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CHAPTER 2

SIMULTANEOUS DETRMINATION OF

ANASTROZOLE AND TEMOZOLOMIDE

IN

TEMOZOLOMIDE CAPSULES 20 MG AND

ANASTROZOLE TABLETS 1 MG

ANALYTICAL METHOD VALIDATION

REPORT

FOR

ASSAY

44

2.1 Introduction

Analytical method of assay for Temozolomide capsules 20 mg and Anastrozole tablets 1 mg

is developed on reverse phase Fast LC. The assay method for Anastrozole tablets 1 mg and

Temozolomide capsules 20 mg has been subjected for validation. All the parameter has been

covered during the validation, which are illustrated in the table no.01.The analytical method

validation has been performed as per ICH guideline Q2 (R1).

This analytical method validation report is prepared and proved capability of analytical

method for its specific nature, accuracy, linearity, preciseness and robust nature.

2.2 Label Claim

Anastrozole Tablet 1 mg

Each Film coated Tablet consisting of Anastrozole …….1 mg

Temozolomide capsules 20 mg

Each Capsule contains consisting of Temozolomide …….20 mg

2.3 References

Validation references

Analytical Method Validation Program as per ICH guideline Q2 (R1)

45

2.4 Analytical method validation program as per ICH guideline

Method validation activities has been conducted as per following validation program,

Table No. 01: Validation program as per ICH guideline

Parameter Assay

Specificity +

Accuracy +

Detection Limit ( LOD ) +

Quantitation Limit (LOQ) +

Linearity +

Range +

Precision

Injection repeatability

Analysis repeatability

Intermediate precision

+

+

+

Solution Stability

Stability of analytical solutions

+

Robustness

Influence of variations of test parameters

+

- Signifies that these characteristic are not evaluated.

+ Signifies that these characteristic are evaluated.

46

2.5 Details of the API, standard, sample and placebo used in validation activity

Table No. 02 : Details of the API, standard, sample and placebo

Sr. No.

Name B.No./Lot

No. % Purity as is

basis Manufactured

date

1 Anastrozole API FX8098 99.7%

2 Temozolomide API IW880085 99.8%

3 Anastrozole working

standard WS/C/A87/01

99.95 % (on as is basis)

NA

4 Temozolomide

working standard TS/C/T23/O1

100.00 % (on as is basis)

NA

5 Anastrozole Tablet 1

mg TANA 81009 NA NA

6 Temozolomide Capsule 20 mg

CTZA91003 NA NA

7 2-Azahyooxanthine Iimpurity standard

474.01.09.03 99.3% NA

8 AIC HCL

ImpurityStandard TS-22/T01 98.5%

NA

9 TMZ II Impurity

Standard TS-22/T03 99.2%

NA

10 Anastrozole Impurity A Impurity standard

TS-21/A5 99.9% NA

11 Anastrozole Impurity B Impurity standard

0098.056-01 98.9% NA

12 Anastrozole Broomo Compound Impurity

standard TS/C/A62/01 99.0%

NA

47

2.6 Details of chemicals and reagents used for validation activity

Table No. 03: List of chemicals and reagents used

Sr.No. Name of

Chemicals / Reagents Batch / Lot No. Grade

Manufacturer

1 Acetonitrile SF0SF60511 HPLC grade Merck

2 Ammonium Acetate ME18DO580153 A R grade Merck

3 Triethylamine SC0S590651 Synthesis Merck

4 Purified water - HPLC grade Millipore

5 Orthophosphoric acid 0174 7006-5 A R grade Qualigens

6 Methanol 1036/1 7003-6 HPLC grade Qualigens

2.7 Details of instrument and equipment used for validation activity

Table No. 04: List of Instruments and equipments used

Sr.No. Name of Instrument /

Equipment Make Software

1 Ultimate 3000 Dionex Chromeleone

2 Waters Waters Empower

3 Weighing Balance Mettler Toledo XS205

5 pH meter Metrohm 780 pH Meter

7 Water Purifier Millipore Gradient A10

8 Vacuum pump ACMEVAC 15P166

9 Sonicator PCi 10.5L 250/SPL

2.8 Detail of LC columns used for validation activity :

1 Column : Inertsil C8

Dimentions : 25 mm x 5-cm, 4.6-µm

Serial No. : ILU9976500341 and ILU9976500768

48

2.9 Method validation parameters for assay by LC method :

SYSTEM SETUP

A. Chromatographic Conditions Column OvenTemperature: 25 C Mobile Phase A Ammonium acetate buffer (0.1%) Mobile Phase B Acetonitrile Flow Rate: 1.0 mL/min Injection Volume: 10 µL Detection: UV at 215 nm Gradient Program:

Time (min.) %A %B

0 95 05 7.0 95 05

12.0 93 07

17.0 50 50

40.0 30 70

45.0 95 05

55.0 95 05

2.9.1 SPECIFICITY AND SYSTEM SUITABILITY

Specificity is the ability to assess unequivocally the analyte in the presence of

components which may be expeced to present.

