of 85% Orthophosphoric Acid in 500mL of water, shake well
to dissolve, make it up to 1000mL with water, subject it to ultra sonication, filter it
through 0.45µm membrane filter.
Mobile Phase A: Buffer : Acetonitrile::90:10% v/v
Mobile Phase B: Buffer : Acetonitrile:10:90% v/v
Diluent: Methanol for Atorvastatin ,
Water: Acetonitrile:50:50% v/v for Clopidogrel and Final dilution with Water :
Acetonitrile: 50:50% v/v
Chromatographic Conditions:
Flow rate 0.4 ml/min
Wavelength 220 nm
Sample temperature NA
Column temperature 30°C
Column Waters BEH C18, 100x2.1mm, 1.7µm
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Gradient Program
Figure 2.4- Chromatogram obtained with experiment No 2
Observation:
Atorvastatin and all its Impurities were eluted between 7 to 13 minutes. Clopidogrel
and all its impurity were eluted within 4 minutes. Difluoro impurity of Atorvastatin
closely eluted with Atorvastatin.
Time %A %B
0.01 85 15
4 70 30
6 50 50
12 40 60
16 40 60
17 85 15
20 85 15
Clopidogrel
Atorvastatin
Difluoro
Experiment 2
Page 67 of 305
Way forward:
Introduction of methanol might help in improving the peak shape and improve
separation. The gap between the two sets of impurities needs to be further reduced by
means of minor modifications in the gradient program.
Experiment 3:
Buffer: Dissolve 2mL of 85% Orthophosphoric Acid in 500mL of water, shake well
to dissolve, make it up to 1000mL with water, subject it to ultra sonication, filter it
through 0.45µm membrane filter.
Mobile Phase A: Buffer : Acetonitrile::90:10% v/v
Mobile Phase B: Buffer :Acetonitrile:Methanol:10:60:30% v/v/v
Diluent: Methanol for Atorvastatin ,
Water: Acetonitrile:50:50% v/v for Clopidogrel and Final dilution with Water :
Acetonitrile: 50:50% v/v
Chromatographic Conditions:
Flow rate 0.4 ml/min
Wavelength 220 nm
Sample temperature Ambient
Column temperature 30°C
Column Waters BEH C18, 100x2.1mm, 1.7µm
Gradient Program
Time %A %B
0.01 85 15
4 70 30
6 50 50
8 45 55
10 35 65
12 30 70
13 85 15
15 85 15
Page 68 of 305
Figure 2.5- Chromatogram obtained with experiment No 3
Observation:
Atorvastatin and all its Impurities were eluted between 9 to 13 minutes. Clopidogrel
showed increased retention with introduction of Methanol. Minor improvement in
resolution of Difluoro impurity and Atorvastatin observed. All peaks related to
Atorvastatin accumulated in 3min width and reduced resolution was observed.
Way forward:
Increasing the percentage of Acetonitrile without changing the Percentage of
Methanol should help logically
Experiment 4:
Buffer: Dissolve 2mL of 85% Orthophosphoric Acid in 500mL of water, shake well
to dissolve, make it up to 1000mL with water, subject it to ultra sonication, filter it
through 0.45µm membrane filter.
Mobile Phase A: Buffer : Acetonitrile::90:10% v/v
Mobile Phase B: Buffer :Acetonitrile:Methanol:10:70:30% v/v/v
Diluent: Methanol for Atorvastatin ,
Water: Acetonitrile:50:50% v/v for Clopidogrel and Final dilution with Water :
Acetonitrile: 50:50% v/v
Page 69 of 305
Chromatographic Conditions:
Flow rate 0.4 ml/min
Wavelength 220 nm
Sample temperature Ambient
Column temperature 30°C
Column Waters BEH C18, 100x2.1mm, 1.7µm
Gradient Program
Figure 2.6- Chromatogram obtained with experiment No 4
Observation:
Atorvastatin and all its Impurities eluted between 9 to 13 minutes. Clopidogrel
showed increased retention with introduction of Methanol. Minor improvement in
Time %A %B
0.01 85 15
4 70 30
6 50 50
8 45 55
10 35 65
12 30 70
13 85 15
15 85 15
EXPERIMEN
T 4
Clopidogrel
Atorvastatin Difluoro
Page 70 of 305
resolution of Difluoro impurity and Atorvastatin was observed. All peaks related to
Atorvastatin showed improved peak shape and separation.
Way forward:
Reducing the percentage of Methanol without changing the Percentage of Acetonitrile
should help in increasing the separation.
Experiment 5:
Buffer: Dissolve 2mL of 85% Orthophosphoric Acid in 500mL of water, shake well
to dissolve, make it up to 1000mL with water, subject it to ultra sonication, filter it
through 0.45µm membrane filter.
