Method Validation for Simultaneous Estimation of Prednisolone and Abiraterone Acetate by RP-HPLC
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1 Journal of Chronotherapy and Drug Delivery ORIGINAL RESEARCH PAPER Method Validation for Simultaneous Estimation of Prednisolone and Abiraterone Acetate by RP-HPLC Divyashree S*, Veena MK, Channabasavaraj KP Department of Pharmaceutical Chemistry, Government College of Pharmacy, Bangalore 5027 (India) * For correspondence divyasheshadri13@gmail.com Key words Prednisolone, Abiraterone acetate, RP- HPLC, Liquid chromatography J Chronother Drug Deliv Vol 7 Issue : Authors Abstract A simple, precise, rapid and accurate Reverse Phase High Performance Liquid Chromatography (RP- HPLC) was developed and validated for the simultaneous estimation of Prednisolone and Abiraterone acetate in bulk drug and the separation was carried out using Phenomenex (250 X 4.6 mm, 5 µ) C18 Column with Methanol as the mobile phase, at a flow rate of 1ml/ min and UV detection at 250 nm. The retention time of Prednisolone and Abiraterone acetate were found to be 2.7 min and 4.2 min and the Limit of Detection and Limit of Quantification were 0.1mcg/ml and 0.4mcg/ml respectively. The calibration curve were linear (R2= ) over a concentration range of mcg/ml. The developed method was validated for parameters like system suitability, specificity, precision, linearity, accuracy, ruggedness and robustness as per ICH guidelines and all the parameters were found to be within the acceptance criteria. Hence the method can be used for routine estimation of Prednisolone and Abiraterone acetate in bulk sample. e-issn:
2 INTRODUCTION Prednisolone (Fig 1a) is a synthetic glucocorticoid, a derivative of Cortisol, used to treat a variety of inflammatory and autoimmune conditions and some cancers. It is the active metabolite of the drug Prednisone and is used especially in patients with liver failure as these individuals are unable to metabolize Presdnisone into active Prednisolone; it is primarily metabolized via the liver enzyme, 11-βhydroxydehydrogenase. Abiraterone acetate is a steroidal anti- androgen, specifically an androgen synthesis inhibitor (Fig 1b), used in combination with Prednisolone/ Prednisone in metastatic castration-resistant prostate cancer (formerly hormone-resistant or hormone-refractory prostate cancer) i.e., prostate cancer not responding to androgen deprivation or treatment with androgen receptor antagonists. 1 Fig 1. Structures of selected drugs - Prednisolone and Abiraterone acetate Literature survey revealed that there were only few methods for estimation of Prednisolone and Abiraterone individually like estimation using UV- Visible Spectroscopy 2,3, RP HPLC method 4, Dual wavelength Spectrophotometric method 5 for Prednisolone acetate, Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry 6, Liquid Chromatographic-Tandem mass spectrometry 7,8, High Performance Liquid Chromatography(HPLC)/UV 9 and HPLC with Fluorescence detector 10 method in rat and human plasma, Liquid Chromatography method for Abiraterone in its degradation products 11, sensitized Spectrofluorimetry method 12 for Abiretarone were reported individually and in combination with other drugs. But no method was developed for the simultaneous estimation of Prednisolone and Abiraterone acetate by RP-HPLC. Hence, in the present study, an attempt has been made to estimate Prednisolone and Abiraterone acetate simultaneously by RP-HPLC. It can be applied for routine analysis in bulk and pharmaceutical dosage forms. MATERIAL AND METHODS Chemicals and Reagents Prednisolone and Abiraterone acetate standards were obtained as gift samples from Biocon and Microlabs. Methanol (HPLC) grade, Class- A apparatus were used throughout the experiement. Preparation of Working Standard Solution of Prednisolone and Abiraterone Acetate Accurately 10 mg of Prednisolone and Abiraterone acetate were weighed and transferred into a 10 ml volumetric flask, dissolved in methanol and the volume is made up to the mark with the same to get a concentration of 0 mcg/ml (Stock A). Sample Preparation Twenty tablets of Prednisolone 5 mg and Abiraterone acetate 5 mg were weighed and their average weights are calculated, and the tablets were powdered. The tablet powder equivalent to 10 mg Prednisolone and Abiraterone acetate were weighed and transferred into a 10 ml volumetric flask, dissolved in methanol and the volume is made up to the mark with the same to get a concentration of 0 mcg/ml. 42 JChrDD 2016 Vol 7 Issue 1