For this study first upon a 10 μl of blank solution (Acetonitrile: water 50:50) and

placebo was injected and ran with the gradient programme. After this 10 μl of

Resolution solution was injected followed by Individual Impurity standard solutions

and injected standard solutions in 6 replicate and the % relative standard deviation (%

R.S.D) of the response peak areas was calculated.

Specificity study solution are prepared as follows,

Blank solution: Acetonitrile: water 50:50.

49

Preparation of Placebo solution for Anastrozole tablets 1 mg:

A Placebo about 0.90 gm was weighedand transferred in to 100 ml volumetric flask

equivalent excluding 10 mg of Anastrozole, to this flask added 60 mL of equal

volumes of Acetonitrile and water respectively and the mixture was subjected to

vigorous shaking for 10 min and diluted up to volume with same solvent. Further

diluted 10 mL of these solutions to 100 ml with Acetonitrile: water 50:50.. The

solution was filtered through 0.45µ nylon filter before analysis. 10 µl of this solutions

were injected in to the HPLC system

Preparation of Placebo solution for Temozolomide capsules 20 mg:

A Placebo about 0.260 gm was weighedand transferred in to 100 ml volumetric flask

equivalent excluding 10 mg of Temozolomide, to this flask added 60 mL of equal

volumes of Acetonitrile and water respectively and the mixture was subjected to

vigorous shaking for 10 min and diluted up to volume with same solvent. Further

diluted 10 mL of these solutions to 100 ml with Acetonitrile: water 50:50. The solution

was filtered through 0.45µ nylon filter before analysis. 10 µl of these solutions were

injected in to the HPLC system.

Preparation of Standard solution

Standard stock solution was prepared by dissolving accurately 50 mg Anastrozole and

50 mg Temozolomide in 100 mL of equal volumes of Acetonitrile and water. 2 ml of

standard stock solution was diluted to 100 ml with Acetonitrile: water 50:50 to obtain

each of 10 µg/mL of Anastrozole and Temozolomide. The solution was filtered

through 0.45µ nylon filter before analysis. A series of solutions containing Anastrozole

and Temozolomide were prepared with mobile phase A and 10 µl of these solutions

were injected in to the HPLC system.

Preparation of Resolution solution

Transferred 2.0 mg of each of AIC HCL (5amino imidazole-4-

carboxamidehydrochloride) impurity, 2-Azahypoxanthine impurity and TMZ II

impurity and Anastrozole Bromo compound, Anastrozole impurity A and Anastrozole

impurity B dissolved in 200 mL of equal volumes of Acetonitrile: water. Further dilute

1 mL of this solution and 2 mL of standard stock solution into 100 mL of Acetonitrile:

water (50:50)

50

Preparation of Impurity solutions for Identification.

AIC HCL (5amino imidazole-4- carboxamidehydrochloride) impurity solution for

Identification

Transferred 2.0 mg of AIC HCL (5amino imidazole-4- carboxamidehydrochloride)

impurity dissolved in 200 mL of equal volumes of Acetonitrile: water. Further dilute 1

mL of this solution 100 mLvolumetric flask and diluted to volume with Acetonitrile:

water (50:50)

2-Azahypoxanthine impurity solution for Identification

Transferred 2.0 mg of 2-Azahypoxanthine impurity dissolved in 200 mL of equal

volumes of Acetonitrile: water. Further dilute 1 mL of this solution 100 mLvolumetric

flask and diluted to volume with Acetonitrile: water (50:50).

TMZ II impurity solution for Identification

Transferred 2.0 mg of TMZ II impurity dissolved in 200 mL of equal volumes of

Acetonitrile: water. Further dilute 1 mL of this solution 100 mLvolumetric flask and

diluted to volume with Acetonitrile: water (50:50).