Mobile Phase A: Buffer : Acetonitrile::90:10% v/v
Mobile Phase B: Buffer :Acetonitrile:Methanol::10:70:20% v/v/v
Diluent: Methanol for Atorvastatin ,
Water: Acetonitrile:50:50% v/v for Clopidogrel and Final dilution with Water :
Acetonitrile: 50:50% v/v
Chromatographic Conditions:
Flow rate 0.4 ml/min
Wavelength 220 nm
Sample temperature Ambient
Column temperature 30°C
Column Waters BEH C18, 100x2.1mm, 1.7µm
Page 71 of 305
Gradient Program
Figure 2.7- Chromatogram obtained with experiment No 5
Observation:
Atorvastatin and all its Impurities were eluted between 9 to 13 minutes. Clopidogrel
showed increased retention with introduction of Methanol. Minor improvement in
resolution of Difluoro impurity and Atorvastatin observed. All peaks related to
Atorvastatin showed improved peak shape and separation. However Clopidogrel
impurities were affected and resolution were reduced.
Time %A %B
0.01 85 15
4 70 30
6 50 50
8 45 55
10 35 65
12 30 70
13 85 15
15 85 15
EXPERIMENT 5
Clopidogrel
Atorvastatin Difluoro
Page 72 of 305
Way forward:
Change in gradient program is not significantly impacting the separation of the
nonpolar set peaks thus the second modification i.e. the pH of the mobile phase needs
to be incorporated. To start with the experimentation needs to be slightly on the lower
side.
Experiment 6:
Buffer: MilliQ Water pH adjusted to 2.0 with 85% Orthophosphoric Acid
Mobile Phase A: Buffer : Acetonitrile::90:10% v/v
Mobile Phase B: Buffer :Acetonitrile : Methanol::10:70:20% v/v/v
Diluent: Methanol for Atorvastatin,
Water: Acetonitrile:50:50% v/v for Clopidogrel and Final dilution with Water :
Acetonitrile: 50:50% v/v
Chromatographic Conditions:
Flow rate 0.4 ml/min
Wavelength 220 nm
Sample temperature Ambient
Column temperature 30°C
Column Waters BEH C18, 100x2.1mm, 1.7µm
Gradient Program
Time %A %B
0.01 90 10
4 70 30
6 50 50
8 45 55
10 35 65
12 30 70
13 90 10
15 90 10
Page 73 of 305
Figure 2.8- Chromatogram obtained with experiment No 6
Observation:
Atorvastatin and all its Impurities eluted between 7 to 12 minutes. Minor
improvement was observed in the separation of Clopidogrel impurities. All peaks
related to Atorvastatin showed improved peak shape and separation. Aim for reducing
the gap of 2 minutes between 5 to 7 minute which can reduce the overall run time.
Way forward:
In continuation to this experiment, the change in pH would have to be performed on
higher side keeping all the other chromatographic conditions same as this experiment.
EXPERIMENT 6
Clopidogrel
Atorvastatin Difluoro
Page 74 of 305
Experiment 7:
Buffer: MilliQ Water pH adjusted to 3.0 with 85% Orthophosphoric Acid
Mobile Phase A: Buffer : Acetonitrile::90:10% v/v
Mobile Phase B: Buffer :Acetonitrile:Methanol::10:70:20% v/v/v
Diluent: Methanol for Atorvastatin,
Water: Acetonitrile:50:50% v/v for Clopidogrel and Final dilution with Water :
Acetonitrile: 50:50% v/v
Chromatographic Conditions:
Flow rate 0.4 ml/min
Wavelength 220 nm
Sample temperature Ambient
Column temperature 30°C
Column Waters BEH C18, 100x2.1mm, 1.7µm
Gradient Program
Time %A %B
0.01 85 15
4 70 30
6 50 50
8 45 55
10 35 65
12 30 70
13 85 15
15 85 15
Page 75 of 305
Figure 2.9- Chromatogram obtained with experiment No 7
Observation:
Retention time of Clopidogrel delayed by 3 minutes where as Imp B of Clopidogrel
eluted faster and merged with void volume peaks.
Way forward:
Introducing ion pairing reagent to the mobile phase can help change the elution
patterns of the peaks in question.
Experiment 8:
Buffer: MilliQ Water pH adjusted to 3.0 with 85% Orthophosphoric Acid + 0.1%
Pentane sulfonic acid sodium salt
Mobile Phase A: Buffer : Acetonitrile::90:10% v/v
Mobile Phase B: Buffer :Acetonitrile:Methanol::10:70:20% v/v/v
Diluent: Methanol for Atorvastatin ,
Water: Acetonitrile:50:50% v/v for Clopidogrel and Final dilution with Water :
Acetonitrile: 50:50% v/v
EXPERIMENT 7
Clopidogrel
Atorvastatin Difluoro
Page 76 of 305
Chromatographic Conditions:
Flow rate 0.4 ml/min
Wavelength 220 nm
Sample temperature Ambient
Column temperature 30°C
Column Waters BEH C18, 100x2.1mm, 1.7µm
Gradient Program
Figure 2.10- Chromatogram obtained with experiment No 8
Time %A %B
0.01 85 15
4 70 30
6 50 50
8 45 55
10 35 65
12 30 70
13 85 15
15 85 15
EXPERIMENT 8
Clopidogrel
Atorvastatin Difluoro
Page 77 of 305
Observation:
Clopidogrel Imp B RT unchanged but separation increased among other impurity.
