Sensitive and rapid titrimetric and spectrophotometric methods for the determination of stavudine in pharmaceuticals using bromate-bromide and three dyes

Four sensitive and rapid methods for the determination of stavudine (STV) in bulk drug and in dosage forms were developed and optimized. In titrimetry, aqueous solution of STV was treated with a known excess of bromate-bromide in HClmedium followed by estimation of unreacted bromine by iodometric back titration. Spectrophotometric methods involve the addition of a measured excess of bromate-bromide in HCl medium and subsequent estimation of the residual bromine by reacting with a fixed amount of methyl orange, indigocarmine or thymol blue followed by measurement of absorbance at 520 nm (method A), 610 nm (method B) or 550 nm (method C). In all the methods, the amount of bromate reacted corresponds to the amount of STV. Calculations in titrimetry were based on a 1:0.666 (STV :KBrO3) stoichiometry and the method was found to be applicable over 3.5-10 mg range. A linear increase in absorbance with concentration of STV was observed in the spectrophotometric methods, and the Beer’s law was obeyed over the concentration ranges 0.125-1.75, 1-10 and 1-9.0 μg mL−1 STV for method A, method B and method C, respectively. The methods when applied to the determination of STV in tablets and capsules were found to give satisfactory results.


INTRODUCTION
Stavudine (STV), chemically known as 2 1 -3 1 -didehydro-2 1 -3 1 -dideoxythymidine (Fig. 1), is a nucleoside analog reverse transcriptase inhibitor (NARTI) active against HIV (The Merck Index 1996).STV is converted intracellularly to triphosphate which stops the DNA synthesis of retroviruses through competitive inhibition of reverse transcriptase and incorporation into viral DNA.It is the fourth antiretroviral drug in the market and is used in the treatment of HIV infection.The drug is official in United States of Pharmacopoeia (The United States Pharmacopoeia 2006).which describes high performance liquid chromatography as assay procedures for bulk drug and tablets, respectively.
Correspondence to: Basavaiah K. E-mail: basavaiahk@yahoo.co.inA number of methods based on high performance liquid chromatography (Bazy et al. 2005, Verweij-van Wissen et al. 2005, Contreras et al. 2004), liquid chromatography-tandem mass spectrometry (Compain et al. 2005, Huang et al. 2004, Raices et al. 2003, Fan et al. 2002, Wiesner et al. 2002), micellar electrokinetic chromatography (Fan and Stewart 2002), radio immuno assay (Tran et al. 2003) and electrophoresis (Pereira et al. 2005) are known for the quantitative determination of STV in biological matrices such as blood plasma, blood serum and human cells.Several chromatographic techniques including HPLC (Dunge et al. 2005, Djurdjevic et al. 2004, Zhang et al. 2003, Sablon et al. 2004, Pai and Desai 2003, Rezk et al. 2003), HPTLC (Wankhede et al. 2005, Kaul et al. 2005) and LC-MS (Volosov et al. 2002), have been used for the determi-nation of STV in pharmaceuticals.The drug has also been assayed by mass spectrometry (Soldin 2004) and UV-spectrophotometry (Sankar et al. 2002a).Despite its long history and established versatility, no titrimetric method has yet been reported for the determination of STV in pharmaceuticals.Visible spectrophotometry, because of its simplicity, speed, sensitivity, reasonable accuracy and precision, and cost-effectiveness, continues to be the preferred technique in laboratories of developing and underdeveloped nations, which can illafford expensive chromatographic and related techniques.Three procedures (Sarma et al. 2002a) have been reported for the assay of STV in pharmaceuticals using KMnO 4 -Fast green FCF, permanganate/periodate-MBTH and iron(III)chloride-ferricyanide as reagents.The same authors (2002b) have used three more reagents, NBS-celestine blue, cobalt thiocyanate and ammonium molybdate for the spectrophotometric determination of STV.A method based on oxidative-coupling reaction (Sankar et al. 2002b) involving the use of iron (III)-MBTH is also found in the literature.But, these methods suffer from one or the other disadvantage like poor sensitivity, heating or extraction step and/or use of expensive chemical/organic solvent.
This paper reports the use of bromate-bromide reagent and three dyes-methyl orange, indigocarmine and thymol blue for rapid and sensitive determination of STV.The methods are based on the bromination/oxidation of the drug by in situ generated bromine followed by estimation of residual bromine by either iodometric back titration or by reacting with a fixed quantity of dye and measuring the change in absorbance.The methods on applying to tablets and capsules yielded satisfactory results and were comparable with those of a reference method.