Anastrozole Bromo compound impurity solution for Identification

Transferred 2.0 mg of Anastrozole Bromo compound impurity dissolved in 200 mL of

equal volumes of Acetonitrile: water. Further dilute 1 mL of this solution 100 mL

volumetric flask and diluted to volume with Acetonitrile: water (50:50)

Anastrozole Impurity A solution for Identification

Transferred 2.0 mg of Anastrozole impurity A dissolved in 200 mL of equal volumes

of Acetonitrile: water. Further dilute 1 mL of this solution 100 mLvolumetric flask and

diluted to volume with Acetonitrile: water (50:50).

Anastrozole Impurity B solution for Identification

Transferred 2.0 mg of Anastrozole impurity B dissolved in 200 mL of equal volumes

of Acetonitrile: water. Further dilute 1 mL of this solution 100 mLvolumetric flask and

diluted to volume with Acetonitrile: water (50:50)

51

Specimen chromatograms for specificity are as follows :

Figure No. 4 : Blank solution chromatogram in specificity

Figure No.5 : Standard solution chromatogram in specificity

Figure No. 6 : Resolution solution chromatogram in specificity

52

Figure No. 7 : Placebo Temozolomide chromatogram in specificity

Figure No. 8: Placebo Anastrozole chromatogram in specificity.

Figure No. 9 : Temozolomide chromatogram for Identification in specificity

53

Figure No. 10 : Anastrozole chromatogram for Identification in specificity

Figure No. 11 : 2-Azahypoxanthine impurity chromatogram for

Identification in specificity

Figure No. 12 : TMZ II Impurity chromatogram for Identification in

specificity

54

Figure No. 13 : AIC HCL Impurity chromatogram for Identification in

specificity

Figure No. 14 : Anastrozole Impurity A chromatogram for Identification in

specificity

Figure No. 15 : Anastrozole Impurity B chromatogram for Identification in

specificity

55

Figure No. 16 : Anastrozole Bromo Compound chromatogram for

Identification in specificity

56

Table No. 05 : System suitability of specificity study

Sr.No System suitability parameter

Results Acceptance criteria

1 Resolution between2-Azahypoxanthine and AIC Impurity

2.00 NLT 2.0.

2 Resolution between Temozolomide and TMZ-II impurity

4.46 NLT 2.0.

3 Resolution between ANZ Impurity A and Anastrozole

7.04 NLT 2.0.

4 Resolution between ANZ impurity B and ANZ Bromo Impurity

2.24 NLT 2.0.

5 RSD Anastrozole peak from five replicate Injections of standard.

0.2 NMT 2.0%.

6 Asymmetry for Anastrozole

1.9 NLT 2.0.

7 Theoretical Plates for Anastrozole

172812 NLT 3000

8 RSD Temozolomide peak from five replicate Injections of standard.

0.1 NMT 2.0%.

9 Asymmetry for Temozolomide.

1.6 NLT 2.0.

10 Theoretical Plates for Temozolomide

7239 NLT 3000

Conclusion : Assay method for Temozolomide capsules 20 mg and Anastrozole

tablets 1 mg is said to specific as per assessment of the specificity study.

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2.9.2 ACCURACY

Accuracy was carried out at 50.0%, 100.0% and 150.0% of the target concentration of Anastrozole (10.0 µg per mL) and Temozolomide (10.0 µg per mL) .Solutions were prepared in triplicate at each level.

Accuracy study solution are prepared as follows,

For blank solution, standard solution preparation, refer the solution preparation under specificity parameter.

For system suitability table refer specificity parameter.

Preparation of accuracy solution as per follows :

Preparation of solutions for Anastrozole tablets 1 mg:

A Placebo about 0.90 gm was weighed and transferred in to 100 ml volumetric flask equivalent excluding 10 mg of Anastrozole, then weighed and added specified quantity of Anastrozole API (quantity as per Table given below) to the same volumetric flask.to this flask added 60 mL of equal volumes of Acetonitrile and water respectively and the mixture was subjected to vigorous shaking for 10 min and diluted up to volume with same solvent. Further diluted 10 mL of these solutions to 100 ml with Acetonitrile: water 50:50.. The solution was filtered through 0.45µ nylon filter before analysis. 10 µl of this solutions were injected in to the HPLC system

Preparation of solutions for Temozolomide capsules 20 mg:

A Placebo about 0.270 gm was weighedand transferred in to 100 ml volumetric flask equivalent excluding 10 mg of Temozolomide, then weighed and added specified quantity of Anastrozole API (quantity as per Table given below) to the same volumetric flask. to this flask added 60 mL of equal volumes of Acetonitrile and water respectively and the mixture was subjected to vigorous shaking for 10 min and diluted up to volume with same solvent. Further diluted 10 mL of these solutions to 100 ml with Acetonitrile: water 50:50. The solution was filtered through 0.45µ nylon filter before analysis. 10 µl of these solutions were injected in to the HPLC system

58

Table No. 06 : Preparation of accuracy levels as per following table:

For Anastrozole

Level No.

