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1 MAY 2014 JULY 2014, Vol. 4, No. 3; E- ISSN: Journal of Chemical, Biological and Physical Sciences An International Peer Review E-3 Journal of Sciences Available online atwww.jcbsc.org Section B: Biological Sciences CODEN (USA): JCBPAT Research Article Development and validation of analytical method for estimation of Spironolactone in oral suspension Rahul CR 1, Jigisha Patel 1, Mayank Bapna 1 * 1* Department of Quality assurance, Shivam Pharmaceutical Studies and Research Centre, Valasan-Anand, Gujarat, India Received: 30 May 2014; Revised: 25 May 2014; Accepted: 15 June 2014 Abstract: A simple, fast, accurate and precise reverse phase high performance liquid chromatographic method has been developed for the estimation of Spironolactone in oral suspension. The chromatographic separation was achieved on Zorbax RX-C18 (150 x 4.6) mm; 5 µm column with an isocratic mixture of methanol: water (60:40). The injection volume was kept at 20µl with mobile phase at a flow rate of 1 ml/min. The wavelength of detection was kept at 240 nm with column temperature at 25 ºC. The retention times for Spironolactone, Methyl paraben and Propyl paraben was found to be 6.61±0.04 min, 2.25±0.04 min and 4.67±0.04 min, respectively. Methyl paraben and Propyl paraben were major excipients of the oral solution. The linearity was obtained in the range of µg/ml, µg/ml and 5-12 µg/ml with correlation coefficient 0.998, and for Spironolactone, Methyl paraben and Propyl paraben, respectively. The proposed method was validated as per ICH guidelines and successfully applied for the determination of investigated drugs in oral suspension. Keywords: Spironolactone, metyl paraben, propyl paraben, oral suspension, RP-HPLC J. Chem. Bio. Phy. Sci. Sec. B, May 2014 July 2014; Vol.4, No.3;
2 INTRODUCTION Spironolactone (SPIRO) 1 : 7α-acetylthio-3-oxo-17α-pregn-4-ene-21, 17-carbolactone is a potassiumsparing diuretic used to diagnose or treat a condition in which there is a too much aldosterone in the body. Aldosterone is a hormone produced by adrenal glands to help regulate the salt and water balance in body. Spironolactone also treats fluid retention (edema) in people with congestive heart failure, cirrhosis of the liver, or a kidney disorder called nephrotic syndrome. It is also used to treat or prevent hypokalemia (low potassium levels in the blood). Methyl paraben (MP) 2 and Propyl paraben (PP) 3 : MP is methyl-4-hydroxybenzoate and PP is propyl- 4-hydroxybenzoate and are used as a preservative in the food, cosmetic, and pharmaceutical industries and they are found naturally in fruits like blueberries where they have antimicrobial activity. They are completely absorbed through the skin or after ingestion, hydrolyzed to p-hydroxybenzoic acid and are rapidly excreted in the urine. Rationale: The drug SPIRO is official in I.P and B.P Detailed literature survey for SPIRO revealed that several methods have been reported for estimation of SPIRO in combination with other drugs like HPLC 4, stability-indicating assay method for SPIRO in extemporaneous preparation 5, HPLC determination of spironolactone and its metabolites in human biological fluids after solid-phase extraction 6, single RP-HPLC assay method for analysis of bulk raw material batches of four parabens that are widely used as preservatives in pharmaceutical and cosmetic products 7, quantitative analysis of methyl and propylparaben by high performance liquid chromatography 8, separation and estimation of methyl and propyl esters of p-hydroxybenzoic acid by gas chromatography 9, LCMS determination of SPIRO and Canrenone 10, simultaneous estimation of metolazone and SPIRO in raw materials, combined tablets and human urine by HPLC 11, RP-HPLC and UV spectrophotometric method for simultaneous determination of SPIRO and torsemide in pharmaceutical dosage form 12, RP-HPLC method for simultaneous estimation of furosemide and SPIRO in their combined tablet dosage form 13, simultaneous estimation of SPIRO and hydroflumethiazide in pharmaceutical dosage form 14, RP- HPLC Method for simultaneous estimation of SPIRO and hydrochlorothiazide in oral solid dosage form 15. Not a single method has been reported for estimation of SPIRO with excipients in oral dosage form, therefore we have developed a method for the estimation of SPIRO and MP and PP in combined liquid dosage form. In the proposed method forced degradation of drug substances and drug product will also be carried out under different stress conditions (acidic, basic, oxidative, thermal, and UV exposure) and the stressed samples will be analyzed by the developed and validated method. MATERIALS AND METHODS Materials: The formulation Spironolactone in oral suspension (label claim: Spironolactone 2 gm/200ml, Methyl paraben 0.4 gm/200ml and Propyl paraben 0.04 gm/200ml), manufactured by Thames laboratory ltd. was procured from analytical research laboratory, Ahmadabad. All the chemicals used were of analytical grade and were purchased from MERCK Chem. Ltd., Mumbai J. Chem. Bio. Phy. Sci. Sec. B, May 2014 July 2014; Vol.4, No.3;
