Research Paper Development of Stability Indicating Reverse Phase HPLC Method for Aripiprazole from Solid Dosage form

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1 International Journal of Pharmaceutical Sciences and Nanotechnology 572 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 2 Issue 2 July - September 2009 Volume 2 Issue 2 July September 2009 Research Paper Development of Stability Indicating Reverse Phase HPLC Method for Aripiprazole from Solid Dosage form Pankaj Nerkar 1 *, Parag Gide 2, Abhishek Chitnis 3, Hitendra Mahajan 4, Surendra Gattani 5 1, 4, 5 Dept. of Pharmaceutics, R.C.Patel College of Pharmacy, Shirpur (Dhule) MS Mumbai Education Trusts College of Pharmacy, Nashik. 3 University of Houston, Texas. ABSTRACT: Aripiprazole is a novel antipsychotic drug, generally used in schizophrenia and known to act as a partial agonist at D 2 and 5 HT 2A receptors. Since stability indicating method is required to discriminate between the intact analyte and degradation products, the present work is aimed at performing purposeful degradation of aripiprazole by exposing it to various ph conditions, oxidative conditions, dry heat and sunlight. Also it describes the final method of analysis of aripiprazole from the tablets. The chromatographic analysis was carried out on HiQSil C 8 column (250 X 4.6 mm, 5 µm), mobile phase was Acetonitrile: Potassium Phosphate buffer (ph =3, 100 mm), (40:60 V/V); injection volume 100 µl, detection was carried out at λ max 225nm. Flow rate was 1.0 ml/min. The developed stability indicating method for aripiprazole was validated as per International Conference on Harmonization (ICH) guidelines. KEYWORDS: Aripiprazole; Stability indicating method; Purposeful degradation; ICH Introduction Aripiprazole is a novel antipsychotic drug, which is available as tablets and in solution for oral administration. Aripiprazole exhibits high affinity for dopamine D 4, serotonin 5- HT 2 C, serotonin 5 HT 7, α1 adrenergic receptors. It has moderate affinity for cholinergic muscarinic receptors. Aripiprazole functions as a partial agonist at the dopamine D 2 and serotonin 5- HT 1 receptors and as an antagonist at serotonin 5- HT 2 A receptor. Literature survey reveals that there are two methods reported for the estimation of aripiprazole from human plasma and rat brain [Kubo M et al., 2005; Shimokawa Y et al., 2055] and rat plasma but no method reported so far for aripiprazole estimation in the presence of its degradation products i.e. stability indicating. The present study describes quantitative estimation of aripiprazole in the presence of its degradation products. This study also deals with the forced degradation of the drug under various stress conditions like hydrolysis (acid, base and neutral), oxidation, photolytic and thermal. Materials and Method Reference standard of aripiprazole was procured from M/S Torrent Drugs and Chemicals, Ahmedabad and used * For correspondence: P. Nerkar, nerkarpankaj@rediffmail.com 572 without further purification. The drug was procured as a gift sample along with Certificate of Analysis. Chemicals like sodium hydroxide, potassium hydroxide, potassium dihydrogen orthophosphate, phosphoric acid, hydrogen peroxide and solvents, like acetonitrile, and methanol were purchased from S. D. Fine Chemicals Limited, Mumbai. Equipment The HPLC system (2000 series) consisted of PU-2080 plus intelligent pump, UV 2075 plus intelligent detector and Rheodyne type injector fitted with 100 μl capacity loop all from JSCO Japan. The column used was HiQSil C 8 (250 x 4.6 mm, 5 μm). The output signal was monitored and processed using Borwin software. Chromatographic Conditions [Snyder L. R. et al., 1997] Aripiprazole (10 μg/ml) was subjected to chromatographic analysis using mobile phases of differing strength employing an injection volume of 100μL, flow rate of 1mL/min, and detection was carried at 225 nm. Purposeful Degradation of Aripiprazole [Singh S et al., 2000] Aripiprazole was subjected to a variety of stress conditions to effect degradation up to about 10 %. The decision tree suggested by Bakshi and Singh [Snyder L. R. et al., 1997]