3 Instrumentation and Chromatographic Conditions The instrument and the chromatographic conditions for the simultaneous estimation of selected drugs are given in Table 1. Table 1. Instrument and chromatographic conditions Chromatographic Conditions Instrument High Performance Liquid Chromatograph LC-20AT Shimadzu SPD- 20A Column C-18 Column Phenomenex (250 X 4.6 mm 5 µ) Detector Wavelength UV Detector 250 nm Injection volume 10 µl Flow rate Mobile phase 1 ml/min Methanol Method Validation The method was validated as per ICH guidelines for the parameters like system suitability, Specificity, Linearity and Range, LOD (Limit of Detection) and LOQ (Limit of Quantification), Precision (System Precision, Method Precision, Intermediate Precision - Ruggedness), Robustness and Accuracy. System Suitability Parameters These tests are based on that the equipment, analytical operations and samples to be analyzed which constitute an integral system that can be evaluated as such. This test ensures that the analytical system is working properly and gives accurate and precise results. Specificity Specificity is the ability to access unequivocally the analyte in the presence of components that might be expected to be present, such as impurities and degradation products. Linearity In the present study, Linearity was carried out using five different concentrations viz 200, 300, 400, 500 and 0 mcg/ml, which was prepared by transferring 2, 3, 4, 5, 6 ml into five different volumetric flasks from Stock A and the volume is made up to the mark with methanol. Peak area and retention time was recorded and linearity was derived by plotting the standard calibration curve for Prednisolone and Abiraterone acetate respectively. Precision The precision of an analytical method is the closeness of agreement among individual test results when the method is applied repeatedly to multiple sampling of homogeneous sample. The precision of an analytical method is usually expressed as the standard deviation or relative standard deviation of series of measurement. System Precision The system precision is checked by using standard substance to ensure that the analytical system is working properly. The retention time and area of six determinants is measured and % RSD is calculated. Method Precision (Repeatability) In method precision, a homogeneous sample of single batch should be analysed six times. This indicates whether a method is giving consistent results for a single batch. JChrDD 2016 Vol 7 Issue 1 43
4 Intra-day Precision Successive five injections of 10 µl of working standard solution of Prednisolone and Abiraterone acetate were injected separately at different intervals in the same day and chromatograms were recorded. The % RSD was calculated for concentration of drug in replicates. Inter-day Precision Successive five injections of 10 µl of working standard solution of Prednisolone and Abiraterone acetate were injected separately on different days and chromatograms were recorded. The % RSD was calculated for concentration of drug in replicates. Intermediate Precision (Ruggedness) Intermediate precision expresses the variations within the laboratories variations (different analyst, different instrument etc) The Intermediate precision was performed for Prednisolone and Abiraterone acetate by different analyst and different instrument on different day. The % RSD was calculated for Intermediate precision. Limit of Detection (LOD) and Limit of Quantification (LOQ) For estimation of LOD and LOQ lower dilutions of Prednisolone and Abiraterone acetate standard were prepared, injected into the chromatograph and response obtained was recorded. Accuracy The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. Accuracy may often be expressed as percentage recovery by the assay of known added amounts of analyte carried out at