STRESS DEGRADATION STUDIES AND DEVELOPMENT OF A VALIDATED UV SPECTROPHOTOMETRIC METHOD FOR MUPIROCIN IN BULK AND PHARMACEUTICAL FORMULATION 1 S.K.Attar*, 2 M.S.Kalshetti, 3 A.S.Bansode, 3 V.D. Shelke 1. Department of Quality Assurance, Adarsh College of Pharmacy, Vita 415311, Maharashtra, India 2. Department of Quality Assurance, D.S.T.S.Mandal s College of Pharmacy, Solapur 413004, Maharashtra, India 3. Department of Quality Assurance Techniques, Sinhgad Institute of Pharmacy Narhe, Pune 411041, Maharashtra, India. ABSTRACT In this work, forced degradation studies of Mupirocin were carried out by developed & validated spectroscopic method. A Simple, accurate, precise and economical spectroscopic method have been developed and validated for the determination of mupirocin in their bulk and ointment formulation. The λmax was found to be 222 nm in ethanol. Beer s law was obeyed in the concentration range of 10-50µg/ml. Good accuracy (99.5), precision (RSD 0.1), LOD (0.06μg/ml), LOQ (0.32μg/ml) and linearity (0.999) were obtained. The result of analysis has been validated as per the ICH (Q2B) guidelines. The stress degradation study includes effect of acid, alkali, hydrogen peroxide, neutral, thermal and photolytic degradation study were carried out according to the ICH requirements (Q1A (R2) and Q1B). The result of stress degradation study shows that the drug was degrading more under acidic, alkaline than oxidation, neutral, thermal and photolytic stress conditions. The proposed validated analytical method and stress degradation study can be used for routine quality control analysis of Mupirocin in bulk and pharmaceutical formulations. Key-words: Spectroscopic method, Mupirocin, Stress Degradation Studies. Corresponding Author: S.K.Attar Department of Quality Assurance, Adarsh College of Pharmacy, Vita 415311, Maharashtra, India INTRODUCTION Mupirocin or pseudomonic acid (9-[[(2E)-4- [(2S,3R,4R,5S)-5-(2S,3S,4S,5S)-2,3-epoxy- 5-hydroxy-4-methylhexyl] 3,4-dyhidroxy- 3,4,5,6-tetrahydro-2H-pyran-2-yl]-3- methylbut-2-enoyl]oxy]nonanoic acid) antibiotic derived from Pseudomonas fluorescens; discovered in 1971 and has in vitro activity against a range of Grampositive and some Gram-negative bacteria. Mupirocin has excellent activity against staphylococci (including methicillinresistant strains), streptococci, and gramnegative organisms such as Haemophilus influenza, Neisseria gonorrhoeae, and Moraxella catarrhalis. It shows high activity against gram-positive staphylococci and streptococci. Mupirocin acts as protein synthesis inhibitor by targeting the isoleucyl-trna synthetase; mupirocin mimics isoleucyl-adenylate. Mupirocin reversibly binds to bacterial isoleucyl-trna synthetase, an enzyme which promotes the conversion of isoleucine and trna to isoleucyl-trna. Prevention of these enzymes from functioning properly Advanced Journal of Pharmacie and Life science Research 19
results in the inhibition of bacterial protein and RNA synthesis. It shows protein binding 75 and half-life 20-40 min [1-4]. Literature survey reveals few spectroscopic, HPLC methods for the estimation of Mupirocin are reported. UV spectrophotometric method for the estimation of mupirocin calcium has been reported [5]. HPLC method for the measurement of mupirocin concentrations in both skin layers and percutaneous samples has been reported [6]. Another HPLC method for estimation of Mupirocin in PEG bases is also reported [7]. Stabilityindicating RP-HPLC [8], Liquid Chromatography-Tandem Mass Spectrometry [9] are reported for the estimation of mupirocin. However there is no method reported for stress degradation of mupirocin by UV spectrophotometry. Materials and Methods: Material Pure samples: Mupirocin was kindly supplied by Glenmark Pharmaceuticals Ltd., Nashik, Maharashtra, India. Marketed Formulation Bactroban (Marketed by GSK) was purchased from an open market for this study which contains Mupirocin 2w/w Instrumentation and chemicals All absorbance measurements were done with Systronics double beam UV- Spectrophotometer 2201 with 10mm matched quartz cell and Borosil glass wares were used for the study. All weighing were done on Shimadzu AY 220 electronic balance, Clean Ultrasonicator (Oscar microclean-103) was also used during the analysis. All the chemicals