INTRODUCTION:

Abacavir sulphate¹ (Abs) (is chemically {(1S, 4R)-4[2-amino – 6-(cyclopropylamino) 9H – Purin – 9-yl)cyclopent -2- enyl} methanol sulphate (Fig.1). The Abs is used in the treatment of HIV as single drug or in combination with other NRTI’S° Abs is metabolised by alcohol dehydrogenase and glucuronyl transferase with renal excretion of metabolites 16 % recovered in stool and 1% unchanged in urine². Oral bioavailability is 83 % (63 - 110), half - life is 1.0 (0.8 – 1.3) hours. The active metabolite Carbovir triphosphate acts as potent HIV - I NRTI. Abs undergoes intracellular phosphorylation by enzyme that do not phosphorylate other FDA approved NRTI. It is monophosphorilated by a pathway involving adenosine phosphotransferrase and is then di and tri phosphorilated, Carbovir triphosphate accumulates and has an intracellular half life of 3 hours the oral bioavailability is high with or without food, Abs is partially metabolised by alcohol dehydrogenase (to form 5’ carboxylic acid) and Glucuronidation (to form 5’ glucuronide)³. A literature survey revealed that several analytical methods were reported for the determination of abacavir sulphate in biological fluids including High Performance Liquid Chromatography (HPLC), IR - absorption spectroscopy^{4, 5- 6}.

No UV spectrometric methods are reported for the estimation of Abs⁷. The aim of the present work is to develop and validate an accurate, specific, economical and reproducible UV Spectrophotometric method for determination for Abs tablets. Drug was found to be freely soluble in 15 % phosphoric acid and distilled water hence this solvent was chosen for proceeding study.

Fig.1. Chemical Structure Abacavir sulphate

MATERIALS AND METHOD:

All the absorbance measurements were made on Systronics – 2201 UV/Visible double beam spectrophotometer with 10 mm matched quartz cells. Whatman filter paper No. 41 was used to filter the solutions. The Abs standard was provided by gift sample from Apotex Research Laboratories, Bangalore. All chemicals were of analytical grade. Commercially available two formulations [Abamune (cipla), Abavir (Hetero Health Care)] were procured from the local market.

Optimization of Parameters

It was fond that abacavir sulphate has λ max at 283 nm by scanning of diluted solution. The selection criteria were based on the minimum strength that gives the maximum absorbance.

Fig.2. Absorption Maximum of Abacavir sulphate

Preparation of Standard Solution:

Stock standard solution of abacavir sulphate was prepared by dissolving 50 mg of the abacavir sulphate in about 10 ml of 15 % phosphoric acid (H₃PO₄), further diluted, and made upto 100 ml with distilled water, to give stock concentrations of 500 µg /ml.

Preparation of calibration curve:

Suitable aliquots of the standard solution of abacavir sulphate (1.0 - 6.0 ml) were taken into 10 ml volumetric flasks, and volume was then made up to the mark with distilled water to prepare a series of standard solutions containing 5-30 µg/ml. Absorbance of the solution was measured at 283 nm against blank. A calibration curve was constructed by plotting average peak are against concentration and regression equation was computed.

Fig.3. Calibration Curve for Abacavir sulphate

Estimation of abacavir sulphate in tablets:

Twenty tablets were taken and weight was calculated. The average weight of the tablets were found out, The quantity of powder equivalent to 300 mg of abacavir sulphate were transferred to 100 ml volumetric flask and mixed with 10 ml of 15 % phosphoric acid (H₃PO₄) and made upto 50 ml with distilled water and the solution was sonicated for 10 minutes. Then volume was made up to 100 ml with the distilled water. The solution was filtered through Whatmann filter paper No: 41. From the filtrate, 1 ml was transferred to 100 ml volumetric flask and the volume was made up to 100 ml with distilled water, 10 ml was transferred to 50 ml volumetric flask and the volume was made up to 50 ml with distilled water. The reference standard of abacavir sulphate was also prepared in the same way at each concentration level and absorbance was noted at 283 nm against blank.

Table 1: Summary of Optical, Regression and Validation Parameters.