2 Pankaj Nerkar et al. : Development of Stability Indicating Reverse Phase HPLC Method for 573 was followed. The acid hydrolysis and base hydrolysis were carried out by preparing four samples. The first was degradation sample in which drug sample was mixed with 10 ml of 0.1 and 1 normal solution of HCl and NaOH in RBF respectively. The same was then heated under reflux on a heating mantle at boiling temperature of 80 0 C for 2 and 4 hrs respectively. The second was zero time sample in which drug (10 mg) was mixed with 10 ml of 0.1 and 1 normal solution of HCl and NaOH in RBF respectively and prepared just before the analysis. The third stressed blank sample comprising of 10 ml of 0.1 and 1 normal solution of HCl and NaOH was heated in RBF on a heating mantle under reflux at boiling temperature of 80 0 C for 2 and 4 hrs respectively. The fourth blank sample made of 10ml of 0.1 and 1 normal solution of HCl and NaOH respectively were kept at normal conditions for 2 and 4hrs respectively. The oxidative degradation, dry heat induced degradation, photolytic degradation and neutral degradation were carried out by preparing three samples, degradation sample, zero time sample and blank sample as described above. In each degradation study the variables concentration, temperature and exposure time were selected in such a way to effect a degradation of less than 10 %. For dry heat and photolytic degradation, methanol was used as a blank sample. For neutral and oxidation degradation 10 ml of water and 3 % H 2 O 2 (V/V) was used as blank sample. For oxidation degradation the degradation sample made of the drug (10 mg) was kept with 3 % H 2 O 2 (V/V) in dark condition for 8 hrs. For neutral degradation the degradation sample was prepared by mixing drug (10 mg) with 10 ml of distilled water in RBF and heating the RBF on heating mantle for 4 hrs. Calibration Experiments [Kiser M. M. et al., 2004; Ermer J. et al., 2005; Nagaraja N. V. et al., 1999] A 100 µg/ml solution of aripiprazole was prepared by dissolving 10 mg of aripiprazole in methanol to make 100 ml. From this, appropriate dilutions were prepared in a mobile phase to get final concentration of µg/ml. These standard solutions were analyzed in three replicates using the above mentioned chromatographic conditions. Description of Final Method Based on the results of optimization of chromatographic conditions and calibration experiments, the final method for analysis of aripiprazole in bulk and a tablet formulation was described. Method Validation [Ermer J. et al., 2005; Nagaraja N. V. et al., 1999; (ICH) 1994; (ICH) 1996] Accuracy and Precision Accuracy and precision were evaluated at three levels by determining % recovery and % relative standard deviation (% R.S.D.) respectively for the results of analysis of aripiprazole from tablets containing 75, 100 and 125 % of the labeled amount of aripiprazole. Twenty placebo tablets were weighed accurately, and powdered. 25, 35 and 45 mg of aripiprazole was mixed with 400 mg of powder of placebo tablets separately in order to get mixtures containing 75, 100 and 125 % of the label claim. The quantity of drug of powder equivalent to one tablet (=117 mg) was weighed separately and analyzed by the developed method. The analysis was performed in triplicate at each level over three days. Percent relative error (% R.E.) was used as measure of accuracy while % R.S.D. which was calculated to determine precision. A t-test [Bolton S. 1990] was performed where the null hypothesis was that the experimental mean was not significantly different from the nominal content of aripiprazole in the tablets. The parameter t was calculated as ± t = (x- μ)/ standard error where x was the experimental mean and μ was the nominal amount of aripiprazole in the tablet. The calculated t value was compared with the tabulated t value at 2 degrees of freedom for α = Calculated t values lesser than tabulated t values indicated lack of significant difference and thereby acceptance of null hypothesis and reflected the accuracy of the method. Specificity Twenty placebo tablets were weighed accurately, triturated and mixed. A quantity equal to an average weight was analyzed by the described method of analysis and the chromatograms were observed for the interfering peaks at the retention time of aripiprazole. The blank tablets exposed to the degradation conditions were also analyzed using optimized chromatographic conditions and resulting chromatograms were inspected for interfering peaks at retention time of aripiprazole. Specificity was also indicated by the separation of aripiprazole from its degradation products. The method was declared specific if there were no interfering peaks at the retention time of aripiprazole and if aripiprazole peak was well resolved from the peaks of all possible degradation products. Robustness Small changes were made in the mobile phase composition and effects of these changes were observed on the chromatographic behavior of aripiprazole in presence of degradation products and excipients. The effects on retention time, resolution and peak shape were observed. Analysis of Marketed Formulation To determine the content of aripiprazole in marketed tablets (label claim 10 mg/ tablet), 20 tablets were weighed, and average weight was calculated. Tablets were