% Level

Prep No.

First dilution Second Dilution

Wt.of Placebo

(mg)

Wt.of API (mg)

Diluted to (mL)

Pipetted

(mL)

Diluted to (mL)

Level 1

50 1 0.90 25.0 100 2 100 2 0.90 25.0 100 2 100

3 0.90 25.0 100 2 100

Level 3

100 1 0.90 50.0 100 2 100

2 0.90 50.0 100 2 100

3 0.90 50.0 100 2 100

Level 5

150 1 0.90 75.0 100 2 100

2 0.90 75.0 100 2 100

3 0.90 75.0 100 2 100

For Temozolomide

Level No.

% Level

Prep No.

First dilution Second Dilution

Wt.of Placebo

(mg)

Wt.of API (mg)

Diluted to (mL)

Pipetted

(mL)

Diluted to (mL)

Level 1

50 1 0.490 25.0 100 2 100 2 0.490 25.0 100 2 100

3 0.490 25.0 100 2 100

Level 3

100 1 0.490 50.0 100 2 100

2 0.490 50.0 100 2 100

3 0.490 50.0 100 2 100

Level 5

150 1 0.490 75.0 100 2 100

2 0.490 75.0 100 2 100

3 0.490 75.0 100 2 100

59

Table No. 07 : % Recovery at difference levels for Anastrozole

Level No.

% Level wrt.

working conc.

Conentr-ation

added in µg per

mL

Conentration recovered in µg per mL

% Recovery

Unrounded % Assay Rounded

1 50

5.1 5.18 101.55 101.6

5.1 5.19 101.74 101.7

5.1 5.19 101.73 101.7

2 100

10.2 10.24 100.35 100.3

10.2 10.28 100.75 100.7

10.2 10.24 100.40 100.4

3 150

15.3 15.03 98.25 98.3

15.3 15.11 98.73 98.7

15.3 15.03 98.25 98.3

Mean % Recovery 100.2

Minimum % Recovery 98.3

Maximum % Recovery 101.7

STDev 1.4

RSD 1.4

60

Table No. 08 : % Recovery at difference levels for Temozolomide

Level No.

% Level wrt.

working conc.

Conentr-ation

added in µg per

mL

Conentration recovered in µg per mL

% Recovery

Unrounded % Assay Rounded

1 50

4.95 4.95 99.91 99.9

4.95 4.94 99.77 99.8

4.95 4.96 100.27 100.3

2 100

9.9 9.90 99.99 100.0

9.9 9.90 99.99 100.0

9.9 9.85 99.49 99.5

3 150

14.85 14.79 99.63 99.6

14.85 14.79 99.59 99.6

14.85 14.79 99.63 99.6

Mean % Recovery 99.8

Minimum % Recovery 99.6

Maximum % Recovery 100.3

STDev 0.251

RSD 0.3

61

Specimen chromatograms of accuracy study are given below,

Figure No. 17: Anastrozole chromatogram in accuracy at 50% level

Figure No. 18 : Anastrozole chromatogram in accuracy at 100% level

Figure No. 19 : Anastrozole chromatogram in accuracy at 150% level

62

Figure No. 20 : Temozolomide chromatogram in accuracy at 50% level.

Figure No. 21 : Temozolomide chromatogram in accuracy at 100% level

Figure No. 22 : Temozolomide chromatogram in accuracy at 150% level

63

Table No. 09 : Mean results of accuracy study for Anastrozole

Sr. No. Parameter Results Acceptance criteria

1 Individual % recovery. % Recovery obtained between 98.3 to 100.2

Between 97.0% to 103.0%.

2 Mean % recovery of all recovery levels.

100.2 Between 98.0% to 102.0%.

3 RSD of recovery of all five recovery levels.

1.4 Not more than 2.0%.

Table No. 10 : Mean results of accuracy study for Temozolomide

Sr. No. Parameter Results Acceptance criteria

1 Individual % recovery. % Recovery obtained between 99.6 to 100.3

Between 97.0% to 103.0%.