and reagents used were of analytical grade (AR) procured from Thomas Baker (chemicals) Pvt. Ltd. Selection of Solvent Solution of mupirocin (10µg/ml) was prepared in different solvents like methanol, ethanol, water. These solutions were scanned in UV Region (200-400 nm) and maximum absorbances were determined for these solutions Preparation of solutions: A primary stock solution of 1mg/ml of mupirocin was prepared in ethanol. The standard solution was prepared by dilution of the primary stock solution with ethanol to obtain working standard of conc. 100 µg/ml. Scanning and Determination of Maximum Wavelength (λmax) In order to ascertain the wavelength of maximum absorption (λmax) of the drug, different solutions of the drugs (10µg/ml, 20µg/ml, 50µg/ml, 100µg/ml) in ethanol was scanned using spectrophotometer within the wavelength region of 200 400 nm against ethanol as blank. The absorption curve showed characteristics absorption at 222 nm for Mupirocin and the absorbance maxima shown in following. (Figure 1) Method Validation 1. Linearity From the Std Stock (100µg/ml) solution, 1, 2, 3, 4 and 5ml were transferred in a series of 10ml volumetric flasks. The volume was made up to the mark with ethanol to obtain the conc. of 10, 20, 30, 40 and 50µg/ml. The Advanced Journal of Pharmacie and Life science Research 20
Adv J Pharm Life sci Res, 20175; ;2:19-28 ISSN 24543535 (On-line) correlation coefficient (r 2 ) of least square linear regression for mupirocin was calculated. (Figure 2) Figure 1: Absorption maxima of mupirocin Figure 2: Calibration Curve of Mupirocin (10-50 µg/ml) at 222 nm Advanced Journal of Pharmacie and Life science Research 21
2. Range: The range of analytical method was decided from the interval between upper and lower level of calibration curves by plotting the curve. 3. Accuracy: Recovery study was carried out by standard addition method by adding the known amount of mupirocin (working standard) to the preanalyzed sample at three different concentration levels i.e. 80, 100, and 120 of assay concentration and percent recoveries were calculated. (Table 1) From the 100 µg/ml Sample Stock solution 1ml was transferred to four different 10ml volumetric flasks separately along with 0, 0.8, 1.0, 1.2ml from the 100 µg/ml Std Stock solution. The volume was made up to the mark with ethanol and respective absorbances were noted, from the above data percent recoveries were calculated. (Table 2) 4. Precision: The precision of the proposed method was ascertained by determination of six replicates of same concentrations of sample and standard for method precision and system precision. Both intraday and interday precisions were carried out. 5. Limit of detection: Detection limit was determined based on the standard deviation of absorbances of same concentrations i.e. standard solution of mupirocin (30 µg/ml) prepared six times and LOD calculated by LOD = 3.3(SD/S) Where, SD- Standard deviation; S- Slope of Curve 6. Limit of Quantitation Quantitation limit was determined based on the standard deviation of absorbances of same concentrations i.e. standard solution of mupirocin (30 µg/ml) prepared six times and LOQ calculated by LOQ = 10(SD/S) Where, SD- Standard deviation; S- Slope of Curve 7. Robustness: Robustness of the method was determined by carrying out the analysis at 2 different temperatures i.e. at room temperature (29 0 c) and 24 0 c. Respective absorbances were noted and the result was indicated by RSD. Assay of mupirocin in ointment formulation: An accurately weighed quantity of ointment equivalent to about 10 mg of mupirocin dissolved in 10 ml of ethanol, sonicated for 10 min and filtered through whatman filter paper No. 41. 1ml of this solution was diluted to 10 ml with ethanol. 3 ml of resulting solution diluted to 10ml with ethanol. The absorbance of this solution was determined to calculate the amount of mupirocin in ointment formulation. (Table 3) Stress Degradation Study: Acid degradation: Solution for acid degradation study was prepared in 0.1 N HCl and the resultant solution were refluxed for 5 hr at 70 0 C in water bath. The specific amount of solution (0.2 ml) was taken out every hour (0-5 hrs) and the volume was made up to 10 ml with methanol. After this, the absorbance was measured by scanning the prepared solution. (Table 4) Advanced Journal of Pharmacie and Life science Research 22