Atorvastatin impurity spread has increased to 5 min and separation is proper but with
improper peak shape
Way forward:
Introduction of Potassium salt buffer along with triethylamine to improve the peak
shape and improve separation of Atorvastatin impurities, where as increasing the
chain length of ion pairing reagent should help in increased retention of Clopidogrel
impurity B. These changes along with change in composition of mobile phase
combinations could bring more separations. Increasing the flow rate would reduce the
run times.
Experiment 9:
Buffer: 10mM KH2PO4 + 0.1% TEA, pH adjusted to 2.5 with OPA and added 0.1%
of 1-Octane Sulphonic acid Sodium salt
Mobile Phase A: Buffer
Mobile Phase B: Buffer: Acetonitrile: Methanol::36:154:10% v/v/v
Diluent: Methanol for Atorvastatin, Water:Acetonitrile:50:50%v/v for Clopidogrel
and Final dilution with Water :Acetonitrile::50:50%v/v
Chromatographic Conditions:
Flow rate 0.4 ml/min
Wavelength 220 nm
Sample temperature Ambient
Column temperature 30°C
Column Waters BEH C18, 100x2.1mm, 1.7µm
Page 78 of 305
Gradient Program
Figure 2.11- Chromatogram obtained with experiment No 9
Time %A %B
0.01 75 25
0.50 75 25
4 50 50
6 40 60
8 30 70
10 20 80
10.5 20 80
10.7 75 25
12 75 25
EXPERIMENT
9
Page 79 of 305
Figure 2.12- Chromatogram obtained with experiment No 9- zoomed
chromatogram
Observation:
All impurities of Clopidogrel and Atorvastatin are well separated except the Difluoro
impurity of Atorvastatin. Run time has been reduced further to 12 minutes and
Clopidogrel Imp B retention increased and no more eluting at the void volume even
the improvement in peak shape can also be observed.
Way forward:
In order to improve the separation of Difluoro impurity and Atorvastatin, different
column should be tried to change the polarity of the stationary phase. Minor
increment in flow rate can further decrease the run time.
Experiment 10:
Buffer: 10mM KH2PO4 + 0.1% TEA, pH adjusted to 2.5 with OPA and added 0.1%
of 1-Octane Sulphonic acid Sodium salt
Mobile Phase A: Buffer
Mobile Phase B: Buffer :Acetonitrile:Methanol::36:154:10% v/v/v
Diluent: Methanol for Atorvastatin ,
Water : Acetonitrile: 50:50% v/v for Clopidogrel and Final dilution with Water
:Acetonitrile ::50:50% v/v
EXPERIMENT
9 ZOOMED CHROMATOGRAM OF
ABOVE AT RT 6 TO 7.4 MINUTES
Page 80 of 305
Chromatographic Conditions:
Flow rate 0.7 ml/min
Wavelength 220 nm
Sample temperature NA
Column temperature 30°C
Column Agilent Eclipse plus RRHD C18, 50x2.1mm, 1.8µm
Figure 2.13- Chromatogram obtained with experiment No 10 impurities spiked.
Gradient table
Time %A %B
0.01 75 25
0.5 75 25
3.0 50 50
7.0 50 50
10.0 20 80
10.5 20 80
10.7 75 25
12.0 75 25
EXPERIMENT 10
Page 81 of 305
Figure 2.14- Chromatogram obtained with experiment No 10- only impurities
Observation:
All impurities of Clopidogrel and Atorvastatin are well separated. Base to base
separation is observed between Difluoro and Atorvastatin. Run time has been reduced
further to 12 minutes
Way forward:
The primary objective of the method has been fulfilled, in order to confirm the
stability indicating capability, the specificity of the method needs to be evaluated by
means of forced degradation study to check the separation of unknown degradation
products.
EXPERIMENT 10 ONLY IMPURITIES OF CLOPIDOGREL AND ATORVASTATIN
Page 82 of 305
Forced degradation study of Atorvastatin and Clopidogrel:
The final method was subjected to forced degradation study to validate the specificity
of the method. API sample were stressed with 2N Acid, 1N Base and 10% peroxide
Solution
Undegraded Sample:
As such sample showed the presence of Clopidogrel impurity C, Atorvastatin Lactone
impurity 1 and Lactone impurity 2 along with Unknown impurity at RT 9.648.
Figure 2.15- Chromatogram of undegraded sample
Undegraded Sample
Page 83 of 305
Acid stressed Sample:
The samples were subjected to 2N HCl acid at 60°C for 2 hours. When stressed at this
condition, Impurity D of Clopidogrel was observed to form in very small amount i.e.
0.04% whereas Lactone Impurity 1 of Atorvastatin showed increased amount to
2.31% from 0.30% (un degraded sample), over 500% increment in the impurity.
However no purity flag was observed and for the main peaks and Purity Angle was
less than Purity Threshold
Figure 2.16- Chromatogram of acid stressed sample
Page 84 of 305
Base Stressed Sample:
The samples were subjected to 1N NaOH base at 60°C for 1 hour. When stressed at
this condition, remarkable increment was observed in Impurity C of Clopidogrel
which was observed to be 26.89% along with this, an unknown impurity was
observed at 0.303 min with was observed to be 0.90%. Atorvastatin was found to be
relatively stable in basic condition. No purity flag was observed and for the main
peaks and Purity Angle was less than Purity Threshold
Figure 2.17- Chromatogram of base stressed sample
1N BASE STRESSED
SAMPLE
Page 85 of 305
Peroxide Stressed Sample:
The samples were subjected to 10% Hydrogen peroxide solution to mimic oxidative
stress condition at 60°C for 1 hour. When stressed at this condition, small amount of
IMP B and IMP D of Clopidogrel along with many unknown impurities were formed.