2 Mean % recovery of all recovery levels.

99.8 Between 98.0% to 102.0%.

3 RSD of recovery of all five recovery levels.

0.3 Not more than 2.0%.

Conclusion: Assay method for Temozolomide capsules 20 mg and Anastrozole tablets 1 mg is found accurate as recovery results and individual recoveries are well within the acceptance criteria.

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2.9.3 LOD AND LOQ SENSITIVITY

The Quantitation limit of an individual analytical procedure is the lowest amount of analyte or known impurity in a sample which can be quantitatively determined with suitable precision and accuracy.

The detection limit of an individual analytical procedure is the lowest amount of analyte or known impurities in a sample which can be detect but not quantified.

Determination of LOD and LOQ solution :

For LOD and LOQ concentration determination, solutions containing Anastrozole

and Temozolomide of different concentration injected and detected signal to noise

ratio instrumentally.

Table No. 11: The Concentrations of LOD and LOQ level experiment.

Concentration For LOD Concentration For LOQ

Anastrozole 0.092 µg/ mL 0.075 µg/ mL

Temozolomide 0.17 µg/ mL 0.16 µg/ mL

Table No. 12: Signal to Noise Ratio for Anastrozole

Table No. 13: Signal to Noise Ratio for Temozolomide

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2.9.4 LINEARITY AND RANGE

The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.

Linearity was carried out with minimum 8 concentration levels in duplicate within a range of LOQ to 150% of the target concentration of analyte by using test sample. Linearity and range of assay method from LOQ to 150.0% of working concentration of anastrozole (Coonc.10.00 µg per mL) and Temozolomide (Coonc.10.00 µg per mL).

Evaluation

Table No. 14 : Linearity study data for Anastrozole

No of Linearity levels

% Level wrt. Working conc.

Concentraton in ppm

Area of Anastrozole

1 0.16 % 0.016 24152

2 10 % 1.011 100016

3 20 % 2.022 200224

4 50 % 5.055 501898

5 75 % 7.583 752937

6 100 % 10.110 1005739

7 125 % 12.638 1253672

8 150 % 15.165 1506657

Table No. 15: Linearity study data for Temozolomide

No of Linearity

% Level w.r.t. Working conc.

Concentration in ppm

Area of Temozolomide

1 0.17 % 0.017 3273

2 10 % 1.051 198599

3 20 % 2.102 392978

4 50 % 5.255 990627

5 75 % 7.883 1488360

6 100 % 10.510 1982557

7 125 % 13.138 2480228

8 150 % 15.765 2975332

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Linearity graph of concentration in µg per mL against area response, plotted on basis of above information is as follows,

Figure No. 23 : Anastrozole Linearity plot of concentration in µg per mL against area responce in assay

Figure No. 24 : Temozolomide Linearity plot of concentration in µg per mL against area responce in assay

Table No. 16: Results of Linearity study

S.R.NO. Anastrozole Temozolomide

1 The linear regression equation

997514x + 7103.2

1,984,462.43x - 1,121.41

2 correlation coefficient 0.999 0.999

Conclusion : Assay method for Temozolomide capsules 20 mg and Anastrozole tablets 1 mg is found linear within the specified range as LOQ to 150% of working level of Anastrozole and Temozolomide

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2.9.5PRECISION :

The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions.

Injection repeatability Analysis repeatability Intermediate precision Injection repeatability

Injection repeatability is established at the start of each experiment. For blank solution, standard solution preparation, refer the solution preparation under specificity parameter.


Analysis repeatability

Analysis repeatability for assay carried out with six preparations of assay samples.

Precision sample solution : Six sample prepared as per follows,

Preparation of sample solution for Anastrozole tablets 1 mg:

A sample about 0.90 gm was weighedand transferred in to 100 ml volumetric flask

(equivalent to 10 mg of Anastrozole), to this flask added 60 mL of equal volumes of

Acetonitrile and water respectively and the mixture was subjected to vigorous shaking for 10

min and diluted up to volume with same solvent. Further diluted 10 mL of these solutions to

100 ml with Acetonitrile: water 50:50.. The solution was filtered through 0.45µ nylon filter

before analysis. 10 µl of this solutions were injected in to the HPLC system.

Preparation of Sample solution for Temozolomide capsules 20 mg:

A sample about 0.270 gm was weighedand transferred in to 100 ml volumetric flask

(equivalent 10 mg of Temozolomide), to this flask added 60 mL of equal volumes of

Acetonitrile and water respectively and the mixture was subjected to vigorous shaking for

10 min and diluted up to volume with same solvent. Further diluted 10 mL of these

solutions to 100 ml with Acetonitrile: water 50:50. The solution was filtered through 0.45µ

nylon filter before analysis. 10 µl of these solutions were injected in to the HPLC system.