MATERIALS AND METHODS
Apparatus: A Systronics Model 106 digital spectrophotometer provided with matched 1-cm quartz cells was used for all absorbance measurements.All chemicals used were of analytical reagent grade and distilled water was used to prepare all solutions.
Reagents and standards: Bromate-bromide mixture (4 mmol L −1 KBrO 3 − 40 × 10 −3 mmol L −1 KBr) was prepared by dissolving accurately weighed 0.668 g of KBrO 3 (Sarabhai M Chemicals, Baroda, India) and 4.76 g of KBr (Indian Drugs and Pharmaceuticals Ltd, Hydrabad, India) in water and diluting to 1 litre in a calibrated flask, and the reagent was used in titrimetric work.A 0.024 mol L −1 sodium thiosulphate solution was prepared by dissolving about 5.96 g of chemical (Sisco Chem.Industries, Mumbai, India) in 1 litre of water.A 10% potassium iodide solution was prepared by dissolving 10 g of salt (Merck Chemicals, Mumbai, India) in 100 mL of water.To prepare 1% starch indicator, 1 g of soluble starch (S.d.Fine Chem., Mumbai, India) was made into paste in water and poured into 100 mL boiling water, boiled for 1 min and cooled.For spectrophotometric investigations, a bromate-bromide solution equivalent 1000 µg mL −1 KBrO 3 and 10-fold excess of KBr was prepared by dissolving accurately weighed 100 mg of KBrO 3 and 1 g of KBr in water and diluting to the mark in a 100 mL calibrated flask.This was then diluted stepwise to obtain working concentrations of 10, 30 and 50 µg mL −1 KBrO 3 for use in method A, method B and method C, respectively.Concentrated hydrochloric acid (S.D. Fine Chem., Mumbai, India; sp.gr.1.18) was diluted appropriately with water to get 5 mol L −1 for spectrophotometric work and it was diluted to get 2 mol L −1 for titrimetry.A 500 µg mL −1 methyl orange dye solution was first prepared by dissolving accurately weighed 58.8 mg of dye (S.D. Fine Chem., Mumbai, India, assay 85%) in water and diluting to 100 mL in a calibrated flask and filtered using glass wool.It was further diluted to obtain a working concentration of 50 µg mL −1 .A stock standard solution equivalent to 1000 µg mL −1 indigo carmine was first prepared by dissolving accurately weighed 112 mg of dye (S.D. Fine Chem., Mumbai, India, 90% dye content) in water and diluting to volume in a 100 mL calibrated flask.The solution was then diluted 5fold to get the working concentration of 200 µg mL −1 .A An Acad Bras Cienc (2008)  80 (2) 1000 µg mL −1 stock standard solution of thymol blue was first prepared by dissolving accurately weighed 100 mg of dye (Loba.Chemie.Mumbai.India, 100% dye content) in water and diluting to volume in a 100 mL calibrated flask.The solution was then diluted five fold to get the working concentration of 200 µg mL −1 .
Standard drug solution: Pharmaceutical grade STV was received from Cipla India Ltd., (99.8% pure), as gift and was used as received.A stock standard solution containing 1 mg mL −1 STV was prepared by dissolving accurately weighed 250 mg of pure drug in water and diluting to the mark in a 250 mL calibrated flask.This solution was used for titrimetric work, and for spectrophotometric work, the same was diluted appropriately with water to get a working concentration of 5 µg mL −1 for method A, and 20 and 40 µg mL −1 for method B and method C, respectively.transferred into a 100 mL Erlenmeyer flask.The solution was acidified by adding 3 mL of 2 mol L −1 hydrochloric acid and diluted to 15 mL with water.Ten mL of bromate-bromide reagent (4 mmol L −1 w.r.t.KBrO 3 ) was pipetted into the flask, the flask was stoppered, the contents mixed and let stand for 10 min with occasional swirling.Finally, 5 mL of 10% potassium iodide solution was added, and the liberated iodine was titrated against 0.024 mol L −1 thiosulphate solution using starch as indicator towards the end point.A blank titration was performed, and the amount of drug in the measured aliquot was calculated from the amount of KBrO 3 reacted with drug.
Spectrophotometric method A: Different aliquots (0.25, 1.0, 1.5,. . .3.5 mL) of a standard 5 µg mL −1 STV solution were transferred into a series of 10 mL calibrated flasks by means of a micro burette and the total volume was adjusted to 4 mL by adding adequate quantity of water.To each flask were added 1 mL each of 5 mol L −1 HCl and bromate-bromide solution (10 µg mL −1 in KBrO 3 ), the last being measured accurately.The flasks were stoppered, content mixed and let stand for 20 min with occasional shaking.Finally, 1 mL of 50 µg mL −1 methyl orange solution was added (accurately measured) and the volume was diluted to the mark with water and mixed well.The absorbance of each solution was measured at 510 nm against a reagent blank after 5 min.