Atorvastatin again showed increase in Lactone 1 impurity to 1.56% from 0.30%,
nearly 400% increment. Small amount of Atorvastatin Tertiary butyl ester Impurity
was also found along with 3 unknown impurities. However no purity flag was
observed and for the main peaks and Purity Angle was less than Purity Threshold
Figure 2.18- Chromatogram of peroxide stressed sample
10% PEROXIDE STRESSED
SAMPLE
Page 86 of 305
Table No 2.5-Forced degradation Atorvastatin
Degradati
on Type
Degradation
Condition
Net
degradat
ion
Purity
angle
Purity
threshold
Acid Exposed for 2hrs with
2N HCl at 60°C 2.4% 0.141 1.040
Base Exposed for 1hr with
1N NaoH at 60°C 0.5% 0.098 1.022
Peroxide Exposed for 1hr with
10% H2O2 at 60°C
3.1%
0.110 1.024
Table No 2.6-Forced degradation Clopidogrel
Degradatio
n Type
Degradation
Condition
Net
degradation
Purity
angle
Purity threshold
Acid
Exposed for 2hrs
with 2N HCl at
60°C
0.4% 0.997 1.173
Base
Exposed for 1hr
with 1N NaoH at
60°C
51% 0.974 1.158
Peroxide
Exposed for 1hr
with 10% H2O2 at
60°C
5.8%
0.809 1.161
Page 87 of 305
Optimized final method:
Related substances method development for Clopidogrel and Atorvastatin by
UPLC
Optimized final method: Clopidogrel & Atorvastatin
Buffer: 10 mM KH2PO4, 1ml of TEA and adjusted pH to 2.50 with OPA, Added
1gm/1000ml of 1-Octane Sulphonic acid Sodium salt
Mobile Phase A: Buffer
Mobile Phase B: Buffer: Acetonitrile::Methanol :: 36:154:10% v/v/v
Diluent: Atorvastatin in Methanol,
Clopidogrel in Water: Acetonitrile::50:50% v/v
Final Diluent: Water: Acetonitrile::50:50% v/v
Chromatographic Conditions:
Flow rate 0.7 ml/min
Column temperature 30°C
Wavelength 220 nm
Inj Volume 2µL
Sample temperature Ambient
Columns Agilent Eclipse plus C18 RRHD, 50X2.1mm, 1.8µm
Gradient program:
Time %A %B
0.01 75 25
0.5 75 25
3.0 50 50
7.0 50 50
10.0 20 80
10.5 20 80
10.7 75 25
12.0 75 25
Page 88 of 305
Diluted standard preparation:
Weighed quantity of 65mg of Clopidogrel Bisulphate and 67mg of Atorvastatin
Calcium working standard or Reference standard to be transferred into a 100ml
volumetric flask, sonicated dissolve and diluted volume with Methanol (Stock-I)
5ml of Stock –I taken in to a 100ml volumetric flask dissolve dilute volume with
diluent-II (Stock-II)
4ml of Stock-II taken in to a 50ml volumetric flask dissolve dilute volume with
diluent-II
Sample preparation:
Weighed and transferred sample equivalent to 50mg Atorvastatin and 75mg of
Clopidogrel in 100ml volumetric flask add 20ml of Methanol and sonicate for 5 min,
add 50ml of diluent-II and sonicated for another 10min with intermediate shaking
made up volume with diluent-II.
Figure 2.19- Chromatogram of API and impurity peaks in optimized method
Page 89 of 305
Conclusion:
The related substances method for determination of related substances for Clopidogrel
and Atorvastatin in a combination tablet has been developed. This method needs to be
validated to confirm the applicability for routine analysis.
Table 2.7- Individual limit of impurities considered for method validation
Clopidogrel Atorvastatin
Imp Name/No Limit Desfluoro impurity 0.10%
Imp A 0.10% Difluoro impurity 0.10%
Imp B 0.10% Lactone impurity 1 0.10%
Imp C 0.10% Lactone impurity 2 0.10%
Imp D 0.10% Tetrabutyl ester
impurity
0.10%
Imp 3 0.10% Oxirane impurity 0.10%
Page 90 of 305
Method validation for Atorvastatin and Clopidogrel
Analytical method validation
Analytical method validation is a process that demonstrates the suitability of the
proposed procedures for the intended purpose. More specifically, it is a process of
establishing documented evidence providing a high degree of assurance with respect
to the consistency of the method and results. It evaluates the product against defined
specifications. The validation parameters viz., specificity, accuracy, precision,
linearity, limit of detection, limit of quantitation, robustness, system suitability have
to be evaluated as per the ICH guidelines for all analytical methods developed by
HPLC.
Validation Characteristics
The following validation characteristics were verified as per the ICH guidelines.
