INTRODUCTION:

Zidovudine (INN) or azidothymidine (AZT) is a nucleoside analog reverse transcriptase inhibitor (NRTI), a type of antiretroviral drug. It is a synthetic drug with pyrimidine nucleoside analogue active against HIV-1, AIDS and pre- AIDS. The chemical name of Zidovudine is 1-(3-azide-2, 3-di deoxy-β-D-ribofuranosyl)-5-methyl Pyrimidin-2, 4(1H, 3H) – dione. Zidovudine also has been referred to as 3′azido-3′-deoxythymidine. It has a molecular formula of C₁₀H₁₃N₅O₄ and a molecular weight of 267.24 g/mol. It has the structural formulaand shown in Fig: 1.

Zidovudine is a white to beige, odorless, crystalline solid and it is soluble in ethanol (95%), sparingly soluble in water^1-3. The drug is officially listed in United States of Pharmacopiea⁴. Several analytical methods that have been reported for the estimation of Zidovudine in biological fluids or pharmaceutical formulations include UV-visible Spectrophotometry, High Performance Liquid Chromatography and HPTLC^5-13. The objective of the work is to develop simple, accurate, precise and economic RP-HPLC method with lesser run time and retention time to estimate the Zidovudine in bulk and pharmaceutical dosage forms.

MATERIALS AND METHOD:

A Shimadzu HPLC model containing LC-20 AT pump, variable wavelength programmable UV/VIS detector and Rheodyne injector was employed for the investigation. All the chemicals used in the investigation were of HPLC grade. The chromatographic analysis was performed on a Venusil XBP C-18 (250 X 4.6mm, 5 mm) column. The mobile phase consisting of Water and Methanol in the ratio of 50:50 v/v was selected. The optimized chromatographic conditions are summarized in Table: 1. The standard solution of Zidovudine was prepared by dissolving 10 mg in 50 ml of mobile phase in 100 ml volumetric flask. The final volume was made up to the mark with mobile phase to give the concentration of 100 µg/ml. The solution was sonicated for 10 min. and filtered using Whatmann filter paper No.41 and used. The various dilutions of Zidovudine in the concentration of 25, 37.5, 50, 62.5 and 75 µg/ml were prepared. The solutions were injected using a 20 µl fixed loop in to the chromatographic system at the flow rate of 1.0 ml/min and the effluents were monitored at 266 nm. The chromatogram was recorded and shown in Fig: 2. The Zidovudine was eluted at 4.47 min. The calibration curves were constructed by plotting average peak area versus concentrations and presented in Fig: 3 and regression equations were computed. The method was extended for determination of Zidovudine in pharmaceutical dosage form. The pharmaceutical dosage forms containing 300 mg and 100 mg strengths were taken.

Twenty tablets of two brands (each containing 300 mg and 100 mg strengths respectively) were weighed and powdered. The powder equivalent to 10 mg of Zidovudine was transferred into 100 ml volumetric flask containing 50 ml of mobile phase and flask was kept for ultrasonication for 15 min and then it was diluted up to the mark with mobile phase and the solution was filtered through Whatmann filter paper No. 41. From the above solution various dilutions were made with the mobile phase, which were analyzed. The concentration of the drug in tablet sample solution was calculated by comparing the peak area of standard chromatogram of Zidovudine. This method was validated as per the ICH guidelines^{14 -16}.

Fig. 1: Chemical Structure of Zidovudine

Table 1: Optimized Chromatographic conditions for the proposed method

Parameters	Optimized conditions
Column	Venusil XBP C-18 (250 X 4.6mm, 5µ)
Mobile phase	Water : Methanol (50 : 50)
Flow rate	1.0 ml / min
Injection volume	20 µl
Detection	266 nm in UV detector
Temperature	Ambient
Retention time	4.47 min
Run time	6 min

Fig. 2: Typical RP-HPLC Chromatogram of Zidovudine by the proposed method.

Table 2: System Suitability Test Parameters for the proposed method

Parameters	Values
Theoretical plates	3439
Asymmetric factor	1.14
Tailing factor	1.22

RESULTS AND DISCUSSION:

A suitability test was applied to representative chromatograms for various parameters. The results obtained were within acceptable limits and presented in Table: 2. Thus, the system meets suitable criteria. The calibration curve was obtained for a series of concentration in the range of 25-75 µg/ml and it was found to be linear. The data of regression analysis of the calibration curves are shown in Table: 3. The precision was determined by taking the sufficient number of aliquots of a homogenous sample. The % RSD was determined and the values were within 2. This shown that the precision of the method was satisfactory. The accuracy studies were calculated by direct comparison from precision and linearity. The % RSD was less than 2.0. This shown that the accuracy values of Zidovudine by the proposed methods are satisfactory. The assay results of tablet were obtained by the proposed method were validated by statistical evaluation and reported in Table: 4. Ruggedness and Robustness were determined and the % RSD values were calculated from precision study, it was found less than 2.0. Limit of detection (LOD) and Limit of quantitation (LOQ) were determined by the proposed method. The results of validation parameters are summarized in Table 5.

Table 3: Regression analysis of the Calibration curve for the proposed method

Parameters	Values
Linearity range (µg/ml)	25-75
Correlation coefficient (r²)	0.9997
Regression equation	Y= 41754 X + 196916
Slope	41754
Intercept	196916

Table 4: Assay Results of Zidovudine tablets using proposed method

Tablets	Labelled amount (mg)	Amount found (mg)	% Recovery ± SD^a
A	300	299.85	99.95 ± 0.09
B	100	99.10	99.10 ± 0.13

^aStandard deviation of six determinations

Fig. 3: Calibration curve at 266 nm of Zidovudine by the proposed method

Table 5: Summary of Validation Parameters for the proposed method

Parameters	Values
Limit of detection (µg/ml)	0.15
Limit of quantitation (µg/ml) ^bPrecision	0.45
System precision (% RSD)	0.45
Method precision (% RSD)	1.60
^bRuggedness ( % RSD) Analyst I Analyst II	1.1 1.3
^bRobustness ( % RSD) Changed condition I ( ratio of mobile phase) Changed condition II ( flow rate)	0.11 0.01

^bMean of six determinations, RSD indicates relative Standard deviation

CONCLUSION:

From the above results, it can be concluded that the method developed in the present investigation are simple, sensitive, accurate, rugged, robust, rapid and precise. Hence, the above said method can be successfully applied for the estimation of Zidovudine in bulk & pharmaceutical dosage forms.

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