RP-HPLC Method for the Simultaneous Estimation of and Abacavir Sulphate in Tablet Dosage Form T.Sudha, 1* V.R.Ravikumar 2 P.V. Hemalatha 2 1.* Department Of Pharmaceutical Analysis 2. Department Of Pharmacognosy, The Erode College of Pharmacy& Research Institute, Erode-638112, Tamilnadu. ABSTRACT A rapid high performance liquid chromatographic methods has been developed and validated for the estimation of and Abacavir simultaneously in combined dosage form, separation was performed on a 5µm C 18 column having dimensions (150X4.6mmid) in isocratic mode, with mobile phase containing a mixture of methanol: water (70:30, v/v/) was used. The Mobile phase was pumped at a flow rate of 1.4 ml/min and eluents were monitored at 275nm. The selected chromatographic conditions were found to separate (R t= 2.549 min) and Abacavir (R t= 3.499 min) having a resolution of 4.13 min. The method was validated in terms of linearity, accuracy, precision, and specificity, limit of detection and limit of quantification. Linearity of and Abacavir were found in the range of 2-12. The percentage recoveries for and Abacavir ranged from 99.18 ±101.00% and 99.83±100.83% respectively. The limit of detection was found to be 0.0268 and 0.0049 for Lam and Aba respectively. Limit of quantification was found to be 0.0184 and 0.0150 for Lam and Aba respectively. The proposed method can be used for the estimation of these drugs in combined dosage form. Key words: Abacavir sulphate,, RP-HPLC, Simultaneous estimation Validation. INTRODUCTION Abacavir Sulphate [1] is a nucleoside reverse transcriptase inhibitor (NRTI) with a activity against human immunodeficiency virus Type I (HIV-I). It is a given a combination with a novel drug a. [2] is a nucleoside reverse transcriptase inhibitor (NRTI) with activity against human immunodeficiency virus type I(HIV-I) and Hepatitis B. Abacavir is chemically (Aba)[1R-4-(2amino -6- cyclopropylamine) purine -9-yl]-1- cyclopent -2enyl] methanol. is chemically (Lam) 4 amino-1-[2r, 5S]-2-(hydroxymethyl)-1, 3- oxathiolan-5yl] pyrimidine-2-one. The drugs prescribed invidually, as well as multicomponent dosage forms available in the market. ISSN : 0976-285X 108
Abacavir Sulphate A number of methods have been published for the estimation of above said analytes. Spectrophotometric estimation of Abacavir sulphate [3]. Spectrophotometric estimation of [4]. in human plasma by RP- HPLC [5]. Titrimetric and spectrophotometric estimation of [6].Methods were reported for simultaneous analysis of Abacavir and in human plasma by LC/MS/MS [7].Determination of Abacavir, & Zidovudine in pharmaceutical tablets human serum and in drug dissolution studies by HPLC [8] was also reported in the literature. However no method has been reported so far, for the estimation of these drugs simultaneously in combined dosage forms. Hence in present study, a new reversed phase high performance liquid chromatography method was developed and validated for the simultaneous estimation of Lam and Aba in tablet dosage form. MATERIALS AND METHODS A Shimadzu HPLC system equipped with LC-10AVP UV- visible detector, a Luna phenomenax, C 18 (150mmX 4.6mm id particle size 5µ) column was used. The chromatographic and integrated data were recorded using winchrom soft ware system. The mobile phase consisted of methanol: water (70: 30% v/v). The flow rate was 1.4ml/min, the wave length was monitored at 275 nm and the injection volume was 20µl. Anatically pure Lam and Aba were obtained as gifted from hetero labs Hyderabad. Tablet formulation (Abamune-L) containing labeled amount of 300mg of Lam and 600mg of Aba were purchased from the local pharmacy. HPLC grade methanol and water were procured from Qualigens India limited Mumbai. Stock solutions Lam and Aba were prepared by separately dissolving 25 mg of Lam and 25 mg of Aba in 25ml of methanol. Further dilutions were made by diluting 2.5 ml mobile phase to obtain 50 solution. Working stock solutions were prepared by diluting the stock solution with mobile phase to obtain final concentration of 2, 4, 6, 8, 10& 12 of both drugs (Lam and Aba). 