3 574 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 2 Issue 2 July - September 2009 triturated and powder equivalent to average weight was weighed. The drug was extracted from the tablet powder with 100 ml methanol. To ensure complete extraction, it was sonicated for 15 min. One ml of supernatant was then diluted up to 10 ml with mobile phase. The resulting solution was injected in HPLC and drug peak area was noted. Regression equation, peak area of the sample and the amount of aripiprazole in the sample was calculated. The amount of aripiprazole per tablet was thus found. Results and Discussion Optimization of Mobile Phase When aripiprazole was subjected to chromatographic analysis using mobile phases of differing strength it was decided to use acetonitrile: potassium phosphate buffer (ph 3, 100 mm)(40:60 V/V), since it gave adequate resolution and good peak shape. (Fig. 1) (Table 1) Purposeful Degradation of Aripiprazole Degradation was observed for aripiprazole during stress conditions like acid, base, neutral, dry heat and oxidation hydrolysis. (Fig. 2-6) The peak area of aripiprazole in stressed sample when compared with the corresponding peak of aripiprazole in zero hour sample revealed a decrease in the area. Also there were no additional peaks at the same retention time in zero sample as well as in blank sample and in the room temperature sample, indicating that the additional peaks were degradation products. Degradation study of the photolytic stressed sample also revealed the photostability of aripiprazole as there was no change between the peak area of stressed sample and the zero hour sample. (Fig.7) (Table 2). Fig. 1 Representative chromatogram of aripiprazole in Acetonitrile: Potassium phosphate buffer (ph 3, 100 mm) (40:60 V/V). R.T Table 1 The chromatographic behavior of aripiprazole in different mobile phases. Mobile phase Retention time (min) Acetonitrile: Water (40:60 V/V) 21.1 Methanol: Potassium phosphate buffer (ph 3, 100 mm)(60:40 V/V) 7.7 Acetonitrile: Buffer (ph 2.4, 100M, potassium phosphate)(40:60 V/V) 5.8 Acetonitrile: Buffer (ph 3, 100 mm, potassium phosphate)(40:60 V/V) 9.2

4 Pankaj Nerkar et al. : Development of Stability Indicating Reverse Phase HPLC Method for 575 Fig. 2 Representative chromatogram of acid treated aripiprazole (0.1 N HCl, refluxed for 2 hrs.) Peak 1: Degradant one R. T. 2.85; Peak 2: Degradant two R.T. 3.80; Peak 3 Degradant three R.T. 5.42; Peak 4: aripiprazole R.T Fig. 3 Representative chromatogram showing base treated aripiprazole (1 N NaOH, refluxed for 4 Hrs.) Peak 1: Degradant one, R.T min; Peak 2: Degradant two, R.T min; Peak 3: Degradant R.T min; Peak 4: aripiprazole, R.T. 9.11min.