System suitability
Specificity
Linearity
Accuracy
Precision
LOD & LOQ
System suitability
This is an integral part of development of a chromatographic method to verify
that the resolution and reproducibility of the system are adequate enough for the
analysis to be performed. It is based on the concept that the equipment, electronics,
analytical operations and samples constituting an integral system could be evaluated
as a whole. Parameters such as plate number (N), asymmetry or tailing factors (As),
relative retention time (RRT), resolution (Rs) and reproducibility (% R.S.D), retention
time were determined. These parameters were determined during the analysis of a
"sample" containing the main components and related substances. System suitability
terms were determined and compared with the recommended limits (1≥As ≤2 and
Rs>1.5).
Page 91 of 305
Specificity
Specificity is the ability of the method to measure the analyte response in
presence of its process related impurities. The specificity of the developed HPLC
method was performed by injecting blank solution and standard solution spiked with
process-related impurities separately The chromatogram of drug with impurities was
compared with the blank chromatogram, to verify the blank interference. No peak was
observed at the retention time of Atorvastatin, Clopidogrel and their impurities. Hence
the method is specific for the determination of Atorvastatin, Clopidogrel in its
combination product.
Precision of Test method
System precision of the method was evaluated by injecting the standard
solution six times and percent relative standard deviation (% R.S.D) for area of
Atorvastatin peak was 3.2% and for Clopidogrel peak it was 1.46%. This proves the
system precision of the test method. The precision of the method for the determination
of impurities related to Atorvastatin and Clopidogrel peaks was studied for
repeatability at 100 % level. Repeatability was demonstrated by analyzing the
standard solution spiked with impurities for six times. The % R.S.D for peak area of
each impurity was calculated. Repeatability for Atorvastatin, Clopidogrel and its
impurities were found to be optimum, thus proves that this method is precise. The
results are given in Table 2.8.
Page 92 of 305
Table 2.8-Precision of Test method
Impurity name
(Atorvastatin)
RRF of
impurities
%Imp of
SPL-1
%Imp of
SPL-2
%Imp of
SPL-3
%Imp of
SPL-4
%Imp of
SPL-5
%Imp of
SPL-6
%
RSD
Atorvastatin 1 0.41 0.43 0.42 0.43 0.45 0.42 3.20
Desfluoro 1.06 0.21 0.21 0.21 0.20 0.21 0.20 2.50
Tert Butyl
Ester 3.39 0.20 0.20 0.20 0.20 0.19 0.20 2.06
Lactone 1.03 0.50 0.51 0.50 0.50 0.50 0.50 0.81
Oxirane 1.05 0.75 0.76 0.75 0.75 0.76 0.74 1.00
Difluoro 0.86 0.50 0.49 0.49 0.49 0.48 0.49 1.29
Impurity name
(Clopidogrel)
RRF of
impurities
%Imp of
SPL-1
%Imp of
SPL-2
%Imp of
SPL-3
%Imp of
SPL-4
%Imp of
SPL-5
%Imp of
SPL-6
%RSD
Clopidogrel 1 0.36 0.35 0.35 0.35 0.36 0.35 1.46
Impurity A 0.66 0.21 0.21 0.20 0.20 0.21 0.20 2.67
Impurity B 2.65 0.20 0.21 0.21 0.20 0.20 0.19 3.73
Impurity C 0.90 0.20 0.20 0.21 0.21 0.21 0.21 2.50
Impurity D 0.95 0.21 0.20 0.21 0.20 0.19 0.21 4.02
Impurity 3 0.90 0.21 0.20 0.20 0.21 0.21 0.20 2.67
Page 93 of 305
Linearity
Standard solutions at different concentration levels ranging from 50% of the spec
level to 300% of the specification limit were prepared and analyzed. In order to
demonstrate the linearity of detector response for Atorvastatin, Clopidogrel and their
impurities, the linearity plot was drawn taking the concentration on X-axis and the
mean peak area on Y-axis. The data were subjected to statistical analysis using a
linear-regression model. The data for correlation coefficients (r) are given in Table
Nos 2.9 to 2.20 and corresponding graphs are presented in Figure Nos No 2.20 to 2.31
Linearity of Atorvastatin Impurities :
Table 2.9-Linearity table for Atorvastatin
Level Atorvastatin
% Concentration Area
50% 1.35 10485
75% 2.05 15987
100% 2.75 21125
125% 3.46 27235
150% 4.13 32758
200% 5.48 42545
correlation 1.000
Figure 2.20- Linearity graph for Atorvastatin
