Lakshmana Rao et.al Indian Journal of Research in Pharmacy and Biotechnology ISSN: (Print) ISSN: (Online)

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1 Development and validation of a stability indicating HPLC method for analysis of Altretamine in bulk drug and pharmaceutical formulations M. Karimulla Santhosh, A. Sreedevi, L. Kalyani, A. Lakshmana Rao * V.V. Institute of Pharmaceutical Sciences, Gudlavalleru, Andhra Pradesh, India. * Corresponding author: dralrao@gmail.com ABSTRACT A simple, sensitive and accurate stability indicating HPLC method has been developed and validated for determination of Altretamine in its bulk form and pharmaceutical formulations. Chromatographic separation was achieved on a Hypersil BDS C18 column (100 mm x 4.6 mm I.D., particle size 5 µm) by a mobile phase consisted of phosphate buffer and acetonitrile (90:10, v/v) with apparent ph of 3.1±0.5 and a flow rate of 1.0 ml/min. The detection wave length was set at 227 nm. An excellent linearity was observed for Altretamine in the concentration range of µg/ml with a correlation coefficient of The retention time was min. The percentage assay of Altretamine was 99.98%. The method developed was validated for accuracy, precision, linearity, ruggedness, robustness, solution stability, selectivity and forced degradation studies like acidic, alkaline, oxidative, thermal, hydrolytic and photolytic stress conditions were performed as per ICH guidelines. The results demonstrated that the method would have a great value when applied in quality control and stability studies of Altretamine. Key Words: HPLC, Altretamine, Stability, Formulation. INTRODUCTION Altretamine (Figure 1) is a synthetic cytotoxic antineoplastic agent (Neil, 2006). Chemically it is N,N,N',N',N'',N''-hexamethyl-1,3,5-triazine-2,4,6- triamine. Altretamine is indicated for use as a single agent in the palliative treatment of patients with persistent or recurrent ovarian cancer following firstline therapy with a cisplatin and/or alkylating agentbased combination (Wiernik, 1992). Altretamine is structurally related to the alkylating agents. Its precise mechanism of action is unknown but hydroxy methyl intermediates in the metabolism process are possibly the reactive species, and may act as alkylating agents (Rhoda, 1995). Altretamine interferes with the growth of cancer cells and slows their growth and spread in the body. Literature survey revealed that few HPLC methods (Ghiorghis, 1991; Barker, 1994) were reported for the determination of Altretamine. But no stability indicating HPLC method was reported. Hence the objective of this method is to develop and validate a simple, rapid and accurate stability indicating HPLC method (Snyder, 1997) in accordance with ICH guidelines (ICH Q2(R1), 2005; ICH Q1A(R2), 2003) for the determination of Altretamine in bulk sample and its pharmaceutical formulations. MATERIALS AND METHODS Chemicals and solvents: The working standard of Altretamine was provided as gift sample from Spectrum Labs, Hyderabad, India. The market formulation CANTRET capsules (Altretamine 50 mg) were procured from local market. HPLC grade acetonitrile and water were purchased from E.Merck (India) Ltd, Mumbai, India. Potassium dihydrogen phosphate, orthophosphoric acid and triethylamine of AR grade were obtained from S.D. Fine Chemicals Ltd, Mumbai, India. Instrumentation: To develop a high performance liquid chromatographic method for quantitative determination of Altretamine using Waters HPLC system on Hypersil BDS C18 column (100 mm x 4.6 mm I.D., particle size 5 µm) was used. The instrument is equipped with an auto sampler and UV detector. A 10 μl rheodyne injector port was used for injecting the samples. Data was analyzed by using Empower 2 software. Chromatographic conditions: A mixture of phosphate buffer ph 3.1 and acetonitrile (90:10, v/v) was found to be the most suitable mobile phase for ideal chromatographic separation of Altretamine. The solvent mixture was filtered through 0.45 μ membrane filter and sonicated before use. It was pumped through the column at a flow rate of 1.0 ml/min. Injection volume was 10 µl and the column was maintained at a temperature of 30 0 C. The column was equilibrated by pumping the mobile phase through the column for at least 30 minutes prior IJRPB 1(6) November December 2013 Page 778