68

Evaluation

Table No. 17: Results of precision study for Anastrozole

Lable Claim of Anastrozole % Assay of Anastrozole

Sample 1 1.00 99.74

Sample 2 0.99 99.33

Sample 3 0.99 99.49

Sample 4 1.00 100.09

Sample 5 1.01 100.84

Sample 6 1.00 100.39

Mean 99.98

S.D 0.57

%RSD 0.57

Table No. 18 : Results of precision study for Temozolomide

Lable Claim of Temozolomide % Assay of Temozolomide

Sample 1 19.09 95.44

Sample 2 19.01 95.05

Sample 3 19.04 95.20

Sample 4 19.16 95.78

Sample 5 19.30 96.49

Sample 6 19.21 96.07

Mean 95.67

S.D 0.55

%RSD 0.57

69

Specimen chromatograms are given below,

Figure No. 25 : Anastrozole sample chromatogram in Precision study.

Figure No. 26 : Temozolomide sample chromatogram in Precision study.

Conclusion: Assay method for Temozolomide capsules 20 mg and Anastrozole tablets 1 mg is found precise during analysis repeatability as RSD is well within acceptance criteria.

70

2.9.6 INTERMEDIATE PRECISION

Intermediate precision is the precision repeated under difference conditions such as by different analyst, on different day, on different instrument and by using different Fast LC column of same brand.

Intermediate Precision is performed by following different conditions,

1. Different analyst

2. Different day

3. Different instrument

4. Different LC column of same dimension and make

Intermediate precision study solution are prepared as follows,

For this study first 10 μl of blank solution (Acetonitrile: water 50:50) and placebo was

injected and ran with the gradient programme. After this 10 μl of Resolution solution was

injected followed by Individual Impurity standard solutions and injected standard solutions in

6 replicate and the % relative standard deviation (% R.S.D) of the response peak areas was

calculated.

Preparation of Placebo solution for Anastrozole tablets 1 mg:

A Placebo about 0.90 gm was weighed and transferred in to 100 ml volumetric flask

equivalent excluding 10 mg of Anastrozole, to this flask added 60 mL of equal volumes of



100 ml with Acetonitrile: water 50:50. The solution was filtered through 0.45µ nylon filter

before analysis. 10 µl of these solutions were injected in to the HPLC system.

Preparation of Placebo solution for Temozolomide capsules 20 mg:

A Placebo about 0.270 gm was weighed and transferred in to 100 ml volumetric flask

equivalent excluding 10 mg of Temozolomide, to this flask added 60 mL of equal volumes of





71

Preparation of Standard solution

Standard stock solution was prepared by dissolving accurately 50 mg Anastrozole and 50 mg

Temozolomide in 100 mL of equal volumes of Acetonitrile and water. 2 ml of standard stock

solution was diluted to 100 ml with Acetonitrile: water 50:50 to obtain each of 10 µg/mL of

Anastrozole and Temozolomide. The solution was filtered through 0.45µ nylon filter before

analysis. and 10 µl of these solutions were injected in to the HPLC system.

Preparation of Resolution solution

Transferred 2.0 mg of each of AIC HCL (5amino imidazole-4- carboxamidehydrochloride)

impurity, 2-Azahypoxanthine impurity and TMZ II impurity and Anastrozole Bromo

compound, Anastrozole impurity A and Anastrozole impurity B dissolved in 200 mL of

equal volumes of Acetonitrile: water. Further dilute 1 mL of this solution and 2 mL of

standard stock solution into 100 mL of Acetonitrile: water (50:50)

Preparation of sample solution for Anastrozole tablets 1 mg:

A sample about 0.350 gm was weighed and transferred in to 100 ml volumetric flask

(equivalent to 10 mg of Anastrozole), to this flask added 60 mL of equal volumes of



100 ml with Acetonitrile: water 50:50.. The solution was filtered through 0.45µ nylon filter

before analysis. 10 µl of this solutions were injected in to the HPLC system.

Prepared six sample preparations for this study.

Preparation of Sample solution for Temozolomide capsules 20 mg:

A sample about 0.270 gm was weighed and transferred in to 100 ml volumetric flask

(equivalent 10 mg of Temozolomide), to this flask added 60 mL of equal volumes of





Prepared six sample preparations for this study.