y = 7831.x - 64.31R² = 0.999
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
0 1 2 3 4 5 6
Atorvastatin
Page 94 of 305
Table 2.10-Linearity table for Desfluoro Impurity
Level Desfluoro
% Concentration Area
50% 0.51 15479
75% 0.75 24025
100% 1.02 30706
125% 1.24 39274
150% 1.51 47123
200% 2.01 61512
correlation 0.999
Figure 2.21- Linearity graph for Desfluoro impurity
y = 30675x + 361.4R² = 0.998
0
10000
20000
30000
40000
50000
60000
70000
0 0.5 1 1.5 2 2.5
Desfluoro Impurity
Page 95 of 305
Table 2.11-Linearity table for Tetra Butyl Ester impurity
Level T-butyl ester
% Concentration Area
50% 1.38 35565
75% 2.06 53794
100% 2.75 69863
125% 3.44 82359
150% 4.12 105739
200% 5.5 142354
correlation 0.998
Figure 2.22- Linearity graph for Tetra Butyl Ester impurity
y = 25698x - 836.0R² = 0.995
0
20000
40000
60000
80000
100000
120000
140000
160000
0 1 2 3 4 5 6
T-Butyl Ester Impurity
Page 96 of 305
Table 2.12-Linearity table for Lactone Impurity
Level Lactone
% Concentration Area
50% 1.24 13489
75% 1.9 18957
100% 2.48 24947
125% 3.15 30268
150% 3.75 36004
200% 5.01 48934
correlation 0.999
Figure 2.23- Linearity graph for Lactone impurity
y = 9366.x + 1401R² = 0.998
0
10000
20000
30000
40000
50000
60000
0 1 2 3 4 5 6
Lactone Impurity
Page 97 of 305
Table 2.13- Linearity table for Difluoro Impurity
Level Difluoro
% Concentration Area
50% 1.25 13685
75% 1.85 19793
100% 2.51 27793
125% 3.2 34689
150% 3.76 41687
200% 5.1 55268
correlation 1.000
Figure 2.24- Linearity graph for Difluoro impurity
y = 10895x + 68.14R² = 0.999
0
10000
20000
30000
40000
50000
60000
0 1 2 3 4 5 6
Difluoro Impurity
Page 98 of 305
Table 2.14- Linearity table for Oxirane Impurity
Level Oxirane
% Concentration Area
50% 1.9 15489
75% 2.8 22782
100% 3.75 29125
125% 4.7 35126
150% 5.63 44651
200% 7.5 60328
correlation 0.998
Figure 2.25- Linearity graph for Oxirane impurity
y = 7949.x - 237.1R² = 0.995
0
10000
20000
30000
40000
50000
60000
70000
0 1 2 3 4 5 6 7 8
Oxirane Impurity
Page 99 of 305
Linearity of Clopidogrel Impurities
Table 2.15- Linearity table for Clopidogrel
Level Clopidogrel
% Concentration Area
50% 1.01 17642
75% 1.49 26289
100% 2 35121
125% 2.52 43665
150% 3.01 52007
200% 4 70256
correlation 1.000
Figure 2.26- Linearity graph for Clopidogrel
y = 17452x + 21.58R² = 0.999
0
10000
20000
30000
40000
50000
60000
70000
80000
0 1 2 3 4 5
Clopidogrel
Page 100 of 305
Table 2.16- Linearity table for Clopidogrel Impurity A
Level Impurity A
% Concentration Area
50% 0.53 5154
75% 0.75 7889
100% 1.03 11590
125% 1.24 13871
150% 1.51 16727
200% 2.01 22147
correlation 0.999
Figure 2.27- Linearity graph for Clopidogrel Impurity A
y = 11446x - 590.7R² = 0.997
0
5000
10000
15000
20000
25000
0 0.5 1 1.5 2 2.5
Impurity A
Page 101 of 305
Table 2.17- Linearity table for Clopidogrel Impurity B
Level Impurity B
% Concentration Area
50% 0.5 23816
75% 0.72 35724
100% 1.12 51633
125% 1.22 58449
150% 1.55 71266
200% 2.05 95354
correlation 0.999
Figure 2.28- Linearity graph for Clopidogrel Impurity B
y = 45457x + 1795.R² = 0.998
0
20000
40000
60000
80000
100000
120000
0 0.5 1 1.5 2 2.5
Impurity B
Page 102 of 305
Table 2.18- Linearity table for Clopidogrel Impurity C
Level Impurity C
% Concentration Area
50% 0.55 16770
75% 0.77 24839
100% 1.08 35025
125% 1.18 38972
150% 1.49 48637
200% 1.98 63443
correlation 0.999
Figure 2.29- Linearity graph for Clopidogrel Impurity C
y = 32586x - 340.9R² = 0.998
0
10000
20000
30000
40000
50000
60000
70000
0 0.5 1 1.5 2 2.5
Impurity C
Page 103 of 305
Table 2.19- Linearity table for Clopidogrel Impurity D
Level Impuriy D
% Concentration Area
50% 0.52 8041
75% 0.76 13674
100% 1.1 24251
125% 1.24 29046
150% 1.48 36584
200% 2.01 55268
correlation 0.998
Figure 2.30- Linearity graph for Clopidogrel ImpurityD
y = 31958x - 10060R² = 0.996
0
10000
20000
30000
40000
50000
60000
0 0.5 1 1.5 2 2.5
Impuriy D
Page 104 of 305
Table 2.20- Linearity table for Clopidogrel Impurity 3
Level Impurity 3
% Concentration Area
50% 0.48 15517
75% 0.77 23218
100% 0.99 31292
125% 1.21 38941
150% 1.52 49547
200% 1.99 65586
correlation 0.999
