Journal of Pharmaceutical and Bioanalytical Science

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1 Journal of Pharmaceutical and Bioanalytical Science Research Article Stability-Indicating RP-HPLC Method for Estimation of Erdosteine and its Degradation Products in Pharmaceutical Dosage Form Anita P. Ayre * and Harshal A. Pawar Dr. L H Hiranandani College of Pharmacy, CHM Campus, Opp. Ulhasnagar Railway station, Ulhasnagar ,Maharashtra,India Received: 12-May-2012; Revised : 20-May-2012; Accepted : 22-May-2012 ; Published online: 01-July-2012 Abstract A simple, selective, precise and accurate stability-indicating high-performance liquid chromatographic method for analysis of Erdosteine and its degradation products in tablet dosage form was developed and validated. The stability indicating ability of the method was determined by performing forced degradation studies which indicated susceptibility of the drug under acidic and neutral hydrolytic conditions. Separation of drug from its degradation products was achieved by isocratic elution using a mobile phase consisting of Acetonitrile : 0.01M Phosphate buffer, ph 5.8 (80:20 v/v)through a HiQ Sil C 18 column (250 X 4.6mm, 5 µm) at a flow rate of 1ml min -1 with UV-detection(270nm) at ambient temperature. Validation of the developed HPLC method was performed using an external standard. The method was found to be linear over a range of 5-50µg ml -1 and demonstrated to be precise, accurate, specific and robust. The results obtained verify that the method can be used for its intended purpose. Key words: Erdosteine, Validation, Degradation product, Mucolytic, RP-HPLC INTRODUCTION A stability-indicating test method is one that accurately and selectively quantifies intact drug in the presence of degradation products and other solution components. The ICH guidelines Q1A on Stability Testing of New Drug Substances and Products emphasizes that the testing of those features which are susceptible to change during storage and are likely to influence quality, safety and/or efficacy must be done by validated stabilityindicating assay methods. Hence these guidelines require conduct of forced degradation studies under a variety of conditions like ph, light, oxidation, dry *Corresponding author Ms.Anita P. Ayre, Dr. L H Hiranandani College of Pharmacy, CHM Campus, Opp. Ulhasnagar Railway station, Ulhasnagar ,Maharashtra,India heat etc. and separation of drug from its degradation product [1]. Erdosteine (±1S-(2-[N-3-(2-oxotetrahydro thienyl)]acetamido)-thioglycolic acid) is a mucolytic agent and is official in Martindale[2]. Erdosteine is a homocysteine-derived thiol derivative (Fig. 1).It Modulates mucus production, viscosity and increases mucociliary transport, thereby improving expectoration and thus it shows mucolytic and antitussive activity[3,4]. Literature survey revealed that no RP-HPLC method has been reported for estimation of Erdosteine and its degradation product in pharmaceutical dosage form [5-7]. Hence, it was attempted to develop a stability-indicating RP-HPLC method for determination of Erdosteine and its degradation product in the tablet formulation. Available online on 2

