INTRODUCTION:

Stavudine is a synthetic nucleoside analogue with activity against HIV-1. The chemical name of Stavudine is 2¹,3¹-didehydro-3¹-deoxythymidine. It has a molecular formula of C₁₀H₁₂N₂O₄ and a molecular weight of 224.22 g / mol and its structure was given in Figure: 1. Stavudine is a white or almost white powder. It is freely soluble in Ethanol (95 %) and sparingly soluble in Water^1-2. The drug is officially listed in monograph of USP³. Several analytical methods that have been reported for the estimation of Stavudine in biological fluids or pharmaceutical formulations include High Performance Liquid Chromatography, Titrimetry and UV-Visible Spectrophotometry^4-14. The objective of the work was to develop simple, accurate, precise and economic RP-HPLC method with lesser run time to estimate the Stavudine in bulk and pharmaceutical dosage forms.

MATERIALS AND METHODS:

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 4.6 mm, 5 mm) column. The mobile phase consisting of 0.01M Ammonium acetate buffer and Methanol in the ratio of 60: 40 v / v was selected.

The optimized chromatographic conditions are summarized in Table.1. The standard solution of Stavudine was prepared by dissolving 10 mg in 100 ml of Mobile phase to give the concentration 100 mcg / ml. The mobile phase and the solution were sonicated for 10 min and filtered using whatman filter paper No.1. The various dilutions of Stavudine in the concentration of 25, 37.5, 50, 62.5 and 75 mcg / 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 265 nm, chromatograms were recorded. The Stavudine was eluted at 3.65 min as shown in Figure: 2. The calibration curve was constructed by plotting average peak area versus concentration and was presented in Figure: 3 with its computed regression equation. The method was extended for determination of Stavudine in pharmaceutical dosage form. The pharmaceutical dosage form containing 30 mg strength was taken.

Twenty capsules each of two different brands (containing 30 mg and 40 mg respectively) were taken, removed the caps and the powder equivalent to 10 mg of Stavudine was transferred into 100 ml volumetric flask containing 50 ml of mobile phase and flask was kept for Ultrasonication for 15 min, then it was diluted up to the mark with mobile phase and the solution was filtered through Whatman filter paper No. 41. From this solution various dilutions were made with the mobile phase, which were analysed. The concentration of the drug in capsule sample solution was calculated by comparing the peak area of standard. The proposed methods were validated as per the ICH guidelines^15-17.

Fig. 1: Chemical Structure of Stavudine

Table 1: Optimized Chromatographic conditions for the proposed method

Parameters	Optimized condition
Column	Venusil XBP C-18 (250 X 4.6 mm,5µ)
Mobile phase	0.01M Ammonium acetate buffer: Methanol (60:40)
Flow rate	1.0 ml / min
Injection volume	20 µl
Detection	265 nm in UV detector
Temperature	Ambient
Retention time	3.6 min
Run time	6 min

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

Fig. 3: Calibration curve of Stavudine by the proposed method.

Table 2: System Suitability Test Parameters for the proposed method

Parameters	Values
Theoretical plates	2740
Asymmetric factor	1.27
Tailing factor	1.19

RESULTS AND DISCUSSION

A suitability test was applied to representative chromatograms for various parameters. The results obtained were within acceptable limits (Table 2). Thus, the system meets suitable criteria. The calibration curve was obtained for a series of concentration in the range of 25-75 mcg / 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 measured in terms of repeatability, which was determined by sufficient number of aliquots of a homogenous sample. The % RSD was found and lying with in 2. This showed that the precision of the method was satisfactory. The accuracy of the method was inferred by establishing the precision and linearity studies of the standard. The % RSD was less than 2.0. This showed that the recoveries of Stavudine by the proposed methods are satisfactory. Ruggedness and Robustness were determined and the % RSD values were calculated from precision study was less than 2.0. Limit of detection (LOD) and Limit of quantitation (LOQ) were determined by the proposed methods. The results of validation parameters are summarized in Table 4. The results of capsule analysis and recovery studies obtained by the proposed method were validated by statistical evaluation and are given in Table: 5.

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

Parameters	Values
Linearity range (mcg / ml)	25-75
Correlation coefficient (r²)	0.999
Regression equation	Y= 56883.709 X + 62506.533
Slope	56883.709
Intercept	62506.533

Table 4: Summary of Validation Parameters for the proposed method

Parameters	Values
Limit of detection (mcg / ml)	0.15
Limit of quantitation (mcg / ml)	0.45
^aPrecision ( % RSD)
System precision	0.22
Method precision	0.57
^aRuggedness ( % RSD)
Analyst I	0.22
Analyst II	0.03
^aRobustness ( % RSD)
Changed condition I ( ratio of mobile phase) 50 : 50 (Buffer : Methanol)	0.22
70 : 30 (Buffer : Methanol)	1.10
Changed condition II ( flow rate of mobile phase) 0.9 ml / min	0.41
1.1 ml / min	0.09

^aMean of six determinations, RSD indicates relative Standard deviation

Table 5: Assay Results of Stavudine capsules using proposed method

Brand used SD^**	Labelled amount (mg)	Amount found (mg)	% Recovery ±
Cap-A	30	29.67	98.90 ± 0.13
Cap-B	40	40.12	100.30 ± 0.20

^**Standard deviation of six determinations

CONCLUSION:

Thus it can be concluded that the method developed in the present investigation was simple, sensitive, accurate, rugged, robust, rapid and precise. Hence, the above said method can be successfully applied for the estimation of Stavudine in pharmaceutical dosage forms.

REFERENCE:

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Received on 15.12.2009 Modified on 19.01.2010

Research J. Pharm. and Tech.3 (3): July-Sept. 2010; Page 770-772