2 to the injection of the drug solution. The detection of the drug was monitored at 227 nm. The run time was set at 6 minutes. Preparation of phosphate buffer ph 3.1: 2.72 grams of potassium dihydrogen phosphate was weighed and transferred into a 1000 ml beaker and dissolved. 1 ml of triethylamine solution was added to the above solution and diluted to 1000 ml with HPLC water. ph was adjusted to 3.1 with orthophosphoric acid solution. Preparation of mobile phase and diluent: 900 ml of phosphate buffer was mixed with 100 ml of acetonitrile and was used as mobile phase. The solution was degassed in an ultrasonic water bath for 5 minutes and filtered through 0.45 µ filter under vacuum. The mixture of 800 ml of water and 200 ml of acetonitrile was used as diluent. Preparation of standard solution: 10 mg of Altretamine was accurately weighed, transferred to 10 ml volumetric flask and is dissolved in 7 ml of the diluent. Sonicated the solution for few minutes to dissolve the drug completely. Then it is filtered through 0.45 μ filter and the volume is made up to 10 ml with diluent to get a concentration of 1 mg/ml stock solution. Further pipetted 1.0 ml of the above stock solution into a 10 ml volumetric flask and diluted up to the mark with diluent to obtain required concentrations. Preparation of sample solution: Twenty commercial capsules were emptied and powdered. A quantity of the powder equivalent to 10 mg of Altretamine was accurately weighed, transferred to 10 ml volumetric flask and is dissolved in 7 ml of the diluent. Sonicated the solution for few minutes to dissolve the drug completely. Then it is filtered through 0.45 μ filter and the volume is made up to 10 ml with diluent to get a concentration of 1 mg/ml stock solution. Further pipetted 1.0 ml of the above stock solution into a 10 ml volumetric flask and diluted up to the mark with diluent to obtain required concentrations of Altretamine in pharmaceutical dosage forms. Inject 10 µl of the above solution into the HPLC system. All experiments were conducted in triplicate. Linearity: Several aliquots of standard solution of Altretamine was taken in different 10 ml volumetric flasks and diluted up to the mark with diluent such that the final concentrations of Altretamine were in the linearity range of µg/ml. Evaluation of the drug was performed with UV detector at 227 nm, peak area was recorded for all the peaks. The response for the drug was linear and the regression equation was found to be y=19094x and correlation coefficient value of Altretamine was found to be The results show that an excellent correlation exists between peak area and concentration of drug within the concentration range indicated. Limit of detection and limit of quantification: The limit of detection (LOD) and limit of quantification (LOQ) of the developed method were determined by injecting progressively low concentrations of the standard solution using the developed HPLC method. The LOD and LOQ for Altretamine were found to be 0.46 μg/ml and 1.39 μg/ml respectively. System suitability: System suitability parameters like retention time, theoretical plates and tailing factor were calculated and compared with standard values. Accuracy: The accuracy of the method was assessed by recovery study of Altretamine in the dosage form at three concentration levels. A fixed amount of preanalyzed sample was taken and standard drug was added at 50%, 100% and 150% levels. The standard concentration was fixed as 100 μg/ml and three concentration levels of 50 μg/ml, 100 μg/ml and 150 μg/ml were added to the standard concentration. Each level was repeated three times. The content of Altretamine per capsule was calculated. The percentage recovery ranges from % and the mean recovery of Altretamine was 99.92% and the recovery values of Altretamine indicate the method is accurate. Precision: The precision was determined for Altretamine in terms of system and method precision. For system precision evaluation, %RSD for Altretamine was 0.32% (limit %RSD < 2.0%). In addition, the method precision was IJRPB 1(6) November December 2013 Page 779