20µl of each solution were injected and the chromatograms were recorded. The calibration graph was plotted by using peak area against concentration. The procedure was repeated for three times to determine the limit of detection and limit of quantification The marketed formulation Abamune-L contains (300mg of Lam & 600mg of Aba). Twenty tablets were weighed accurately finely powered and mixed. The average mass per tablet was determined. The powder equivalent to 25mg of each was accurately weighed and added a minimum quantity of methanol to dissolve the substance the total volume as brought to 25ml with more methanol (1000) in a volumetric flask. The solution were sonicated for 10 minutes and then filtered through Whatmann no: 41. to separate out insoluble excipients. Collected the filterate after rejecting the first portion of the filterate. From the clear solution further dilution was made by diluting 2.5ml into 50ml with mobile phase to obtain 50. Further dilution was made by diluting 3ml to 10ml with mobile phase to obtain 6. 20µl of each solution was injected and the chromatogram were recorded. The analysis was repeated for six times. The content of the drug was calculated from the peak area recorded. ISSN : 0976-285X 109
The accuracy of the method was confirmed by recovery studies. The recovery was performed at three different concentrations (80%,100%,120%) were added to fixed amount each of the drug were determined by the proposed method further the precision of the developed method was confirmed by interday and intraday analysis. RESULTS AND DISCUSSION A simple, precise, accurate, RP-HPLC method has been developed for the estimation of Lam and Aba in bulk and in tablet formulation. A Shimadzu HPLC system with Luna phenomenax C 18 column was used for analysis optimization of mobile phase was performed based on resolution, tailing factor and peak area obtained for both Lam and Aba. The mobile phase was methanol: water (70: 30 v/v/) was gave two symmetric and well resolved peaks for both Lam and Aba. The resolution between Lam and Aba was found to be 4.13minutes which indicates good separation of both the components. The retention time for Lam and Aba were 2.259 and 3.499 respectively (Fig-1). The UV detection was carried out as 275nm as Lam and Aba showed very good absorbance at this wavelength. An chromatogram of Lam and Aba shown in fig-1.the linearity of an analytical method to its ability to elicit test results that are directly (or) by a well defined mathematical transformation proportional to the concentration of analyte samples with in a given range. The linearity of the method was observed with in the expected range demonstration its suitability for analysis. The system suitability parameters like tailing factor, asymmetric factor, number of theoretical plates were calculated and these values were compared with the standard limit as per USP [9].It was found that the values were with in the limits. The values are shown in table-1. The linearity concentrations range of 2 to 12. The correlation coefficient r 2 value (n=3) for Lam and Aba were found to be 0.9996 and 0.9998 respectively. The results were shown in table-2. The value of intercept was less than 2% of the response of 100% of the test concentration in all cases indicates functional linear relationship between the concentration of analyte and area under the peak area. The proposed method was validated as per ICH guidelines [10, 11] Limit of quantification was found to be 0.0814 and 0.0150 for Lam and Aba respectively. Limit of detection was found to be 0.0268 and 0.0049 for Lam and Aba respectively. The values were shown in table -2. Precision is the degree of reproducibility or repeatability of the method under normal operating condition. The method passed the test for repeatability as determined by % RSD [12] of the peak area of six replicate. The percentage concentration of Lam and Aba were found to 100.01±0.5440 and 99.93±0.2290. The low% RSD value indicated that the method has good precision. The results of analysis were shown in table-3. Further the precision of the developed method