Figure 2.31- Linearity graph for Clopidogrel Impurity 3
y = 33662x - 1697.R² = 0.998
0
10000
20000
30000
40000
50000
60000
70000
0 0.5 1 1.5 2 2.5
Impurity 3
Page 105 of 305
Accuracy of test method
Accuracy of the test method was determined by analyzing Atorvastatin, Clopidogrel
drug substance spiked with impurities at five different concentration levels of 50 %,
75%, 100 %, 125%, 150% and 200% of each at the specified limit. The mean
recoveries of all the impurities were calculated individually and are represented in the
below tables individually for Atorvastatin, Clopidogrel and all the impurities
Table No 2.21-Accuracy of Atorvastatin Desfluoro Impurity
% of Impurities Amount added
(in µg/ml)
Amount found
(in µg/ml)
% Recovery of
impurity
50% 0.512 0.521 102.0
75% 0.752 0.734 97.3
100% 1.024 0.991 97.1
125% 1.247 1.217 97.6
150% 1.515 1.526 100.7
200% 2.011 2.021 100.5
Table No 2.22-Accuracy of Atorvastatin Tertiary Butyl Ester
% of
Impurities
Amount added
(in µg/ml)
Amount found
(in µg/ml)
% Recovery of
impurity
50% 0.501 0.520 104.0
75% 0.770 0.751 97.4
100% 1.041 0.981 94.2
125% 1.210 1.211 100.0
150% 1.551 1.520 98.1
200% 2.013 2.014 100.0
Page 106 of 305
Table No 2.23-Accuracy of Atorvastatin Lactone
% of
Impurities
Amount added
(in µg/ml)
Amount found
(in µg/ml)
% Recovery of
impurity
50% 1.242 1.222 98.4
75% 1.91 1.884 98.9
100% 2.481 2.497 100.4
125% 3.151 3.254 103.2
150% 3.754 3.725 99.2
200% 5.012 5.002 99.8
Table No 2.24-Accuracy of Atorvastatin Difluoro
% of
Impurities
Amount added
(in µg/ml)
Amount found
(in µg/ml)
% Recovery of
impurity
50% 1.257 1.198 95.2
75% 1.854 1.99 102.7
100% 2.517 2.488 98.8
125% 3.201 3.151 98.4
150% 3.760 3.681 97.9
200% 5.105 4.908 96.1
Page 107 of 305
Table No 2.25-Accuracy of Atorvastatin Oxirane
% level
Amount added
(in µg/ml)
Amount found
(in µg/ml)
% Recovery of
impurity
50% 1.901 1.801 94.7
75% 2.810 2.911 103.9
100% 3.751 3.713 98.9
125% 4.705 4.760 101.3
150% 5.630 5.614 99.6
200% 7.512 7.717 102.7
Table No 2.26-Accuracy of Clopidogrel Impurity-A
% level
Amount added
(in µg/ml)
Amount found
(in µg/ml)
% Recovery of
impurity
50% 0.509 0.570 111.8
75% 1.019 1.045 102.0
100% 2.039 2.020 99.1
125% 3.058 3.011 98.4
150% 4.078 4.106 100.5
200% 6.518 6.232 101.8
Table No 2.27-Accuracy of Clopidogrel Impurity B
% Level Amount added
(in µg/ml)
Amount found
(in µg/ml)
% Recovery of
impurity
50% 0.501 0.541 108.0
75% 0.721 0.684 94.4
100% 1.122 1.101 98.2
125% 1.224 1.189 96.7
150% 1.558 1.524 98.1
200% 2.056 2.165 105.4
Page 108 of 305
Table No 2.28-Accuracy of Clopidogrel Impurity C
% level Amount added
(in µg/ml)
Amount found
(in µg/ml)
% Recovery of
impurity
50% 0.551 0.497 89.1
75% 0.771 0.754 97.4
100% 1.083 0.964 88.9
125% 1.184 1.211 101.7
150% 1.490 1.481 99.3
200% 1.981 2.052 103.5
Table No 2.29-Accuracy of Clopidogrel Impurity D
% level
Amount added
(in µg/ml)
Amount found
(in µg/ml)
% Recovery of
impurity
50% 0.527 0.557 105.8
75% 0.765 0.701 92.1
100% 1.102 1.150 104.5
125% 1.241 1.251 100.8
150% 1.480 1.522 102.7
200% 2.011 2.023 100.5
Table No 2.30-Accuracy of Clopidogrel Impurity 3
% level
Amount added
(in µg/ml)
Amount found
(in µg/ml)
% Recovery of
impurity
50% 0.481 0.518 106.3
75% 0.777 0.684 88.3
100% 0.991 0.930 93.9
125% 1.216 1.181 97.5
150% 1.524 1.502 98.7
200% 1.991 2.023 101.5
Page 109 of 305
Limit of detection (LOD) and limit of quantitation (LOQ)
Limit of detection or LOD is the lowest level at which the impurity or API peak can
be observed or in other words can be distinguished from that of the system noise.
Limit of quantitation or LOQ is the lowest level at which the impurity or API can be
quantitatively estimated with an acceptable accuracy. This estimation was performed
by means of the slope method. The calculation was carried by means of the following
formula.
SLOD 3.3
Where = standard deviation of intercept
S = slope of the calibration curve
SLOQ 10
Where = standard deviation of intercept
S = slope of the calibration curve
The high level of sensitivity of the method can be observed by means of low levels of
the LOD and LOQ values.