Linearity range (µg/ml)	5 -30 µg/ml
λ max	283 nm
Beer’s law limit (µg/ml)	5-30 µg/ml
Regression equation	Y = 0.032 × -0.0055
Slope	0.032
Intercept	0.0294
Correlation coefficient	0.9998
Accuracy (%recovery)	99.46 %
LOD (µg/ml)	0.5671
LOQ (µg/ml)	1.7187

METHOD VALIDATION:

Accuracy of the method was determined by the recovery studies in the tablets formulations of the abacavir sulphate. Recovery studies were carried out by addition of known quantities of standard drug solution to pre-analyzed sample at three different concentrations. Also the experiment was repeated three times in a day to determine intra-day precision and on three different days to determine inter-day precision. The percentage relative standard deviation (%RSD) was calculated at each concentration level. The values of method validation are given in Table. 2. The proposed method obeys beer’s law in the concentration range of 5-30 µg/ml. In this method, the correlation coefficient (r²) was found to be 0.9998, the slope was 0.0294. Limit of Detection (LOD) and Limit of Quantization (LOQ) were calculated by repeating the blank measurements six times at 283 nm. LOD and LOQ for abacavir sulphate were found to be 0.5671 µg/ml and 1.7181 µg/ml respectively. It can be concluded that the developed method is sensitive.

Table: 2. Precision of Proposed Method

Concentration (µg/ml)	Concentration found on
	Intra-day		Inter-day
	S.D	%RSD	S.D	%RSD
1 2 3	1.9344 4.3019 3.0262	0.6395 1.440 1.0086	3.4526 3.6913 3.8942	1.1569 1.2304 1.3080

RESULTS AND DISCUSSION:

The proposed method is simple and precise and do not suffer from any interference due to common excipients of tablets. Beer’s law is obeyed in concentration range of 5-30 µg/ml. Method were validated in terms of accuracy and precision. The accuracy of the methods was proved by performing recovery studies in the commercially available formulations. Values greater than 99% indicate that proposed method is accurate for the analysis of drug. The precision of the method was checked in terms of Inter-day and Intra-day, where methods were repeated on three different days and also repeated on three different time periods in same day. The results given in Table 2 shows % RSD of less than 2 % at each level clearly indicate that the method is precise enough for the analysis of the drug. Summary of Optical, Regression and Validation Parameters are shown in Table 1. Commercial formulation of abacavir sulphate tablets was successfully analyzed by the proposed and reference methods. The values obtained by the proposed and reference methods are presented in Table 3.

Table 3: Analysis of Abacavir sulphate in Tablet Formulations

S. No

Brand

Labeled claim

(mg/tablet)

Amount found

(mg/tablet)

% Reco

very

% RSD

Abamune

Abavir

300

300.48

300.66

100.16

100.22

1.7019

1.2317

ACKNOWLEDGEMENT:

Our sincere thanks to Dr. V. Ganesan, M.Pharm., Ph.d., Principal, Er. P. Ganapathi, President, and Mr. A. Natarajan, Secretary and Correspondent, The Erode College of Pharmacy and Research Institute, Veppampalayam, Erode -638 112, Tamilnadu.

REFERENCES:

1. The Merck Index 14^th Edition 2006, Merck Research Laboratories page No. 1

2. The Pharmacological Basis of Therapeutics Joel G. Hardman. LEE .E. Limbird 10^th Edition International Edition Page No. 1924.

3. The Pharmacological Basis of Therapeutics 10^th International Edition – Alfred Goodman Gilman Page No. 1359 -1360.

4. Indian Pharmacopoeia 2007 volume I - Page No. 143,245,673.

5. Chiral LC for Separation of the Enantiomers of Abacavir Sulfate - U. Seshachalam et al Chromatographia 2006, 64, December Page No. 11 – 12.

6. Determination of Abacavir, Lamivudine and Zidovudine in Pharmaceutical Tablets, Human Serum and in Drug Dissolution Studies by HPLC. A. Savaser, S. Goraler, et al Chromatographia 2007, 65, March. Page No. 5 – 6.

7. Spectrometric Estimation of Abacavir Sulphate in Pharmaceutical Formulations –N. Appala Raju et al - E-J chem. E journal of Chemistry vol.5 No.3, July 2008 Page No. 511 -514.

Received on 05.04.2010 Modified on 11.05.2010

Research J. Pharm. and Tech.3 (4): Oct.-Dec.2010; Page 1122-1124