3 studied and the %RSD for Altretamine was 0.77% (limit %RSD < 2.0%). Ruggedness and robustness: The ruggedness of the method was determined by carrying out the experiment on different instruments by different operators using different columns of similar types. Robustness of the method was determined by making slight changes in the chromatographic conditions like changes in flow rate and mobile phase composition. It was observed that there were no marked changes in the chromatograms, which demonstrated that the HPLC method so developed is rugged and robust. Solution stability: The stability of solution under study was established by keeping the solution at room temperature for 24 hrs. The result showed no significant change in concentration and thus confirms the stability of the drug in the mobile phase used for the analysis. Analysis of the marketed formulations: The proposed method was applied for the determination of Altretamine in pharmaceutical formulatons of Altretamine capsules. 10 µl of each standard and sample solution were injected and from the peak area of Altretamine, amount of drug present in samples were computed. The result of assay undertaken yielded 99.98% of label claim of Altretamine. The assay obtained is more than 99% and no interference of impurity peak observed in Altretamine peak. Acidic degradation studies: To 1 ml of stock solution of Altretamine, 1 ml of 2N hydrochloric acid was added and refluxed for 30 mins at 60 0 C. The resultant solution was diluted to obtain 100 µg/ml solution and 10 µl solution were injected into the system and the chromatograms were recorded to assess the stability of sample. Alkaline degradation studies: To 1 ml of stock solution of Altretamine, 1 ml of 2N sodium hydroxide was added and refluxed for 30 mins at 60 0 C. The resultant solution was diluted to obtain 100 µg/ml solution and 10 µl solution were injected into the system and the chromatograms were recorded to assess the stability of sample. Oxidative degradation studies: To 1 ml of stock solution of Altretamine, 1 ml of 20% hydrogen peroxide (H 2 O 2 ) was added separately. The solutions were kept for 30 mins at 60 0 C. The resultant solution was diluted to obtain 100 µg/ml solution and 10 µl solution were injected into the system and the chromatograms were recorded to assess the stability of sample. Thermal degradation studies: The standard Altretamine solution was placed in oven at C for 6 hrs to study thermal degradation. The resultant solution was diluted to obtain 100 µg/ml solution and 10 µl solution were injected into the system and the chromatograms were recorded to assess the stability of the sample. Hydrolytic degradation studies: Stress testing under hydrolytic conditions was studied by refluxing the s t a n d a r d A l t r e t a m i n e s o l u t i o n in water for 6 h r s at a temperature of 60ºC. The resultant solution was diluted to obtain 100 µg/ml solution and 10 µl solution were injected into the system and the chromatograms were recorded to assess the stability of the sample. Photolytic degradation studies: The photolytic stability of the drug Altretamine was studied by exposing the standard Altretamine solution to UV light by keeping the beaker in UV chamber for 7 days or 200 Watt hours/m 2 in photo stability chamber. The resultant solution was diluted to obtain 100 µg/ml solution and 10 µl solution were injected into the system and the chromatograms were recorded to assess the stability of sample. RESULTS AND DISCUSSION In the present work, a simple, accurate and precise stability indicating HPLC method has been optimized, developed and validated for the determination of Altretamine in pharmaceutical formulations with UV detector by using Hypersil BDS C18 column (100 mm x 4.6 mm I.D., particle size 5 µm) in isocratic mode with mobile phase composition of phosphate buffer ph 3.1: acetonitrile (90:10, v/v) and ph adjusted to 3.1 with orthophosphoric acid. The use of phosphate buffer and acetonitrile in the ratio of 90:10, v/v resulted in peak with good shape and resolution. IJRPB 1(6) November December 2013 Page 780

4 The flow rate was 1.0 ml/min and the drug component was measured with UV detector at 227 nm. The results of optimized HPLC conditions were shown in Table 1. The method was linear in the range of µg/ml for Altretamine with correlation coefficient of The linearity results were shown in Table 2 and the linearity curve of Altretamine was shown in Figure 2. The % recoveries of Altretamine were found in the range of % and the % mean recovery was found to be 99.92% for Altretamine, which indicate the method is accurate. The results of recovery studies were shown in Table 3. The %RSD for system precision and method precision for Altretamine were found to be 0.32 and 0.77, which indicate the method is precise. The results of precision studies were shown in Table 4 and Table 5. The retention time of Altretamine was min, cuts down on overall time of sample analysis and the method was more cost effective as it utilizes very less quantity of mobile phase. The number of theoretical plates was 4253 and tailing factor was 1.49 for Altretamine, which indicates efficient performance of the column. Typical chromatogram of drug Altretamine was shown in Figure 3. Selectivity of the method was demonstrated by the absence of any interfering peaks at the retention time of the drug. The limit of detection and limit of quantification for Altretamine were found to be 0.46 μg/ml and 1.39 μg/ml, which indicate the sensitivity of the method. A system suitability test was performed to evaluate the chromatographic parameters and the summary of system suitability parameters were shown in Table 6. Validated method was applied for the determination of Altretamine in commercial formulations. The % assay was found to be 99.98% for Altretamine and the assay results were shown in Table 7. HPLC studies of Altretamine under different stress conditions indicated the following degradation behavior. In acidic degradation, the degradation product of Altretamine was appeared at retention time of min and the % degradation is 9.08%. In alkaline degradation, the degradation product of Altretamine was appeared at retention time of min and the % degradation is 6.96%. In oxidative degradation, the degradation product of Altretamine was appeared at retention time of min and the % degradation is 7.40%. In thermal degradation, the degradation product of Altretamine was appeared at retention time of min and the % degradation is 5.27%. In hydrolytic degradation, the degradation product of Altretamine was appeared at retention time of min and the % degradation is 0.21%. In photolytic degradation, the degradation product of Altretamine was appeared at retention time of min and the % degradation is 1.05%. The results of analysis are given in Table 8. The typical chromatograms of degradation behavior of Altretamine in different stress conditions are shown in Figure 4 to Figure 9. Figure.1. Molecular structure of Altretamine IJRPB 1(6) November December 2013 Page 781