5 576 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 2 Issue 2 July - September 2009 Fig 4 Representative chromatogram showing water treated aripiprazole. Peak 1 Degradant 1, R. T. 4.34; Peak 2: Degradant 2, R.T. 5.38, Peak 3: aripiprazole R.T. 9.60; Peak 4: Degradant 3 R.T (water reflux for 4 hrs.). Fig 5 Representative chromatogram of Oxidative degradation study of aripiprazole. Peak 1: Hydrogen peroxide, R.T. 2.46; Peak 2: Aripiprazole R.T. 9.15; Peak 3: Degradant 1, R.T (3% H 2 O 2, for 8 hrs.).

6 Pankaj Nerkar et al. : Development of Stability Indicating Reverse Phase HPLC Method for 577 Fig. 6 Representative chromatogram of dry heat degradation of aripiprazole. (80 0 C for 8 hrs) Peak 1: Degradant 1, R.T. 7.34; Peak 2: aripiprazole, R.T Fig 7 Representative chromatogram of degradation in photolytic condition of aripiprazole. (48 hrs in sunlight) Peak 1: aripiprazole R.T Conditions Time (in Hrs) Drug peak area at zero Hrs Table 2 Summary of degradation products of aripiprazole. Drug peak area of stressed sample Retention time of degradant (mins) % Degradation 1 N HCl ,3.8& N NaOH ,5.5& Water ,5.3& H 2 O 2, 3 % Dry Heat 80 0 C Sun light No Degradant No Degradation

7 578 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 2 Issue 2 July - September 2009 Calibration Experiments The data obtained in the calibration experiments when subjected to linear-regression analysis showed a linear relationship between peak areas and concentrations in the range of μg /ml (Fig. 8). A plot of residual obtained by plotting the difference between back calculated areas and experimental areas against concentration, showed a random scatter and the variance of the peak areas was homogenous over the linear range (Fig. 9) (Table 3) y = x R 2 = Fig 8 Calibration curve for aripiprazole Calibration standard Nominal Concentration μg/ml Fig 9 Residuals plot for aripiprazole. Table 3 Linearity data for Aripiprazole. Peak Area of replicate (μv.sec) Mean peak area (μv.sec) Standard deviation of peak area Equation Y = X R

8 Pankaj Nerkar et al. : Development of Stability Indicating Reverse Phase HPLC Method for 579 Description of Final Method Twenty tablets of aripiprazole (label claim 10 mg) were weighed and powdered. Powder was mixed thoroughly and quantity equivalent to 10 mg of aripiprazole was weighed and mixed with 80 ml of methanol. The mixture was sonicated for 10 min. and volume was made up to 100 ml with methanol. 1 ml of the resulting solution was diluted to 10 ml with the mobile phase [i.e. Acetonitrile: Potassium phosphate buffer (ph 3, 100 mm) (40:60 V/V)]. This final dilution was then chromatographed at a detection wavelength of 225 nm; the column used was HiQSil C8 column (250 X 4.6 mm, 5 µm); flow rate was 1 ml/min; injection volume was 100 µl. The peak area was referred to calibration equation y = x to get concentration of aripiprazole from sample and thus the content in tablets. Method Validation Specificity The HPLC chromatograms recorded for the matrix (blank tablets exposed to the degradation conditions) showed no peaks at the retention time of aripiprazole and also the representative chromatograms of degradation samples in various stress conditions (Figures 2-7) showed that aripiprazole was well resolved from its degradation peaks, indicating the specificity of the method. Accuracy and Precision The results of intra and inter-day variation of aripiprazole at three different concentrations levels (75 %, 100 %, and 125 % of label claim) are depicted in (Table 4). The data indicates that the maximum %relative error at 75%, 100% and 125% was 2.89, 2.7 and respectively while the maximum % relative standard deviation was 2.25, 2.9 and 2.29 respectively indicating that the method has acceptable accuracy and precision. Also, the calculated t-values were lesser than the tabulated t-value of 4.3 for α = 0.05 at two degrees of freedom. This indicated that the experimental values were not significantly different from the nominal which reflected the accuracy of the method. Further, when the data given in Table 4 was subjected to one way ANOVA to get estimates of within and between day variability, the following observations were made The calculated F value was lesser than the tabulated F 2,6 (α = 0.01) of indicating that the inter day variability was not significantly different from the intra day variability at 1% level of significance(table 5). Table 4 Results of accuracy and precision studies for Aripiprazole. Day Amount added (in mg) Amount found (in mg) Mean % relative error % RSD Calculated t- values