72

Evaluation

Table No. 19 : Results of Intermediate precision study for Anastrozole

Lable Claim of Anastrozole

% Assay of Anastrozole

Sample 1 1.02 101.68

Sample 2 1.00 100.29

Sample 3 0.99 99.29

Sample 4 1.00 100.50

Sample 5 1.01 100.74

Sample 6 1.01 100.60

Mean 100.52

S.D 0.77

%RSD 0.76

Table No. 20 : Results of Intermediate precision study for Temozolomide

Lable Claim of Temozolomide % Assay of Temozolomide

Sample 1 19.46 97.30

Sample 2 19.19 95.97

Sample 3 19.00 95.02

Sample 4 19.23 96.17

Sample 5 19.28 96.40

Sample 6 19.25 96.26

Mean 96.19

S.D 0.74

%RSD 0.76

73

Comparative result of precision and intermediate precision results

Table No. 21 : Comparison between % Assay in precision study and intermediate precision study

for anastrozole

Sr.No.

% Assay values in Precision for anastrozole

(Analyst I)

% Assay values in Intermediate Precision for

anastrozole

(Analyst II)

1 99.74 101.68

2 99.33 100.29

3 99.49 99.29

4 100.09 100.50

5 100.84 100.74

6 100.39 100.60

Average 99.98 100.52

SD 0.57 0.77

RSD 0.57 0.76

% Absolute difference 0.5

For Temozolomide

Sr.No.

% Assay values in Precision for Temozolomide

(Day I)

% Assay values in Intermediate Precision for Temozolomide

(Day II)

1 95.44 97.30 2 95.05 95.97

3 95.20 95.02

4 95.78 96.17

5 96.49 96.40

6 96.07 96.26

Average 95.67 96.19 SD 0.55 0.74

RSD 0.57 0.76 % Absolute difference 0.5

74


Figure No. 27 : Anastrozole sample chromatogram in Intermediate Precision study.

Figure No. 28 : Temozolomide sample chromatogram in Intermediate Precision study.

Conclusion: Assay method for Temozolomide capsules 20 mg and Anastrozole tablets 1 mg is found rugged during analysis of precision and intermediate precision as % absolute difference is well within acceptance criteria.

75

2.9.7 SOLUTION STABILITY

Solution stability study for standard solution has been conducted for 72 hours by keeping the solution on bench top at normal illuminated laboratory condition.

1. Sample solution stability at bench top condition (At normal illuminated laboratory condition)

This study has done for 72 hours for standard and sample solutions.

Solution stability study solutions are prepared as follows :

For standard solution preparation, refer the solution preparation under specificity parameter


Standard solution at initial time point :

Calibration standard solution from specificity is used for solution stability.

Test solution at initial time point :

Test solution from specificity is used for solution stability.

Standard and test solution stability summary :

Solution stability study for standard and test solution has been established by calculating % relative difference of the standard area and test area at each time interval with respect to initial solutions area.

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Evaluation

Table No. 22 : Solution stability study of standard solution at bench top condition for Anastrozole (i.e. at normal illuminated laboratory condition) as given below

Solution stability for Standard solution

Status for standard solution

Area response for Anastrozole peak

% Recovery Relative % Difference

Initial 1002568 100.0

6 hours 1003613 100.1 0.21

24 hours 1002037 99.9 0.05

72 hours 1003776 100.1 0.24

Table No. 23 : Solution stability study of Sample solution at bench top condition for Anastrozole (i.e. at normal illuminated laboratory condition) as given below


Status for Sample solution

Area response for Anastrozole peak


Initial 1011034 100.0

6 hours 1009787 99.9 0.12

24 hours 998827 98.8 1.21

72 hours 1006823 99.6 0.42

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Table No. 24: Solution stability study of Sample solution at bench top condition for Temozolomide (i.e. at normal illuminated laboratory condition) as given below



Area response for Temozolomide peak


Initial 1981865 100.0 -

6 hours 1976779 99.7 0.26

24 hours 1977816 99.8 0.20

72 hours 1977832 99.8 0.20

Table No. 25: Solution stability study of Sample solution at bench top condition for Temozolomide (i.e. at normal illuminated laboratory condition) as given below



Area response for Temozolomide peak


Initial 1956086 100.0 -

6 hours 1963322 100.4 0.74

24 hours 1956890 100.0 0.08

72 hours 1968727 100.6 1.29

Results:

% Relative difference between area of standard solution upto 72 hours on bench top under normal illuminated laboratory condition with respect to area of initial area is within limit i.e. less than 2.0%.