Spectrophotometric method B:
Varying aliquots (0.5,1.0, . . .5.0 mL) of a standard 20 µg mL −1 STV solution were transferred into a series of 10 mL calibrated flasks by means of a micro burette and the total volume was brought to 5 mL by adding water.To each flask were added 1 mL of 5 mol L −1 hydrochloric acid and 1.5 mL of bromate-bromide solution (30 µg mL −1 in KBrO 3 ) by means of a micro burette.The content was mixed well and the flasks were kept aside for 10 min with intermittent shaking.Finally, 1 mL of 200 µg mL −1 indigo carmine solution was accurately measured and added to each flask, the volume was diluted to the mark with water, mixed well and absorbance measured against a reagent blank at 610 nm after 5 min.
Spectrophotometric method C: Different aliquots (0.25, 0.5, 1.0,. . .2.25 mL) of a standard 40 µg mL −1 STV solution were transferred into a series of 10 mL calibrated flasks by means of a micro burette and the total volume was adjusted to 3 mL by adding adequate quantity of water.To each flask were added 1 mL each of 5 mol L −1 HCl and bromate-bromide solution (50 µg mL −1 in KBrO 3 ), the last being measured accurately.The flasks were stoppered, content mixed and let stand for 15 min with occasional shaking.Finally, 1 mL of 200 µg mL −1 thymol blue solution was added (accurately measured) and the volume was diluted to the mark with water and mixed well.The absorbance of each solution was measured at 550 nm against a reagent blank after 5 min.In all the three spectrophotometric method, a standard graph was prepared by plotting the absorbance versus the concentration of STV.The concentration of the unknown was read from the calibration graph or computed from the regression equation derived using Beer's law data.

PROCEDURE FOR DOSAGE FORMS
Procedure for tablets/capsule: Twenty tablets/contents of capsules were weighed and ground into a fine powder.An amount of powder equivalent to 250 mg of STV was weighed into a 250 mL calibrated flask, 60 mL of water added and the mixture shaken for 20 min; then the volume was made up to the mark with water, mixed well and filtered using a Whatman No. 42 filter paper.First 10 mL portion of the filtrate was discarded and a suitable aliquot was then subjected to analysis by titrimetric method.The tablet extract (1 mg mL −1 ) was diluted suitably with water to get working concentrations of 5, 20 and 40 µg mL −1 for method A, method B and method C, respectively before subjecting to analysis by spectrophotometric methods.

RESULTS AND DISCUSSION
The acidified solution of bromate and bromide behaves as an equivalent solution of bromine and has been widely used for the determination of many organic and inorganic substances.The present methods make use oxidising/brominating ability, and bleaching action of in situ generated bromine on the dyes used.
The proposed spectrophotometric methods are indirect and are based on the determination of the residual bromine (in situ generated) after allowing the reaction between STV and a measured amount of bromine to be complete.The bromine was determined by reacting it with a fixed amount of methyl orange, indigo carmine or thymol blue dye.The methods make use of bleaching action of bromine on the dyes, the decolouration being caused by the oxidative destruction of the dyes.