the different levels (80%, % and 120%). Robustness The robustness of an analytical method is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage. The evaluation of robustness should be considered during the development phase and depends on the type of the procedure under study. It should show the reliability of an analysis with respect to deliberate variations in method parameters such as: 1. Influence of variations in flow rate 2. Influence of variations in change in composition of mobile phase 3. Influence of variations in wavelength Assay The proposed method was used to estimate the total drug content present in marketed dosage form. The assay was done in sample tablet and compared with the standard drugs and the percentage recovery was calculated. RESULTS AND DISCUSSION From literature survey it was found that no UV Spectrophotometric, HPLC Methods have been reported for simultaneous estimation of Prednisolone and Abiraterone acetate. It was felt necessary to develop and validate new method for estimation of Prednisolone and Abiraterone acetate which could be applicable for their analysis in marketed dosage formulation. The isobestic point of Prednisolone and Abiraterone acetate was found to be 250 nm and hence it was selected as the ƛ max for simultaneous estimation. A RP-HPLC method was developed on a C-18 Column Phenomenex (250 X 4.6 mm 5 µ) using isocratic mode with mobile phase as Methanol at flow rate of 1mL/min. The linearity range was found to be in the concentration range of mcg/ml for Prednisolone and Abiraterone acetate with percentage curve fitting within acceptance criteria for both the drugs. Retention time of Prednisolone and Abiraterone acetate were found to be 2.7 min and 4.2 min, respectively. 44 JChrDD 2016 Vol 7 Issue 1
5 For determination of linearity, peak area and retention time were recorded and linearity was derived by plotting the standard calibration curve for Prednisolone and Abiraterone acetate as shown in Fig 2. The linearity and regression coefficient value is both for Prednisolone and Abiraterone acetate in the concentration range of mcg/ml. Fig 2. Calibration curves of Prednisolone and Abiraterone acetate showing linearity in the selected concentration range Table 2. System suitability parameters for the developed RP-HPLC method for selected drugs System Suitability Factor Prednisolone Abiraterone Acetate Acceptance Criteria Theoretical Plates > 2000 HETP Tailing factor < 2 Resolution > 2 JChrDD 2016 Vol 7 Issue 1 45
6 Fig 3 shows the graph of system suitability and Table 2 shows the results of system suitability study. The results suggest that the developed method is suitable and specific. Fig 3. System suitability study showing peaks of selected drugs The results of precision repeatability studies, Ruggedness and Accuracy studies of Prednisolone and Abiraterone acetate are given in Table 3 to 6. The results were within the acceptable limits showing that the developed method was accurate and precise. Table 3. Results of precision of Prednisolone and Abiraterone acetate using the developed HPLC method Precision Parameters Prednisolone %RSD Abiraterone %RSD Acceptance Criteria System Precision < 2% Method Precision < 2% Intraday Precision < 2% Interday Precision < 2% Table 4. Results of ruggedness study Drug Analyst-I/Instrument-I Analyst-/ Instrument- Acceptance Criteria Prednisolone 102.6% 109.7% 90% to 110% Abiraterone acetate 107.4% 105.4% 90% to 110% 46 JChrDD 2016 Vol 7 Issue 1
7 Table 5. Results of accuracy study of Prednisolone Level Replicate Standard 80% I I % I I 120% I I Sample Peak Area Total found Amount of standard recovered (mcg) % Recovery Table 6. Results of accuracy study of Abiraterone acetate Level Replicate Standard Sample Peak Area Total found Amount of standard recovered (mcg) % Recovery 80% I I % I I 120% I I The LOD determined for Prednisolone and Abiraterone acetate was found to be 0.1 mcg/ml and LOQ for Prednisolone and Abiraterone acetate was found to be 0.4 mcg/ml. The results are given in Table 7. Table 7. Results of limit of detection (LOD) and limit of quantification (LOQ) studies Parameter Prednisolone peak Area Abiraterone Peak Area Prednisolone concentration Abiraterone concentration LOD LOQ Table 8. Recovery studies for Prednisolone and Abiraterone acetate Level Mean Per cent Recovery Prednisolone Mean Per cent Recovery Abiraterone Acceptance criteria 80% 105.5% 108.4% 90%-110% % 106.4% 108.1% 90%-110% 120% 106.9% 107.2% 90%-110% JChrDD 2016 Vol 7 Issue 1 47
