1. Analytical method development and Validation of Temozolomide in a Tablet Dosage form by Reverse Phase HPLC By Anusha. Karna 12Z51S0407 1

2. Summary of Presentation Abstract Introduction Aim and Objective Drug profile Literature Review Plan of work Materials and Methods Results and Discussion Conclusion 2

3. ABSTRACT ABSTRACT 3

4. Temozolomide (Temodar and Temodal) is an oral alkylating agent used for the treatment of Refractory anaplastic astrocytoma -- a type of cancerous brain tumour. Temozolomide is also indicated for relapsed Grade III anaplastic astrocytoma and not indicated for, but as of 2011 used to treat oligodendroglioma brain tumors in some countries, replacing the older (and less well tolerated) PCV (Procarbazine- Lomustine-Vincristine) regimen.oligodendrogliomaProcarbazine LomustineVincristine 4

5. According to the Literature survey it was found that few analytical methods such as (HPLC, UV-Visible Analysis and LC-MS) were reported for the estimation of Temozolomide. The objective of the proposed method is to develop simple and accurate methods for the determination of Temozolomide by RP-HPLC method in pharmaceutical dosage forms & it’s stability Indicative studies. 5

6. INTRODUCTION Analytical methods development and validation play important roles in the discovery, development, and manufacture of pharmaceuticals. The official test methods that result from these processes are used by quality control laboratories to ensure the identity, purity, potency, and performance of drug products.

7. HPLC 7

8. What is HPLC? HPLC stands for High Performance Liquid Chromatography (Sometimes referred to as High Pressure liquid Chromatography) High-Performance Liquid Chromatography was developed in 1960s as faster way to do column chromatography It is a form of Liquid chromatography which is used to separate mixture of compounds that are dissolved in a solution. It is a powerful tool used in analysis, it yields high performance and high speed when compared to the traditional columns chromatography because of the forcibly pumped mobile phase. It is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of mixture 8

9. Principle of HPLC Principle of HPLC Separation is PARTITIONING 9

10. Modes of HPLC 10

11. 11

12. Advantages of HPLC Advantages over traditional chromatography include:  Speed  Adaptability  Resolution  Sensitivity  Columns reusable 12

13. Temozolomide 13

14. DRUG Profile Name : Temozolomide Type :small molecule Description :Temozolomide (Temodar and Temodal) is an oral alkylating agent used for the treatment of refractory anaplastic astrocytoma (a type of cancerous brain tumour). Temozolomide is not active until it is converted at physiologic pH to the active form,5(3-methyltriazen-1-yl)imidazole-4- carboxamide (MTIC). Melting Point :212 °C Synonyms :Methazolastone, Temozolamide, Temozolodida [Spanish], Temozolomidum [Latin] Brand names :Temodal, Temodar Chemical Formula :C 6 H 6 N 6 O 2 IUPAC Name :3-methyl-4-oxo-3H,4H-imidazo[4,3- d][1,2,3,5]tetrazine-8- carboxamide 14

15. Literature Review Lakshmanarao*, g. Tarakaramesh and j.v.l.n.s. rao A simple, rapid, sensitive and precise high performance liquid chromatographic method has been developed for the estimation of temozolomide in pharmaceutical dosage forms. The percentage recovery ranges from 98.99-102.02 %. The proposed method was validated by determining sensitivity, accuracy, precision and linearity. The proposed method is simple, fast, accurate, precise and reproducible hence can be applied for routine quality control analysis of temozolomide in pharmaceutical formulations Abdul razak*, et.al., an uv spectrophotometric method for the quantitative determination of temozolomide (tmz) in bulk and capsule was developed in present work. The parameters linearity, precision, accuracy, limit of detection and limit of quantitation were studied according to international conference on harmonization guidelines. 15

16. Literature Review (Contd) Y.Rama Chandra reddyet. al., Anastrozole tablets were subjected to different ICH prescribed stress conditions of thermal, hydrolysis, humidity, photolysis and oxidation stress. The drug was found to be stable for all the stressed conditions except for oxidation. V.n. daphalet.al., a fast, sensitive and accurate reverse phase liquid chromatographic method was developed and validated for the simultaneous determination of anastrozole and Temozolomide in an anticancer dosage form. 16

17. PLAN OF WORK The work is planned to carry out as given below Collection of relevant literature. To undertake solubility study &analytical study of Temozolomide& to develop initial UV & chromatographic conditions. Optimisation of initial chromatographic conditions. Analytical method validation of developed stability indicating method. Quantitative determination of Temozolomide in pharmaceutical dosage form using the method developed and validated. 17

18. Materials and Instrumentation Instruments: HPLC – METHANOLS Model NO.486 series Compact System Consisting of Inertsil-C18 ODS column. Electronic balance (SARTORIOUS) Digital pH meter(POLOMAN) Sonicator( FAST CLEAN) 18