TABLE 1: SUMMARY OF UV SPECTROPHOTOMETRIC METHOD Sr.No. Parameters Values 1. Beer s law limit (µg/ml) 10-50 2. Regression equation (y = mx+c ) y = 0.020x 3. Correlation coefficient (r 2 ) 0.999 4. Slope (m) 0.020 5. Intercept (c) 0.0 7. Linearity 0.999 8. Limit of Detection (µg/ml) 0.06 9. Limit of Quantitation (µg/ml) 0.2 Precision (RSD) 10. -Intraday 0.1 -Interday 1.0 11. Accuracy ( recovery) 98-100 12. Percent mean recovery for mupirocin ointment 100.4 TABLE 2: RECOVERY STUDY DATA OF MUPIROCIN Level of recovery Abs Amount recovered (µg/ml) mean recovery 80 0.454 22.7 98.12 100 0.496 24.8 99.5 120 0.535 26.7 99.1 TABLE 3: ASSAY OF FORMULATION Concentration of formulation Amount estimated label claim 50 µg/ml 50.2 µg/ml 100.4 Advanced Journal of Pharmacie and Life science Research 23
TABLE 4 : STABILITY RESULTS FOR ACID DEGRADATION Time i Hrs. Abs conc. Assay of degraded degradatio n Analyte at o h.4315 21.57 100 0 Analyte at 1 h 0.4109 20.54 95.22 4.77 Analyte at 2 h 0.3633 18.16 84.19 15.8 Analyte at 3 h 0.3332 16.66 77.23 22.76 Analyte at 4 h 0.3039 15.19 70.42 29.57 Analyte at 5 h 0.2887 14.14 65.55 34.44 Alkali degradation: Solution for alkali degradation study was prepared in 0.1 N NaOH. To make soluble the drug, few drops of methanol was added. Then, this solution was refluxed for 5 hr at 70 o c in water bath. Initially at 0 hr take 0.2 ml of this solution and the volume was made up to 10 ml with methanol and then, withdrawing the specific amount of solution every hour. After this, the absorbance was measured by scanning the prepared solution in a UV spectrophotometer. (Table 5) Neutral degradation: Solution for neutral degradation study was prepared in double distilled water. To make soluble the drug, few drops of methanol was added. Then, this solution was refluxed for 5 hr at 70 o c in water bath. Initially at 0 hr take 0.2 ml of this solution and the volume was made up to 10 ml with methanol and then, withdrawing the specific amount of solution every hour. After this, the absorbance was measured by scanning the prepared solution. (Table 6) Oxidation with H 2 O 2 Solution for oxidation degradation study was prepared in 3 H 2 O 2. To make soluble the drug, few drops of methanol was added. Then, this solution was placed in cupboard for 5 hr. Initially at 0 hr take 0.2 ml of this solution and the volume was made up to 10 ml with methanol and then, withdrawing the specific amount of solution every hour. After this, the absorbance was measured by scanning the prepared solution. (Table 7) Thermal degradation: About 10 mg of mupirocin was taken in cleaned petridish and exposed to dry heat at 70 C in an oven for 5 hr. Then a solution of 20 µg/ml with methanol was prepared from 0 to 5 hrs. After this, the absorbance was measured by scanning the prepared solution. (Table 8) Advanced Journal of Pharmacie and Life science Research 24
TABLE 5 : STABILITY RESULTS FOR ALKALI DEGRADATION Time in Hrs. Abs conc. Assay of degraded degradation Analyte at o h 0.3977 19.88 100 0 Analyte at 1 h 0.3719 18.59 93.51 6.48 Analyte at 2 h 0.2941 14.7 73.94 26.05 Analyte at 3 h 0.2345 11.72 58.95 41.04 Analyte at 4 h 0.2128 10.64 53.52 46.47 Analyte at 5 h 0.1758 8.79 44.21 55.78 TABLE 6 : STABILITY RESULTS FOR NEUTRAL DEGRADATION Time in Hrs. Abs conc. Assay of degraded Degradation Analyte at o h 0.4472 22.36 100 0 Analyte at 1 h 0.4259 21.29 98.34 1.66 Analyte at 2 h 0.4129 20.64 92.3 7.7 Analyte at 3 h 0.4112 20.56 91.94 8.06 Analyte at 4 h 0.3941 19.7 85.87 14.13 Analyte at 5 h 0.3716 18.58 83.09 16.9 TABLE 7 : STABILITY RESULTS FOR OXIDATIVE DEGRADATION Time in Hrs. Abs conc. Assay of degraded degradation Analyte at o h 0.4107 20.53 100 0 Analyte at 1 h 0.4004 20.02 97.51 2.48 Advanced Journal of Pharmacie and Life science Research 25