3 Instruments: Following instruments with given specification were used for estimation of SPIRO from oral suspension, HPLC (Shimadzu) LC2010CHT with SPDM20A diode detector, LC solution software was applied for data collecting and processing, ph meter (ELICO), digital balance (Sartorius-0.1 mg 205 gm) and FTIR Spectrophotometer (Brukeroptics).The chromatographic separation was achieved on a Zorbax RX-C18 (150 x 4.6) mm; 5µm as a stationary phase with isocratic elution. The mobile phase, methanol: water (60:40) was pumped at a flow rate of 1 ml/min. The samples were analysed by a PDA detector at 240 nm with the injection volume of 20 µl. METHOD DEVELOPMENT Preparation of standard solution for Spironolactone: Accurately weighed 250 mg of standard SPIRO was transferred to 50 ml volumetric flask and dissolved in 30 ml of diluent and shaken vigorously. The volume was made up to the mark with diluent to give a solution containing 5000 µg/ml of SPIRO. From this 20 ml of solution was taken out and diluted up to 50 ml with diluent in a volumetric flask to give solution of 2000 µg/ml of SPIRO. Further, 2.5 ml of this solution was taken and diluted up to 10 ml with diluent to give a working standard solution containing 500µg/ml of SPIRO. Preparation of standard solution for MP: Accurately weighed 100 mg of MP was transferred to a 50 ml volumetric flask and dissolved in 30 ml diluent. The flask was then shaken and volume was made up to the mark with diluent to give a solution containing 2000 µg/ml MP. Ten ml of this solution was taken and diluted up to 50 ml with diluent in volumetric flask to give solution of 400 µg/ml of MP. Further, 2.5 ml solution was taken and diluted up to 10 ml with diluent to give working standard solution containing 100 µg/ml MP. Preparation of standard solution for PP: Accurately weighed 20 mg of PP was transferred to a 50 ml volumetric flask and dissolved in 30 ml of diluent. The flask was then shaken and the volume was made up to the mark with the diluent to give a solution containing 400 µg/ml of PP. Five ml of this solution was taken and diluted up to 50 ml with the diluent to give a solution of 40 µg/ml of PP. Further, 2.5 ml of this solution was taken and diluted up to 10 ml with diluent to give a working standard solution containing 10 µg/ml of PP. Preparation of sample solution: One ml of oral suspension was taken and diluted up to 20 ml with the diluent. According to label claim 1 ml of suspension contains 10 mg SPIRO, 2 mg MP and 0.2 mg PP therefore, after dilution 1 ml of suspension in 20 ml of diluent will contain 500 µg/ml, 100 µg/ml and 10 µg/ml of SPIRO, MP and PP, respectively. Calibration curve: Calibration curves constructed were linear over the concentration range of µg/ml, µg/ml and 5-12 µg/ml for SPIRO, MP AND PP, respectively. Calibration curves were prepared using ratio of analyte peak area to internal standard peak versus concentration of analytes. The calibration curves are shown in Figure 1, 2 and J. Chem. Bio. Phy. Sci. Sec. B, May 2014 July 2014; Vol.4, No.3;
4 Figure 1: Linearity curve of Spironolactone Figure 2: Linearity curve of Methyl Paraben. Figure 3: Linearity curve of Propyl paraben J. Chem. Bio. Phy. Sci. Sec. B, May 2014 July 2014; Vol.4, No.3;