19. Chemicals: Acetonitrile HPLC Grade Raw Material: Temozolomide Working Standard 19

20. METHOD DEVELOPMENT FOR HPLC The objective of this experiment was to optimize the assay method for estimation of Temozolomide based on the literature survey made. So here the trials mentioned describes how the optimization was done. 20

21. Trial: 1 Mobile Phase: Pure Acetonitrile 100% Preparation of Standard Solution: 10mg of f Temozolomide drug was weighed and dissolved in 10ml of Mobile phase and taken in 10ml of volumetric flask and sonicated for 20 minutes to get 1000ppm and 1 ml was taken from this and diluted to 10 ml with mobile phase Chromatographic Conditions: Flow rate : 1.0ml/min Column : Inertsil- C18 ODS column Detector wavelength : 254nm Column temp : Ambient Injection volume : 10µl Run time : 10min Retention time : 3.2 21

22. Observation: Theoretical plates are less, peak shape is not good and asymmetry is more than limit. The trial 1 chromatogram result was shown as above 22

23. Trail: 2. Trail: 2. Mobile Phase: Acetonitrile and Methanol were mixed in the ratio of 90:10V/V and sonicated Preparation of Standard Solution: 10mg of Temozolomide drug was weighed and dissolved in 10ml of Mobile phase and taken in 10ml of volumetric flask and sonicated for 20 minutes to get 1000ppm and 1 ml. was taken from this and diluted to 10ml.with mobile phase Chromatographic Conditions: Flow rate : 1ml/min Column : Inertsil-C18 ODS column Detector wavelength : 254nm Column temp : Ambient Injection volume : 10µl Run time : 10min Retention time : 4.0 23

24. Observation: The retention time is same and asymmetry is high, peak tailing occurs. The trial2 chromatogram result was shown as above 24

25. Trail: 3 Mobile Phase: Acetonitrile and Methanol were mixed in the ratio of 80:20 V/V and sonicated Preparation of Standard Solution: 10mg of Temozolomide drug was weighed and dissolved in 10ml of Mobile phase and taken in 10ml of volumetric flask and sonicated for 20 minutes to get 1000ppm and 1 ml. was taken from this and diluted to 10ml.with mobile phase Chromatographic Conditions: Flow rate : 1.0ml/min Column : Inertsil- C18 ODS column Detector wavelength : 254nm Column temp : Ambient Injection volume : 10µl Run time : 10min 25

26. Observation: Retention time is less and asymmetry is high. The trial 3chromatogram result was shown as above 26

27. OPTIMIZED METHOD Mobile Phase: Acetonitrile and Methanol were mixed in the ratio of 60:40 and sonicated 27 Parameters Method Stationary phase (column) Inertsil -ODS C 18 (150 x 4.6 mm) Mobile Phase Acetonitrile:Methanol (70:30) Flow rate (ml/min)1.0 ml Run time (minutes)10 Column temperature (°C)Ambient Volume of injection loop (  l) 10 Detection wavelength (nm)254 Drug RT (min)3.06

28. Chromatogram of standard 28

29. Preparation of Solutions Preparation of stock solution: 10mg of Temozolomide drug was weighed and dissolved in 10ml of Mobile phase and taken in 10ml of volumetric flask and sonicated for 20 minutes to get 1000ppm and 2 ml. was taken from this and diluted to 10ml.with mobile phase. Preparation of working standard solution: The stock solution equivalent to 25ppm to 150ppm were prepared, sonicated and filtered through 0.45µ membrane. Preparation of sample drug solution for pharmaceutical formulations: Twenty tablets containing Temozolomide of each marketed formulation were taken and powdered. The powder equivalent to 500 mg of the active ingredient was accurately weighed and taken in a 100ml volumetric flask containing 50 ml mobile phase and sonicated for 15 minutes and the solution was made up to volume with mobile phase and filtered through 0.45micron membrane. 29

30. Calculation 30

31. Validation Parameters: The Developed method was validated as per ICH Q2 (R1) guideline.  SYSTEM SUITABILITY  ACCURACY  SYSTEM PRECISON  PRECISION  LINEARITY AND RANGE  LOD AND LOQ  ROBUSTNESS 31

32. 1. SYSTEM SUITABILITY: A Standard solution was prepared by using Temozolomide working standard as per test method and was injected Five times into the HPLC system. The system suitability parameters were evaluated from standard chromatograms by calculating the % RSD from five replicate injections for Temozolomide, retention times and peak areas. OBSERVATION: The %RSD for retention times and peak areas were found to be within the limit.refer table: 1 As sown in fig 6 – 10. 32

33. Data of System Suitability InjectionRTPeak AreaUSP Plate countUSP Tailing 13.006 4437.5151 101681.106 23.0114439.6279102141.109 33.0004437.5151102001.110 43.0084440.1612101981.107 53.0074446.1712102101.108 Mean 3.0064 4440.19810198 1.108 SD 0.004037 3.1749------- % RSD 0.134291 0.0715------- 33