Analyte at 2 h 0.38 19 92.54 7.46 Analyte at 3 h 0.3375 16.87 82.17 17.82 Analyte at 4 h 0.3205 16.02 78.03 21.97 Analyte at 5 h 0.3037 15.18 73.94 26.05 TABLE 8 : STABILITY RESULTS FOR THERMAL DEGRADATION Time in Hrs. Abs conc. Assay of degraded Degradation Analyte at o h 0.4002 20.01 100 0 Analyte at 1 h 0.3819 19.09 95.41 4.59 Analyte at 2 h 0.3565 17.82 89.05 10.95 Analyte at 3 h 0.3129 15.64 78.16 21.84 Analyte at 4 h 0.2904 14.52 72.56 27.44 Analyte at 5 h 0.2562 12.81 64.01 35.99 TABLE 9 : STABILITY RESULTS FOR PHOTOLYTIC DEGRADATION Time in Hrs. Abs conc. Assay of degraded Degradation Analyte at o h 0.4086 20.4 100 0 Analyte at 1 h 0.352 17.66 85.56 14.14 Analyte at 2 h 0.3312 16.56 81.17 18.82 Analyte at 3 h 0.3143 15.7 78.39 21.6 Analyte at 4 h 0.308 15.4 76.84 23.15 Analyte at 5 h 0.3055 15.27 76.19 23.8 Advanced Journal of Pharmacie and Life science Research 26
TABLE 10 : SUMMARY OF STRESS DEGRADATIONS RESULTS Stress Condition Time (Hours) Assay of Degraded Alkaline Degradation 5 hrs. 44.21 Acidic Degradation 5 hrs. 65.55 Oxidative Degradation 5 hrs. 73.94 Neutral Degradation 5 hrs. 83.09 Photolytic Degradation 5 hrs. 76.19 Thermal Degradation 5 hrs. 64.01 Photolytic degradation: About 10 mg of mupirocin was taken in cleaned Petridis and exposed to dry heat at 70 C in an oven for 5 hr. Then a solution of 20 µg/ml with methanol was prepared from 0 to 5 hrs. After this, the absorbance was measured by scanning the prepared solution. (Table 9) Results and Disscusion: Mupirocin being UV absorbing has been successfully employed for its quantitative determination by UV spectrophotometric method. Being freely soluble in ethanol, stock solutions and working standards were made in ethanol. The λ max of the drug for analysis was determined by taking scan of the drug sample solution in the entire UV region (200-400 nm). The correlation coefficient of the standard curves for the drug was 0.999 (Graph). The commercial dosage form showed 100.4 recovery by this method which was within the specified limits of content uniformity. The proposed method showed absorption maxima at 222 nm and obeyed Beer s law in the concentration range of 10-50 µg/ml. The limit of detection (LOD) was found to be 0.06 µg/ml and limit of quantification (LOQ) to be 0.2 µg/ml. The percentage recovery value indicates no interference from excipient used in formulation. The low value of percentage relative standard deviation shows that the developed method was precise. All statistical data prove validity of proposed method, which can be applied in industries for routine analysis of mupirocin from ointment. A simple, precise, rapid and accurate stability indicating UV method for determination of mupirocin from pure and its dosage forms has been developed and validated. Recoveries in formulation were in good agreement with their respective label claims. The proposed methods can be used for the routine determination of mupirocin in bulk and pharmaceutical dosage forms. The proposed UV- Spectrophotometric method has been evaluated over the linearity, accuracy, precision, robustness, LOD and LOQ. The proposed method was found to be convenient and effective for the quality control and stability studies of mupirocin. The results obtained from the stress testing show that the drug substance is particularly unstable under acidic and alkaline condition as compared to neutral, oxidation, thermal and photolytic degradation. Therefore, care should be taken in the manufacturing process and during storage of this in Advanced Journal of Pharmacie and Life science Research 27
order to avoid degradation, because if the drug is degraded could result in diminution of the therapeutic activity and safety. ACKNOWLDGEMENT: The authors extend their sincere thanks to Glenmark Pharmaceutical Ltd. Nasik, India for providing gift sample of pure mupirocin. References: 1. The United State Pharmacopoeia (USP30-NF25), National Publishing Philadelphia, Asian, 2007: 2690-2691. 2. Drug Bank of Mupirocin (Available from: http://www.drugbank.ca/drugs) 3. Martindale, The complete drug reference, 2009: 302 4. The Merck Index, An encyclopedia of chemical, drugs and biological, 2006: 6302. 5. Bageshwar DV, Pawar AS, Khanvilkar VV, KadamVJ: Int J Pharmacy Pharm Sci 2010; 2: 86 6. Echevarria L, Blanco J, Campanero A, Santoyo S, Ygartuay P: J chromatogr. B 2003; 796: 233 7. Porter RS, Chen TK: J.chromatogr. A 1996; 732: 399 8. Jagota NK, Stewart JT, Warren FW, John PM: J Clin Pharm Ther 1992; 17: 181 9. Haider, Lee TD, Barton JD, Coates ARM, Mantle PG: Biomarkers 2011;16: 422 10. ICH Harmonized Tripartite Guideline, Text on Validation of Analytical Procedure Advanced Journal of Pharmacie and Life science Research 28