METHOD DEVELOPMENT
Titrimetry: Direct titration of STV with in situ generated bromine was not successful.However, the reaction between the two was found to occur when the two were allowed to stand for some time, thus enabling the indirect titrimetric determination of STV.Hence, several factors like nature of acid and its concentration, reaction time, and the excess of reagent were optimized.Reproducible and stoichiometric results were obtained when 0.16 to 0.32 mol L −1 hydrochloric acid concentration was maintained.Hence, 0.24 mol L −1 acid concentration for the oxidation step and the iodometric back titration was used in the assay.Reaction was complete in 10 min and yielded stoichiometry of 1:0.666 (STV: KBrO 3 ), and contact times up to 20 min had no effect on the stoichiometry of the reaction.A constant molarratio was obtained when excess of reagent was not more than 2 times the theoretical amount.Under the optimum conditions, 3.5-10 mg of STV could be determined with good accuracy and precision with reaction stoichiometry of 1:0.666.Spectrophotometry: STV, when added in increasing concentrations to a fixed concentration of in situ generated bromine, consumes the latter proportionally and there occurs a concomitant fall in the concentration of An Acad Bras Cienc (2008)  bromine.When a fixed concentration of dye is added to decreasing concentrations of bromine, a concomitant increase in the concentration of dye results.Consequently, a proportional increase in the absorbance at the respective λ max is observed with increasing concentration of STV.
Preliminary experiments were performed to fix the upper concentrations of the dyes that could be determined spectrophotometrically, and these were found to be 5, 20 and 20 µg mL −1 for methyl orange, indigo carmine and thymol blue respectively.A bromate concentration of 1 µg mL −1 in the presence of excess of bromide was found to bleach the red colour due to 5 µg mL −1 methyl orange whereas 4.5 and 5.0 µg mL −1 bromate was required to destroy the blue and violet colour due to 20 µg mL −1 each of indigocarmine and thymol blue, respectively.Hence, different concentrations of STV were reacted with 1 mL of 10 µg mL −1 KBrO 3 in method A, 1.5 mL of 30 µg mL −1 KBrO 3 in method B and 1 mL of 50 µg mL −1 KBrO 3 in method C respectively, followed by determination of residual bromine as described under the respective procedure.
For both steps, i.e., the reaction between STV and bromine, and the determination of the latter by reacting with the dye, HCl medium was found to be ideally suited.One mL of 5 mol L −1 acid in a total volume of about 5-7 mL was used in all the methods and the same quantity of acid was maintained for the bleaching step.Reaction times of 20, 10 and 15 min are not critical for method A, method B and method C. respectively, and any delay up to 30 min did not affect the absorbance reading.A 5 min standing time was found necessary for the complete bleaching of the dye colour by the residual bromine.The absorbance of each dye colour was constant for several hours even in the presence of reaction product.
Analytical data: A linear correlation was found between absorbance at λ max and concentration of STV in the ranges given in Table I.The graphs showed negligible intercept as described by the regression equation: (where Y = absorbance of 1-cm layer of solution; a = intercept; b = slope and X = concentration in µg mL −1 ).Regression analysis of the Beer's law data using the method of least squares was made to evaluate the slope (b), intercept (a) and correlation coefficient (r) for each system and the values are presented in Table I.The optical characteristics such as Beer's law limits, molar absorptivity and Sandell sensitivity values of all the three methods are also given in Table I.The limits of detection (LOD) and quantitation (LOQ) calculated according to ICH guidelines (ICH Harmonised Triplicate Guideline 1996) are also presented in Table I and reveal the very high sensitivity of the methods.

Method validation:
To evaluate the accuracy and intraday precision of the methods, pure drug solution at three different levels (concentrations) was analysed, each determination being repeated seven times.The relative error (%) and relative standard deviation (%) were less than 3.0 and indicate high accuracy and precision of the methods (Table III).For a better picture of reproducibility on a day-to-day basis, a series of experiments was performed in which standard drug solution at three different levels was determined each-day for five days with all solutions being prepared afresh each day.The day-to-day relative standard deviation values were in the range of 2.0-3.5% and represent the best appraisal of repeatability of the proposed methods.

APPLICATION
Three brands of STV tablets/capsules in 30 and 40 mg strength are currently available in the Indian market.The validity of the methods was checked by applying them to assay in two brands of capsules and one brand of tablets.Table IV gives the results of assay and reveal that there is close agreement between the results obtained by the proposed methods and the label claim.The results were also compared statistically with those obtained by a literature method (Sankar et al. 2002a) by applying Student's t-test for accuracy and F-test for precision.At the 95% confidence level, the calculated t-and F-values did not exceed the tabulated values (t = 2.77 and F = 6.39) suggesting that the proposed methods are as accurate and precise as the literature method.
The accuracy and validity of the proposed methods were further ascertained by performing recovery experiments.Pre-analysed tablet/capsule powder was spiked with pure STV at three different levels and the total was found by the proposed methods.Each determination was repeated three times.The recovery of pure      = Mean value of five determinations.Tabulated value of t at 95% confidence level is 2.77 and tabulated F-value at the same level confidence is 6.39 both for four degrees of freedom.
An Acad Bras Cienc (2008)   for stavudine and are superior to the existing HPLC and UV-spectrophotometric methods.They rely on the use of simple and cheap chemicals, and inexpensive techniques but provide a sensitivity comparable to that achieved by sophisticated and expensive technique like HPLC.These advantages coupled with good accuracy and precision make the methods highly suitable for routine use in laboratories as a part of industrial quality control.

TABLE II Quantification and regression characteristics of spectrophotometric methods.
Y = a+bX, where Y is the absorbance and X concentration in µg mL −1 .S a = Standard deviation of intercept.S b = Standard deviation of slope. *

TABLE III Intra-day accuracy and precision of the methods.
*In titrimetry taken/found/range, SD and SEM are in mg while in spectrophotometric methods the they are in µg mL −1 .**Mean value of seven determinations SD = Standard deviation; SEM = Standard mean of error; RSD = Relative standard deviation; and CL = Confidence limits at 95% confidence level for six degrees of freedom.

TABLE V Results of recovery study via standard-addition method.
Mean value of three determinations.In titrimetry STV in dosage form/pure STV added are in mg.In spectrophotometric methods the same are in µg. *