% Relative difference between areas of test solution up-to 72 hours on bench top under normal illuminated laboratory condition with respect to area of initial test solution area is within limit i.e. less than 2.0%.

Conclusion :

Standard solution is stable up-to 72 hours at normal illuminated laboratory condition on bench top.

Test solution is stable up-to 72 hours at normal illuminated laboratory condition on bench top.

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2.9.8 ROBUSTNESS

9.8.1 Influence of variation of test parameters

Table No. 26 : List of parameters studied with deliberated changes

Sr.

No. Parameters Deliberate changes

1 Flow rate of mobile phase. 0.9 And 1.1 (i.e. ± 0.1 mL per minute).

2 Change in column temperature. 23°C and 27°C (i.e. ± 2.0°C).

For preparation of blank, standard and placebo solutions refer the solution preparation under specificity parameter and for test solution refer precision parameter.

Robustness 1

Table No. 27: Results of Robustness at the flow rate of 0.9 ml /mim for sample solution.

Sr. No. % Assay of Anastrozole % Assay of Temozolomide

1 99.86 98.75

2 100.46 99.06

3 100.47 97.63

Mean % Assay value for Robustness Study

100.26 98.48

Mean % Assay value for Precision Study

99.98 95.67

% Absolute difference 0.54 1.33

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Robustness 2

Table No. 28 : Results of Robustness at the flow rate of 1.1 ml /mim for sample solution.

Sr. No. % Assay of Anastrozole % Assay of Temozolomide

1 99.45 99.88

2 100.90 100.03

3 100.17 100.05


100.17 98.38


99.98 95.67


Robustness 3

Table No. 29 : Results of Robustness at the column oven temperature 23°C sample solution.

Sr.No. % Assay of Anastrozole % Assay of Temozolomide

1 98.02 98.63

2 97.72 98.66

3 98.53 97.78


98.09 98.35


99.98 95.67


80

Robustness 4

Table No. 30 : Results of Robustness at the column oven temperature 23°C sample solution.

Sr.No. % Assay of Anastrozole % Assay of Temozolomide

1 99.94 98.90

2 100.11 98.41

3 100.87 97.93


100.30 98.41


99.98 95.67


Conclusion: Robustness studies signified that the results of the method remained unaffected

by small, deliberate changes in the flow rate and column temperature.


Robustness I :- High flow rate

Figure No. 29 : Resolution chromatogram in Robustness I study.

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Figure No. 30: Mix Standard chromatogram in Robustness I study.

Figure No. 31: Anastrozole sample chromatogram in Robustness I study.

Figure No. 32 : Temozolomide sample chromatogram in Robustness I study.

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Robustness II: - Low flow rate

Figure No. 33 : Resolution chromatogram in Robustness II study.

Figure No. 34 : Mix Standard hromatogram in Robustness II study.

Figure No. 35 : Anastrozole Sample chromatogram in Robustness II study.

83

Figure No. 36 : Temozolomide Sample chromatogram in Robustness II study.

Robustness III :- High column Temperature.

Figure No. 37 : Resolution chromatogram in Robustness III study.

Figure No. 38 : Mix Standard chromatogram in Robustness III study.

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Figure No. 39 : Temozolomide Sample chromatogram in Robustness III study.

Figure No. 40 : Anastrozole Sample chromatogram in Robustness III study.

Robustness IV :- Low column Temperature.

Figure No. 41 : Resolution chromatogram in Robustness IV study.

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Figure No. 42: Mix Standard chromatogram in Robustness IV study.

Figure No. 43: Anastrozole Sample chromatogram in Robustness IV study.

Figure No. 44: Temozolomide Sample chromatogram in Robustness IV study.

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2.9.9. CONCLUSION

The advantages of the proposed method involve a simple procedure for sample preparation

and relatively short time of analysis. Apart from this, it can be used for assays of Anastrozole

and Temozolomide in biological fluids or in pharmacokinetic investigations. The proposed

method was validated by testing its linearity, accuracy, precision, limits of detection, and

limit of quantitation. Robustness and stability of solutions. The results of the analysis of

pharmaceutical dosage forms by the proposed methods are highly reproducible, reliable, and

are in good agreement with the label claims of the drug. The additives usually present in the

pharmaceutical formulations of the assayed samples did not interfere with Anastrozole and

Temozolomide. It may be said that the proposed methods are precise, sensitive, and accurate,

so that these can be used as standard Pharmacopeial methods for the simultaneous

determination of Anastrozole and Temozolomide using the HPLC systems with PDA

detector.

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