Table 2.31-LOD and LOQ of Atorvastatin Impurities
Impurity Name LOQ LOD
Desfluoro 0.008% 0.003%
Tert Butyl Ester 0.017% 0.004%
Lactone 0.010% 0.003%
Oxirane 0.020% 0.005%
Difluoro 0.010% 0.003%
Page 110 of 305
Table 2.32-LOD and LOQ of Clopidogrel Impurities
Impurity Name LOQ LOD
Impurity A
Tetrahydro pyridine (C-748) 0.010% 0.002%
Impurity B
Cyclic Amide 0.003% 0.001%
Impurity C
Acid Impurity 0.016% 0.004%
Impurity D
Dehydro Impurity 0.011% 0.002%
Impurity 3
Methyl Ester Impurity 0.017% 0.002%
Conclusion:
A method for determination of Clopidogrel, Atorvastatin, and their related substances
has been successfully developed by UPLC. This method is having lot of advantages
owing to its extremely short run time. This method has also been validated as per
ICH guidelines. The method has demonstrated the stability indicating capability as it
has complied the acceptance criteria of separating all the unknown degradation
products arising from various stress studies, namely acid, base and peroxide.
The method is found to be specific, precise, linear and accurate in the range of its
intended application. This method is suitable for use in routine analysis in any quality
control laboratory and if applied will prove to be extremely beneficial for the
organization and the end user i.e. the patient.
Page 111 of 305
References:
1. M.R Law.;NJ Wald.; AR Rudnicka.; BMJ 326 (7404): 1423(June 2003).
2. PW Wilson.; RB D'Agostino.; D Levy.; AM Belanger.; H Silbershatz.; WB Kannel
97 (18): 1837–47. (May 1998)
3. P Jones.; S Kafonek.; I Laurora, D Hunninghake. American Journal of Cardiology
81 (5): 582–7. (March 1998)
4. P Savi.; J-L Zachayus.; N Delesque-Touchard.;C Labouret.; C Herve´.; M-F
Uzabiaga.; J-M Pereillo.; J-Ml Culouscou.; F.Bono.;P Ferrara, and J-M Herbert.
Proceedings of the National Academy of Sciences of the USA 103 (29): pp11069–
11074. (July 2006)
5. S Rossi, Australian Medicines Handbook. Adelaide: Australian Medicines
Handbook; (2006).
6. D Ml Randall.; KE Neil. Disease management. 2nd
ed. London: Pharmaceutical Press.
159. (2004)
7. S. Tetik.; Ak Koray.;S Isbir.; E Eksioglu-Demiralp.;S Arsan.;O Iqbal.;T Yardimci.;
Clin Appl Thromb Hemost 16 , 2 , pp189-198, (April 2010)
8. S Ertürka.; E S Aktaşa.; L Ersoya.; S Fıçıcıoğlub.; Journal of Pharmaceutical and
Biomedical Analysis 33, 5, pp 1017–1023(2003)
9. BG Chaudhari , N M Patel, P BShah Chemical and Pharmaceutical Bulletin Vol. 55
No. 2 P 241-246 (2007)
10. AA Kadav.; DN Vora.; Journal of Pharmaceutical and Biomedical Analysis
48, 1, 10, pp 120–126 (September 2008)
11. R K Seshadri.; M M Desai.; T V Raghavaraju.;D Krishnan.; D V Rao. and IE
Chakravarthy.; Sci Pharm, 78(4) pp821–834, (December 2010)
12. D N Vora.; A A Kadav.; Journal of Liquid Chromatography & Related
Technologies
31,18 (2008)
13. A. Mitakos.; I. Panderi.; Journal of Pharmaceutical and Biomedical Analysis 28, 3–
4, 15, pp 431–438 (May 2002)
14. S S. Singh, K Sharma, D Barot, P. RMohan, V B. Lohray, Journal of
Chromatography B, pp 821, 2, 25, pp 173–180 (July 2005)
Page 112 of 305
15. J-M Pereillo.; M Maftouh.; A.; Andrieu.; M-F Uzabiaga.; O.Fedeli.; P Savi,
M.Pascal, J-M Herbert, J-P Maffrand and C Picard, Drug Metabolism and disposition,
30, 11 pp 1288-1295 (2002)
16. RV Nirogi.; V N Kandikere.; M Shukla.; K Mudigonda.; S Maurya.; R Boosi.;
Rapid Communications in Mass Spectrometry, 20, 11, pp 1695–1700, (15 June 2006)
17. K Anandakumar.; T Ayyappan.; V Raghu Raman.; T Vetrichelvan.; A.S.K Sankar.;
D Nagavalli.; Indian Journal of Pharmaceutical sciences, 69,4, pp597-599(2007)
18. H O Kaila.; MA Ambasana1 and A K Shah.; International Journal of ChemTech
Research, 3, 1, pp 459-465, (2011)
19. X Yang, S Liu, J Sun, X Liu, Y Sun, Z He, Chromatographia, 70, 1-2, pp 259-263
(July 2009)
20. M K Javed, Z Iqbal, A Khan, A Khan, Y Shah & L Ahmad, Journal of Liquid
Chromatography & Related Technologies, 34, 18 (2011)