9 580 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 2 Issue 2 July - September 2009 Table 5 Lable claim at 75, 100 and 125 %. Source Degrees of freedom SS MS F At 75 % Lable claim. Between Days Within Days Total At 100% Lable claim Between Days Within Days Total At 125 % Lable claim Between Days Within Days Total Robustness There was no significant change in the retention time of aripiprazole and its degradation products after introducing small changes in mobile phase composition indicating robustness of the method. Linearity and Range These parameters were evaluated in calibration experiments (Table 3). Analysis of Marketed Formulation The chromatograms of the drug samples extracted from tablets did not show a change in the retention time. There was no interference from the excipients, which are commonly present in the tablets. The drug content was found to be % with a % RSD of 2.46 as shown in (Table 6) Therefore it may be concluded that, degradation of aripiprazole had not occurred in the marketed formulations. The % low RSD value indicated the suitability of the method for the routine analysis of aripiprazole in pharmaceutical formulation. Conclusion Stability indicating reversed phase high performance liquid chromatography method for aripiprazole is developed and validated as per ICH guidelines. The analysis of aripiprazole in the presence of degradants is possible now. This method can also be used for further impurity profiling of the drug. As the retention time of aripiprazole is 9.2 min, it can be used for the determination of aripiprazole from the tablets too. From the purposeful degradation study it is clear that the molecule is quite stable to sun light and it shows degradation in acidic, basic, oxidative, dry heat and neutral hydrolysis. Acknowledgement We are thankful to the Principal, Vilasrao Kadam, Bharati Vidyapeeth s College of Pharmacy, Navi Mumbai, for the facilities. References Bolton S., Pharmaceutical Statistics, Practical and Clinical Applications, J. Swarbrick, Ed., Volume 44, 2 nd Edition, Marcel Dekker, New York, (1990) Ermer J., McB J. H., Miller, Eds., Method Validation in Pharmaceutical Analysis, A Guide to Best Practice, Wiley VCH, Weinheim, (2005) International Conference on Harmonization (ICH), Validation of Analytical Methods: Definitions and Terminology, ICH Q2 A, (1994). International Conference on Harmonization (ICH), Validation of Analytical Methods: Methodology, ICH Q2 B, (1996) Kiser M. M., Dolan J. W. Selecting the Best Curve Fit, LC-GC Europe, 2, 17 (3) (2004) Kubo M., Mizooku Y., Hirao Y., Osumi T., Development and validation of an LC- MS/MS method for the quantitative determination of aripiprazole and its main metabolite, OPC , in human plasma, J. of Chroma. B, 822(2005)

10 Pankaj Nerkar et al. : Development of Stability Indicating Reverse Phase HPLC Method for 581 Nagaraja N. V., Paliwal J. K., Gupta R. C., Choosing the calibration model in assay validation, Journal of Pharmaceutical and Biomedical Analysis, 20 (1999) Shimokawa Y., Akiyama H., Kashiyama E., Koga T., High performance liquid chromatographic methods for the determination of aripiprazole with ultraviolet detection in rat plasma and brain, application to the pharmacokinetic study, J. of Chroma. B, 821 (2005) Singh S., Bakshi M., Guidance on the conduct of stress tests to determine inherent stability of drugs, Pharmaceutical technological Asia September/ October 2000, Snyder L. R., Kirkland J. J., Glajch J. L., Practical HPLC Method Development, 2 nd Edition, John Wiley and Sons, New York, (1997)