5 Figure.2. Calibration curve of Altretamine Figure.3. Typical chromatogram of Altretamine Figure.4. Acidic degradation chromatogram of Altretamine Figure.5. Alkaline degradation chromatogram of Altretamine IJRPB 1(6) November December 2013 Page 782

6 Figure.6. Oxidative degradation chromatogram of Altretamine Figure.7. Thermal degradation chromatogram of Altretamine Figure.8. Hydrolytic degradation chromatogram of Altretamine Figure.9. Photolytic degradation chromatogram of Altretamine IJRPB 1(6) November December 2013 Page 783

7 Table.1. Optimized chromatographic conditions of Altretamine Parameter Condition Mobile phase Phosphate buffer:acetonitrile (90:10, v/v) ph 3.1±0.5 Diluent Water:acetonitrile (80:20, v/v) Column Hypersil BDS C18 column (100 mm x 4.6 mm, 5 μm) Column temperature 30 0 C Wave length 227 nm Injection volume 10 µl Flow rate 1.0 ml/min Run time 6 min Table.2. Linearity results of Altretamine Concentration (μg/ml) Area Table.3. Recovery results of Altretamine Level Concentration Concentration added (μg/ml) found (μg/ml) % Recovery Mean recovery 50% % 100% % 99.92% 150% % Table.4. System precision data of Altretamine S. No. Area of Altretamine Average SD %RSD 0.32 Table.5. Method precision data of Altretamine S. No. Area of Altretamine Average SD %RSD 0.77 Table.6. System suitability parameters of Altretamine Parameter Results Linearity range (μg/ml) Correlation coefficient Theoretical plates (N) 4253 Tailing factor 1.49 LOD (μg/ml) 0.46 LOQ (μg/ml) 1.39 Retention time (min) IJRPB 1(6) November December 2013 Page 784

8 Table.7. Assay results of Altretamine Formulation Label claim Amount found %Assay CANTRET 50 mg mg 99.98% Stress conditions Table.8. Degradation studies of Altretamine Area of % peak Degradation Degradation time % of active drug present after degradation Standard Drug Acidic 30 mins % 90.92% Alkaline 30 mins % 93.04% Oxidative 30 mins % 92.60% Thermal 6 hours % 94.73% Hydrolytic 6 hours % 99.79% Photolytic 7 days % 98.95% CONCLUSION The present study represents the first report that deals with the development of a stability indicating HPLC method for determination of Altretamine. This study is a typical example for development of a stability indicating assay established by following the recommendations of ICH guidelines. The proposed method showed acceptable accuracy, precision, selectivity and wide linear concentration range. The results of analysis proved that the method is suitable for the determination of Altretamine in bulk and capsule dosage forms without any interference from the degradation products and it is recommended for routine quality control analysis of the Altretamine in pharmaceutical formulations. REFERENCES Barker IK, Crawford SM and Fell AF, Determination of Altretamine in human plasma with highperformance liquid chromatography, Journal of Liquid Chromatography B, 660(1), 1994, Ghiorghis A and Talebian AH, High-pressure liquid chromatography separation of potential impurities of Altretamine, Journal of Liquid Chromatography, 14(12), 1991, ICH Harmonised Tripartite Guideline, Stability Testing of New Drug Substances and Products, Q1A(R2), International Conference on Harmonization, 2003, ICH Harmonised Tripartite Guideline, Validation of analytical procedures: Text and methodology, Q2(R1), International Conference on Harmonization, 2005, Neil OJM, The Merck Index, An Encyclopedia of Chemicals Drug and Biologicals, 14 th Ed., Merck Research Laboratories, Division of Merck and Co. Inc., White House Station, NJ, 2006, 57. Rhoda LC and Diana F, Altretamine: A review of its pharmacodynamic and pharmacokinetic properties and therapeutic potential in cancer chemotherapy, Drugs, 49(6), 1995, Snyder LR, Kirkland JJ and Glajch JL, Practical HPLC Method Development, 2 nd Ed., New York, John Wiley and Sons, 1997, Wiernik PH, Hexamethylmelamine and low or moderate dose cisplatin with or without pyridoxine for treatment of advanced ovarian carcinoma: a study of the eastern cooperative oncology group, Cancer Investigation, 10(1), 1992, 1-9. IJRPB 1(6) November December 2013 Page 785