is confirmed by interday and intraday analysis. The result show good agreement with the label claim of the formulation. To evaluate the accuracy of the method, known amount of the pure drug was added to the previously analysed solution containing pharmaceutical formulation and the mixture was analysed by the proposed method and the recoveries were calculated. The percentage recovery of Lam and Aba were found in the range 99.18 ±101.00% and 99.83±100.83% respectively. The %RSD value for Lam and Aba were found to be 0.9413 Sand 0.5004 respectively. The low percentage RSD value indicated that there is no interference due to the excipients used in formulation. Hence the accuracy of the method was confirmed. The results were shown in table-4. Specificity ability to asses unequivocally the analyte in the presence of components they may be expected to be present in the sample matrix. CONCLUSION In the present study the attempt has been undertaken to develop most simple, economical, sensitive and accurate analytical HPLC methods for the simultaneous estimation of these drugs without their prior separation. The method was validated and found to be simple, sensitive, accurate and precise percentage of recovery shows that the method is free from interference of the excipients used in the formulation. Therefore the proposed method can be used for routine analysis of and Abacavir in their combined dosage form. REFERENCES [1] Indian Pharmacopoeia, vol II, Govt. of India, Ministry of Health & Family Welfare, Sector-3, RajNagar, Ghaziabed, 201002, 2007, 674. [2] Indian Pharmacopoeia, vol II, Govt. of India, Ministry of Health & Family Welfare, Sector-3,RajNagar, Ghaziabed,201002, 2007, 1276 [3] Ramanamurthy, Hiremath, S., AppalaRaju, N., The Indian pharmacist 2006, l5, 91-92. [4] Shalini,S., Shanooja,V.P., Abdul Jameel,S., Basima., Digest Journal of Nanomaterials and Biostructure 2009, 4(2), 357-360 [5] Kano, E.K., DosReisSerra, C.H., Koono, E.E., Andrade s, Int J Pharm, 2005, 297, 73-79. [6] Basavaiah, K., Somasekar, Indian Journal of chemical technology. 2006, 13, 7-11. [7] Noel, A.Gomes, Ashutosh, Journal pharm biomed anal, 2008, 48(3), 918-26. [8] Savaser, A., Goraler, S., Chromatagraphia, 2007, 65, 5-6. ISSN : 0976-285X 110
[9] The United States of Pharmacopoeia, National formulary USP23, Asian Edition, Rockville MD, United States Pharmacopoeial convension,inc,1995,a-125 [10] International conference on Harmonization guidance for Industry In: Q2A Text on validation of Analytical methods. Switzerland, IFPMIA: 1994, 1, 4. [11] International conference on Harmonization guidance for Industry In: Q2B Text on validation of Analytical methods. Switzerland, IFPMIA: 1996, 1-8. [12] Gupta, S.C., Kapoor, V.K., Fundamentals of mathematical statistics. 9 th edition. Sultan chand and sons. New Delhi, 1994, 2.6, 3.2-3.8 FIG.1: A TYPICAL HPLC CHROMATOGRAM OF LAM AND ABA TABLE-1 SYSTEM SUITABILITY PARAMETERS Values Parameters Abacavir Asymmetric factor Tailing factor Capacity factor HETP THPL 0.9090 0.9523 2.13 0.0678 2218.0 0.9000 1.0000 3.85 0.1316 1139.11 Resolution 4.13 minutes ISSN : 0976-285X 111
TABLE-2 METHOD VALIDATION PARAMETERS Values Parameters Abacavir Linearity range Correlation coefficient Regression equation Slope Intercept Limit of detection Limit of Quantification 2-12 0.9996 Y=656789.2X120048.2 656789.2 120048.2 0.0268 0.0184 2-12 0.9998 Y=564930.8X+29627.68 564930.8 29627.68 0.0049 0.0150 TABLE-3: ASSAY OF LAM AND ABA Formulation Drugs Label claim found (mg) %purity Average % SD %RSD Abamune-L 300mg 301.42 300.21 299.33 300.69 301.45 297.14 100.47 100.07 99.77 100.23 100.48 99.04 100.01 0.5440 0.7173 Abacavir 600mg 601.06 600.07 597.26 599.97 600.04 598.92 100.17 100.11 99.54 99.99 100.00 99.82 99.93 0.2290 0.6271 ISSN : 0976-285X 112
TABLE-4: RECOVERY STUDIES OF LAM AND ABA Label claim Present Added Found Recovered % Recovery Average % SD %RSD Lam 300mg 2.44 2.99 3.64 5.46 6.01 6.66 2.42 3.66 99.18 101.00 100.54 100.24 0.9436 0.9413 Aba 600mg 4.81 5.99 7.23 7.83 9.01 10.25 4.85 5.98 7.26 100.83 99.83 100.41 100.35 0.5024 0.5004 ISSN : 0976-285X 113