5 METHOD VALIDATION The proposed method was validated in accordance with ICH guidelines for accuracy, precision, LOD, LOQ, linearity and percentage recovery. Linearity: Linearity study was carried out for SPIRO, MP and PP at six different concentration levels. Calibration curves constructed were linear over the concentration of µg/ml, µg/ml and 5-12 µg/ml for SPIRO, MP and PP, respectively. Evaluation of these drugs was performed with UV detector at 240 nm and peak area was recorded for all the peaks. The correlation coefficient for SPIRO, MP and PP were found to be 0.998, and 0.999, respectively. Limit of detection and limit of quantification: The LOD and LOQ of the developed method were determined by injecting progressively low concentration of the standard solutions using the developed HPLC method. The LOD for SPIRO, MP and PP were found to be µg/ml,1.335 µg/ml and µg/ml respectively. The LOQ for SPIRO, MP and PP were found to be µg/ml, µg/ml and µg/ml, respectively. Accuracy: The accuracy of the method was assessed by recovery studies of SPIRO, MP and PP in combined dosage form at three concentration levels. A fixed amount of pre-analyzed sample was taken and standard drug was added at 50%, 100% and 200% levels. Each level was repeated for three times as shown in Table-1. The percentage recoveries of SPIRO, MP and PP were %, % and 101.8% which shows that there is no interference from excipients and the lower values of RSD of assay indicate the method is accurate. Table-1: Accuracy studies for the proposed method. Spiked level Amount added (µg/ml) Found (µg/ml) %Recovery SPIRO MP PP SPIRO MP PP SPIRO MP PP 50% % % Table-2: Intra-day and Inter-day precision for the analysis of SPIRO, MP and PP. Sr. No. Intraday precision Inter-day precision SPIRO MP PP SPIRO MP PP Avg SD %RSD J. Chem. Bio. Phy. Sci. Sec. B, May 2014 July 2014; Vol.4, No.3;
6 Precision: The precision for the developed method was determined in terms of intraday and inter-day precision. For intraday precision evaluation, a standard solution of fixed concentration was injected at various time intervals on a particular day and % RSD for SPIRO, MP and PP were found to be 0.64%, 0.82% and 0.62%, respectively (limit %RSD < 2.0%). In addition, the inter-day precision was studied by injecting the same concentration of standard solution on consecutive days and the %RSD for SPIRO, MP and PP were found to be 0.65%, 1.19% and 0.84%, respectively (limit %RSD < 2.0%). The results are shown in Table-2. Assay: Twenty microlitre of sample solution was injected and from the peak area of SPIRO, MP and PP amount of each drug in sample was computed. The results of the assay (Table-3) undertaken, yielded %, % and 101.8% of SPIRO, MP and PP, respectively. Table-3: Assay results of proposed methods. Drug Conc. In 200ml Conc. taken for Conc. %Assay Dosage Form assay Found SPIRO 2gm 500µg/ml MHB 0.4gm 100µg/ml PHB 0.04gm 10µg/ml Table-4: Force degradation study of SPIRO, MP and PP. Degradation Condition % ASSAY % DEGRADATION SPIRO MP PP SPIRO MP PP 0.1N HCl 46.42% 92.92% 75.48% 53.58% 7.08% 24.52% 0.1N NaOH 41.64% 89.59% 67.26% 58.36% 10.41% 32.74% 3% H2O % 83.97% 51.37% 67.76% 16.03% 48.63% Photo/UV 38.9% 71.09% 46.59% 61.1% 28.91% 53.41% Thermal 49.47% 96.58% 79.38% 50.53% 3.42% 20.62% STABILITY INDICATING STUDY Force degradation studies: Whole stability indicating RP-HPLC assay method for simultaneous determination of SPIRO, MP and PP were done using above developed RP-HPLC method. In order to establish stability-indicating nature of the method, standards of drugs, drug product and diluent were subjected to various stress conditions to conduct force degradation studies. Stress studies were carried out under the conditions of acidic, basic, oxidative, thermal and UV exposure. Several trials with different severity of each stressed condition were conducted, the results of which are shown in Table J. Chem. Bio. Phy. Sci. Sec. B, May 2014 July 2014; Vol.4, No.3;
7 RESULTS AND DISCUSSIONS Estimation of SPIRO was carried out using optimized HPLC method. The method was linear in the range of µg/ml, µg/ml and 5-12 µg/ml for SPIRO, MP and PP, respectively. The linearity curves for SPIRO, MP and PP are shown in Figure 1, Figure 2 and Figure 3. The samples were analysed by a PDA detector at 240 nm with the injection volume of 20 µl. The resultant peaks were good in shape and resolution (Figure 4). The percentage recoveries of SPIRO, MP and PP were %, % and 101.8% which shows that there is no interference from excipients and the lower values of RSD of assay indicate the method is accurate. The % RSD of SPIRO, MP and PP for intraday precision study were found to be 0.64%, 0.82% and 0.62% respectively (limit %RSD< 2.0%). and % RSD of SPIRO, MP and PP for inter-day precision study were found to be 0.65%, 1.19% and 0.84%, respectively.(limit %RSD < 2.0%). Figure 4: Typical chromatogram of standard for Spironolactone, Methyl Paraben and Propyl Paraben. The retention time of SPIRO, MP and PP was found to be 6.61min, 2.25min and 4.67min, respectively with an asymmetry factor of 1.06 for SPIRO, 1.13 for MP and 1.01 for PP, which indicates efficient performance of the column. The LOD for SPIRO, MP and PP were found to be µg/ml, µg/ml and µg/ml respectively. The LOQ for SPIRO, MP and PP were found to be µg/ml, µg/ml and µg/ml, respectively which indicate good sensitivity of the proposed method. Assay studies of the proposed method indicate %, % and 101.8% recovery for SPIRO, MP and PP, respectively. The results of the assay are shown in Table-4. No interfering peaks were found in the chromatogram of the formulation within the run time indicating that excipients used in formulations did not interfere with the estimation of the drug by the proposed HPLC method J. Chem. Bio. Phy. Sci. Sec. B, May 2014 July 2014; Vol.4, No.3;