2 concentration of 1mg/ml. Standard solution was then prepared by dilution of the stock solution with mobile phase to give a solution of concentration 10 g/ml. MATERIALS AND METHODS Materials Fig.1. Chemical structure of Erdosteine Erdosteine, an active pharmaceutical ingredient (API) with a purity of 99.2% w/w was obtained from Macleods Pharmaceuticals Ltd., Palghar as a gift sample. It was used without further purification. All chemicals and reagents used were of HPLC/AR grade and were purchased from S. D. Fine Chemicals Ltd., Mumbai. HPLC Equipment The HPLC system employed was JASCO (2000 series) comprising of JASCO PU-2080 plus intelligent pump, JASCO UV-2075 plus intelligent detector and Rheodyne sample injector fitted with 100 l capacity loop. The software used was Borwin version 1.5. Chromatographic Conditions The chromatographic resolution was achieved on a reverse phase HiQ Sil C18 column (250 X 4.6mm, 5µm) column, using a mobile phase consisting of Acetonitrile : 0.01M Phosphate buffer, ph 5.8 (80:20 v/v) pumped at a flow rate of 1ml/min. It was filtered through 0.45 m Millipore filter and was degassed by sonicating for 20mins. The column was maintained at 25 C and the wavelength was monitored at 270nm. The injection volume was 100 l. Preparation of Standard Solution A stock solution of Erdosteine reference standard was prepared in methanol at a Forced Degradation Studies of Erdosteine All degradations were done at a drug concentration of 1mg/ml. For acid degradation, the drug was dispersed in 1N HCL solution and refluxed for 2h at 1000C. For degradation under alkaline condition, the drug was dissolved in 1N NaOH solution and refluxed for 8h at 1000C. For oxidative degradation, the drug was kept in contact with 30% V/V hydrogen peroxide at ambient temperature in the dark for 24h. The drug powder was kept in an oven at 800C for 48h to study thermal degradation. Also, the drug was dispersed in water and refluxed for 4h at 1000C for neutral hydrolysis. The photolytic degradation study of the drug was performed by exposing the drug to direct sunlight for 48h. After each study the mixtures were diluted with the mobile phase to a final concentration of 5µg/ml in terms of the drug and subjected to chromatographic analysis. Method Validation Linearity The stock solution of Erdosteine reference standard having a concentration of 1mg/ml was diluted suitably with the mobile phase to get concentrations of 5, 10, 20, 30, 40, and 50µg/ml. These were analyzed in three replicates to get the drug peak areas that were then plotted against concentration and subjected to least square linear regression analysis. System Suitability Testing System suitability testing is used to verify that the resolution and reproducibility of the system are adequate for the analysis to be performed. Parameters such as theoretical plates, tailing factor, resolution and Reproducibility (%RSD for area of Available online on 3

3 six replicates) are determined and compared against the specifications. Accuracy and Precision Accuracy and precision were evaluated at three levels by determining % recovery and % relative standard deviation (% RSD) respectively for the results of recovery of Erdosteine from pre-analyzed tablet powder spiked with Erdosteine at three levels. The analysis was performed in triplicate at each level over three days and intra-day and inter-day variations were evaluated. Specificity The specificity of the method was inferred from the resolution of the drug peak from that of the possible degradation products produced in the forced degradation experiments. Specificity was also studied by analyzing Placebo before and after subjecting to forced degradation. Lack of interfering peaks at the retention time of the drug was taken as indication of specificity. Robustness Small changes were made in the mobile phase composition and effects of these changes were observed on the chromatographic behavior of Erdosteine in presence of degradation products and excipients. The effects on retention time, resolution and peak shape were observed. RESULT AND DISCUSSION Development and optimization of stabilityindicating HPLC method An isocratic method was found suitable to optimize the separation of major degradation products formed under various stress conditions. The best resolution was obtained with an initial run of a mixture of Acetonitrile: 0.01M Phosphate buffer, ph 5.8 (80:20 v/v); the retention time was observed to be 4.52 min (Fig. 2). Fig. 2. Representative chromatogram of Erdosteine standard RT: 4.52 min. Degradation behaviour When samples obtained after forced degradation were subjected to chromatographic analysis, it was found that there was no change in the peak area or appearance of peaks of degradation products in case of samples subjected to degradation under alkaline, oxidative, dry heat and photolytic conditions. However, there was reduction in drug peak area and appearance of additional peaks in case of samples subjected to degradation under acidic and neutral hydrolytic conditions. (Fig. 3 and Fig. 4). The results of forced degradation studies are summarised in Table 1. Available online on 4