34. ACCURACY (RECOVERY): A study of Accuracy was conducted. Drug Assay was performed in triplicate as per test method with equivalent amount of Temozolomide into each volumetric flask for each spike level to get the concentration of f Temozolomide equivalent to 50%, 100%, and 150% of the labeled amount as per the test method. The average % recovery of Temozolomide was calculated. ACCEPTANCE CRITERIA: The mean % recovery of the Temozolomide at each spike level should be not less than 98.0% and not more than 102.0%. 34

35. Data of Accuracy Concentration % of spiked level Amount added (ppm) Amount found (ppm) % Recovery Statistical Analysis of % Recovery 50% Sample 124.982599.82MEAN99.82 50% Sample 223.892598.89 50% Sample 324.892598.98%RSD0.82 100 % Sample 150.4750100.92MEAN100.3 100 % Sample 250.4550100.83 100% Sample 351.4650100.56%RSD1.62 150% Sample 176.0375101.38MEAN101.5 150% Sample 275.7875101.50%RSD0.62 150% Sample 375.8675101.42 35

36. Observation The recovery results indicating that the test method has an acceptable level of accuracy. 36

37. PRECISION: Repeatability: System precision: Standard solution prepared as per test method and injected five times. Method precision: Prepared six sample preparations individually using single as per test method and injected each solution. ACCEPTANCE CRITERIA: The % relative standard deviation of individual Temozolomide, from the six units should be not more than 2.0%. The assay of Temozolomide should be not less than 98% and not more than 102.0%. OBSERVATION: Test results are showing that the test method is precise. Refer tables 2 and 3 for system precision and for method precision. 37

38. Data of Repeatability (System precision) Injection Peak Areas of Temozolomide %Assay Concentration 50ppm 14435.56 100.56 24437.58 100.88 34435.56 100.78 44440.15 100.06 54445.13 101.02 Statistical Analysis Mean4438.796 100.06 SD62.64 52.3 % RSD1.23 0.09 38

39. LINEARITY OF TEST METHOD: A Series of solutions are prepared using Temozolomide working standard at concentration levels from 25ppm to 150 ppm of target concentration.Measure the peak area response of solution at Level 1 and Level 6 six times and Level 2 to Level 5 two times. ACCEPTANCE CRITERIA: Correlation Coefficient should be not less than 0.9990. % of y- Intercept should be ±2.0. % of RSD for level 1 and Level 6 should be not more than 2.0%. OBSERVATION: The linear fit of the system was illustrated graphically. The results ar 39

40. Linearity Plot (Concentration Vs Response) 40

41. LIMIT OF DETECTION AND QUANTITATION (LOD and LOQ): From the linearity data calculate the limit of detection and quantitation, using the following formula.  LOD = 3.3 σ S σ = standard deviation of the response S = slope of the calibration curve of the analyte. LOQ = 10 σ S σ = standard deviation of the response S = slope of the calibration curve of the analyte. 41

42. From the linearity plot the LOD and LOQ are calculated: LOD= 3.3 σ S 3.3×3.43 = ------------------ =0.13 87.06 LOQ = 10 σ S 10×3.43 = --------- = 0.40 87.46 42

43. ROBUSTNESS: a) Effect of variation of flow rate: A study was conducted to determine the effect of variation in flow rate. Standard solution prepared as per the test method was injected into the HPLC system using flow rates, 1.0ml/min and 1.2ml/min. The system suitability parameters were evaluated and found to be within the limits for 1.0ml/min and 1.2ml/min flow. Temozolomide was resolved from all other peaks and the retention times were comparable with those obtained for mobile phase having flow rates 1.0ml/min. ACCEPTANCE CRITERIA: The Tailing Factor of Temozolomide standards should be NMT 2.0 for Variation in Flow. 43

44. Robustness: Data for Effect of variation in flow rate: Robustness: Data for Effect of variation in flow rate: Flow 0.8 mlStd Area Tailing factor Flow 1.0 mlStd Area Tailing factor Flow 1.2 mlStd Area Tailing factor 6079.401.1064882.351.1104076.021.123 5895.631.1104970.641.1124167.621.125 5935.371.1124900.201.1104138.321.124 6056.361.1184924.731.1114140.311.124 6059.631.1174781.371.1124098.211.123 Avg6005.0811.112Avg4891.861.111Avg4124.101.1238 SD74.9770.0044SD62.6970.00089SD32.6830.0007 %RSD1.2480.4003%RSD1.2810.0804%RSD0.79250.0065 44

45. CONCLUSION A sensitive& selective RP-HPLC method has been developed & validated for the analysis of TemozolomideAPI. Further the proposed RP-HPLC method has excellent sensitivity, precision and reproducibility. The result shows the developed method is yet another suitable method for assay, purity which can help in the analysis of Temozolomide in different formulations. 45

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