8 The mean percentage recovery for Prednisolone and Abiraterone acetate at three different levels was found to be within the limits (90-110%) (Table 8). CONCLUSION A simple, rapid and reliable RP-HPLC method has been established for the simultaneous estimation of Prednisolone and Abiraterone acetate either alone and in formulation. The method has several advantages like rapid, simple, no need for special reagents, high sensitivity. This makes the method suitable for routine analysis of the drugs in quality control laboratories. ACKNOWLEDGEMENT The authors would like to thank the Principal, GCP, HOD of Pharmaceutical Chemistry Department, GCP, Chief Scientific Officer and Scientific Officer Drug testing Laboratory, Bangalore, for providing laboratory facilities to carry out this work and Biocon for providing gift sample of Prednisolone and Abiraterone acetate. DECLARATION OF INTEREST It is hereby declared that this paper does not have any conflict of interest. REFERENCES 1. Auchus RJ, Yu MK, Nguyen S, Mundle SD. Use of prednisone with abiraterone acetate in metastatic castration-resistant prostate cancer. Oncologist. 2014; 19(12): Ashok R, Prakash PP, Selvan RT. Development and validation of analytical method for estimation of Prednisolone in bulk and tablet using UV- Visible Spectroscopy. Int J Pharm Pharm Sci. 2011; 4(3): Kashyap R, Subrahmanyam EVS, Sharbaraya AR. Development and validation of UV spectroscopy method for the estimation of prednisolone in bulk and dosage form. Int J Chem Pharmaceut Res. 2012; 4(2): Kurakula M, Mohd AB, Samhuidrom, Rao AP, Diwan PV. Estimation of Prednisolone in Proliposomal formulation using RP HPLC method. Int J Res Pharmaceut Biomed Sci. 2011; 2(4): Hina BP, Sejal KP. Dual wavelength Spectrophotometric method for simultaneous estimation of Gatifloxacin sesquihydrate and Prednisolone acetate in combined dosage form. Int J Pharmaceut Chem Biol Sci 2013; 3(2): Wani TA. Highly sensitive Ultra-Performance Liquid Chromatography- tandem Mass Spectrometry method for the determination of Abiraterone in Human Plasma. Anal Methods. 2013; 9: Gurav S, Punde R, Farooqui J, Zainuddin M, Rajagopal S, Mullangi R. Development and validation of a highly sensitive method for the determination of Abiraterone in rat and human plasma by LC-MS- ESI: application to a pharmacokinetic study. Biomed Chromat. 2012; 26(6): Martins V, Yasmin A, Nicola W, Florence R. A validated Liquid Chromatographic-Tandem Mass Spectroscopy method for the quantification of Abiraterone acetate and Abiraterone in human plasma. J Chromt B Analyt Technol Biomed Life Sci. 2006; 843(2): Kumar SV, Rudresha G, Gaurav S, Zainuddin M, Dewang P, Kethiri RR et al. Validated RP-HPLC/UV method for the quantification of Abiraterone in rat plasma and its application to pharmacokinetic study in rats. Biomed Chromat. 2013; 27(2): Belleville T, Noe G, Huillard O, Thomas-Schoemann A, Vidal M, Goldwasser F et al., A HPLCfluorescence method for the quantification of Abiraterone in plasma from patients with metastatic castration-resistant prostate cancer. J Chromat B Analyt Technol Biomed Life Sci. 2015; 989: Khedra A, Darwishb I, Bamanea F. Analysis of Abiraterone stress degradation behavior using Liquid Chromatography coupled to ultraviolet detection and electrospray ionization Mass Spectrometry. J Pharmaceut Biomed Anal. 2013; 74: Gong A, Zhu X. β-cyclodextrin sensitized Spectrofluorimetry for the Determination of Abiraterone acetate and Abiraterone. J Fluoresc. 2013; 23(6): ICH. Q3A Text on validation of analytical procedure. Geneva: International Conference on Harmonization; JChrDD 2016 Vol 7 Issue 1
9 14. ICH, Q3B Validation of analytical procedures: methodology. Geneva: International Conference on Harmonization; Received: February 15, 2016; Accepted: February 27, 2016 JChrDD 2016 Vol 7 Issue 1 49
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