Method Development for Related substances of Atorvastatin ...shodhganga.inflibnet.ac.in/bitstream/10603/8657/7/07_chapter 2.pdfPage 49 of 305 4. NMR Study: The 1H and 13C NMR (Fig

Page 44 of 305

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

http://www.sigmaaldrich.com/catalog/product/SIGMA/C0614?lang=en&region=IN

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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

http://www.sciencedirect.com/science/article/pii/S0731708503004084

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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

http://www.trc-canada.com/Structures/A791850.png

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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.

Page 57 of 305

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

Page 60 of 305

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:

Page 62 of 305

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

Page 63 of 305

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

Page 64 of 305

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

Page 65 of 305

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:





Mobile Phase B: Buffer : Acetonitrile:10:90% v/v






Wavelength 220 nm

Sample temperature NA



Page 66 of 305

Gradient Program


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:





Mobile Phase B: Buffer :Acetonitrile:Methanol:10:60:30% v/v/v






Wavelength 220 nm




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


Observation:


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:





Mobile Phase B: Buffer :Acetonitrile:Methanol:10:70:30% v/v/v




Page 69 of 305



Wavelength 220 nm




Gradient Program


Observation:

Atorvastatin and all its Impurities eluted between 9 to 13 minutes. Clopidogrel


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:





Mobile Phase B: Buffer :Acetonitrile:Methanol::10:70:20% v/v/v






Wavelength 220 nm




Page 71 of 305

Gradient Program


Observation:



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


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 B: Buffer :Acetonitrile : Methanol::10:70:20% v/v/v

Diluent: Methanol for Atorvastatin,





Wavelength 220 nm




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


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


Page 74 of 305

Experiment 7:

Buffer: MilliQ Water pH adjusted to 3.0 with 85% Orthophosphoric Acid



Diluent: Methanol for Atorvastatin,





Wavelength 220 nm




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


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






EXPERIMENT 7

Clopidogrel


Page 76 of 305



Wavelength 220 nm




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

EXPERIMENT 8

Clopidogrel


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



Wavelength 220 nm




Page 78 of 305

Gradient Program


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




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



Wavelength 220 nm

Sample temperature NA


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 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




Wavelength 220 nm

Inj Volume 2µL


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


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


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


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


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


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


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


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


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


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


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


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

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Documents

Method Development for Related substances of Atorvastatin ...shodhganga.inflibnet.ac.in/bitstream/10603/8657/7/07_chapter 2.pdfPage 49 of 305 4. NMR Study: The 1H and 13C NMR (Fig