8 CONCLUSION The developed HPLC method is simple, specific, accurate and precise for the simultaneous estimation of SPIRO, MP and PP in oral liquid suspension. The developed method provides good resolution between SPIRO, MP and PP. It was successfully validated in terms of linearity, accuracy, precision, LOD, LOQ and recovery in accordance with ICH guidelines. Thus the described method is suitable for routine analysis and quality control of pharmaceutical preparations containing these drugs in combinations. REFERENCES 1. British Pharmacopoeia commission London HMSO. Publication.British Pharmaco. 2009, 1, 2, Indian Pharmacopoeia, Volume-III, Electronic edition, Government of India, Ministry of Health and Welfare, Indian Pharmacopoeia commission, Ghaziabad. 2007, Indian Pharmacopoeia Volume-III, Electronic edition, Government of India, Ministry of Health and Welfare, Indian Pharmacopoeia commission, Ghaziabad. 2007, K.S. Alexander, S.S. Vangala and D. David, An Improved High-Performance Liquid Chromatography Assay for Spironolactone tablet Analysis, Journal of Drug Development and Industrial Pharmacy. 1998, 24, L. Sriphong, K. Dejpisuttitham and P. Ketwang Development of stability- indicating assay method for spironolactone in extemporaneous preparation, Goodman & Gilman s The pharmacological Basis of Therapeutic, Brunton McGraw-Hill, 2006,12, F. Varin, T.U. Minh and B. France High-performance liquid chromatographic determination of spironolactone and its metabolites in human biological fluids after solid-phase extraction,journal of Chromatography B Biomedical Sciences and Applications. 1992, 574, S. Kumar, S. Mathkar, C. Romero and A.M. Rustum Development and Validation of a Single RP-HPLC Assay Method for Analysis of Bulk Raw Material Batches of Four Parabens that are Widely Used as Preservatives in Pharmaceutical and Cosmetic Products, Journal of Chromatographic Science. 2011, 49, A.F. Fitzpatrick and A.D. Coopert Quantitative Analysis of Methyl and Propylparaben by High Performance Liquid Chromatography, Society of cosmetic Chemistry. 2010, 26, J.D. Sam, Separation and estimation of methyl and propyl esters of p-hydroxy benzoic acid by gas chromatography, J. of Pharmaceutical Sci. 2006, 54, V. Laurian, I. Silvia, LCMS Determination of Spironolactone and Canrenone, CROATICA CHEMICA ACTA. 2011, 84, I.M. Walashet, Simultaneous determination of metolazone and spironolactone in raw materials, combined tablets and human urine by high performance liquid chromatography. Early applied demonstrations of new analytical methods with clearsocietal impact. 2013, 5, R. Laxman and S. Bhardrwaj. Devlopment and validation of RP-HPLC and ultraviolet spectrophotometric methods for simultaneous determination of 2203 J. Chem. Bio. Phy. Sci. Sec. B, May 2014 July 2014; Vol.4, No.3;
9 spironolactone and torsemide in pharmaceutical dosage form International Journal of Research in Ayurveda and Pharmacy. 2010,1, M. Bhojani, Development and Validation of RP-HPLC Method for Simultaneous Estimation of Furosemide and Spironolactone in their Combined Tablet Dosage Form Journal of Pharmaceutical Sci. and Bioscientific Reseach. 2012, 2, L.R. Khanchandani. Development and validation of liquid chromatographic method for simultaneous estimation of Spironolactone and Hydroflumethiazide in pharmaceutical dosage form Novus International Journal of Chem. 2013, 2, G.S. Devika, Isocratic RP-HPLC Method for Simultaneous Estimation of Spirinolactone and Hydrochlorthiazide in Oral Solid Dosage Form, Research Journal of Pharmacy and Technology. 2012, 5, * Corresponding author: Mayank Bapna; Department of Quality assurance, Shivam Pharmaceutical Studies and Research Centre, Valasan-Anand, Gujarat, India 2204 J. Chem. Bio. Phy. Sci. Sec. B, May 2014 July 2014; Vol.4, No.3;
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