4 Fig. 3. Representative chromatogram of acid treated Erdosteine (1N HCL, refluxed for 2h at C) Peak 1: degradant one, RT: min; Peak 2: degradant two, RT: min; Peak 3: Erdosteine, RT: min. Fig. 4. Representative chromatogram of Erdosteine after hydrolysis (Refluxed in H 2 O for 4h at C). ) Peak 1: degradant one, RT: min; Peak 2: degradant two, RT: min; Peak 3: Erdosteine, RT: min; Peak 4: degradant three, RT: min. Table 1: Summary of results of forced degradation studies Conditions Drug peak area at zero time (µv.sec) Drug peak area of stressed sample (µv.sec) Retention time(s) of degradation products (mins) % Degradation Refluxed in 1N NaOH for 8h at C No Degradant No Degradant Refluxed in 1N HCL for 2h at C & Refluxed in H 2 O for 4h at C , & Exposed to direct sunlight for 48h No Degradant No Degradant Kept in 30% H 2 O 2 for 24h No Degradant No Degradant Kept in oven at 80ºC for 48h No Degradant No Degradant Available online on 5

5 Method Validation Linearity Six-point calibration curve was obtained in the concentration range of 5-50µg/ml for Erdosteine based on three independent determinations performed at each concentration. The response for the drug was linear and the calibration equation was y = 60875x with R 2 = as shown in Fig. 5. Fig.5. Calibration curve for Erdosteine System Suitability Testing The results of system suitability and system precision are presented in Table 2. Accuracy and Precision The accuracy results showed excellent recovery at each added concentration ranging from %. Method precision was within the limits of acceptance criteria showing %RSD value less than 2. The results of accuracy and precision are summarised in Table 3. Table 2: System Suitability Testing and System Precision Parameter System Precision Retention Time ( in minutes) of Erdosteine Resolution of Erdosteine from degradants Value % RSD for six replicate injections of standard : < 2 About 4.5 >2 Theoretical Plates >2000 Tailing Factor <1.5 Specificity The HPLC chromatograms recorded for the blank and placebo exposed to the degradation conditions showed no peaks at the retention time of Erdosteine and also the representative chromatograms of stressed samples under various stress conditions (Fig. 3 & Fig. 4) showed that Erdosteine was well resolved from its degradation products, indicating the specificity of the method. Robustness There was no significant change in the retention time, resolution and peak shape of Erdosteine and its degradation products after introducing small changes in the mobile phase composition. Table 3: Intra and inter-day accuracy and precision through recovery study Day Amount added (% w/w) Amount found (% w/w) % Mean Recovery % RSD Available online on 6

6 CONCLUSION The isocratic RP-HPLC method developed for quantitative determination of Erdosteine and its degradation products in pharmaceutical dosage form is rapid, precise, selective and robust. The validation of method shows satisfactory data for the parameters tested. The method well separated the drug from its degradation products. The developed method is thus stability indicating and can be used successfully for the estimation of Erdosteine and its degradation products in pharmaceutical dosage form. ACKNOWLEDGEMENTS The authors wish to thank Macleods Pharmaceuticals Ltd. for providing gift sample of Erdosteine. REFERENCES 1. ICH Harmonized Triplicate Guideline: Stability Testing of New Drug Substances and Products Q1A (R2), ICH Steering Committee, Step 4 of ICH process, Feb Martindale: The complete drug reference, 31st edition, KL Dechant, S.Noble, Erdosteine, Drugs, (1996),52, Committee, Step 4 of ICH process, Feb R.W.Negro, Erdosteine: Antitussive and Antiinflammatory Effects, Lung, 186, (2008), S Savu, M Mitrea, L Silvestro, C Mancini, HPLC with on-line mass spectrometry detection application to elucidate Erdosteine metabolism: preliminary study, Int J Clin Pharmacol Ther., 38(8), (2000): N Muramatsu, T Toyo'oka, K Yamaguchi, S Kobayashi, High-performance liquid chromatographic determination of Erdosteine and its optical active metabolite utilizing a fluorescent chiral tagging reagent, R-(-)-4- (N,N-dimethylamiosulfonyl)-7-(3- aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole,j Chromatogr B Biomed Sci Appl., 719 (1-2),(1998): R.K. Nanda, J. Gaikwad, A. Prakash, Spectrophotometric estimation of Erdosteine, International Journal of Pharm Tech Research,1(3), (2009): Copyright: 2012 Anita P. Ayer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Available online on 7