Development of Rapid UV Spectrophotometric Method for the Estimation of Efavirenz in Formulations

Simple, sensitive, accurate, precise and rapid ultraviolet (UV) spectrophotometric method was developed for the estimation of efavirenz in pure form, its formulations and stability samples. For the estimation of efavirenz, solvent system employed was 1% w/v sodium lauryl sulphate (SLS) and wavelength of detection (λdet) was 247 nm. The developed method was used to estimate the total drug content in two commercially available oral formulations of efavirenz and recovery studies were also carried out. Sample recovery in both the formulations using the above method was in good agreement with their respective labeled claims, thus suggesting the validity of the method and non-interference of formulation excipients in the estimation. The developed method was found to be stability specific and were validated as per ICH guidelines-2005, USP-2000 and statistical methods.

Literature survey reveals that only HPLC methods are available for the estimation of efavirenz alone, in combination with other drugs, in its dosage form and in plasma [5][6][7][8][9][10] however, no UV spectrophotometric method was found in literature.The present investigation has been undertaken to develop simple UV spectrophotometric method for the estimation of efavirenz in pure form and its formulations.

Experimental
A Hitachi-U2000 spectrophotometer with a pair of matched quartz cells was to measure absorbance of the resulting solutions.Efavirenz was gifted by Strides Pharma.All the other reagents used were of analytical grade.

Preparation of standard curve
A 100 µg/mL stock solution of efavirenz was prepared in 1%w/v SLS by first dissolving 10 mg of the drug in 10 mL of methanol and then, made up to the final volume with 1%w/v SLS.
The λ max of efavirenz was determined by scanning suitable dilutions with high correlation coeffiecient.From the stock solution, various standard dilutions were made to obtain solutions of 1,2,3,4 and 5 µg/mL.Their respective absorbance values were measured at fixed λ max with parameter set at 0.5 nm for bandwidth as well as data pitch.Average absorbance values, standard deviation and % coefficient of variance at each concentration were calculated.One way ANOVA test for linearity was carried out by picking five sets of calibration curves on random basis.

Sample preparation (Bulk drug)
Efavirenz 50 mg was accurately weighed and taken into 100 mL volumetric flask containing 10 mL of methanol and further it was made upto 100 mL with 1.5% w/v sodium lauryl sulphate solution.This solution was further serially diluted with 1.5% w/v sodium lauryl sulphate solution to get 10, 20, 40, 50,100 µg/mL solution.The efavirenz content was further determined by measuring the absorbance at 247nm.

Sample preparation (Dosage forms)
Twenty tablets were weighed and powdered; the powder equivalent to 50 mg of efavirenz was weighed accurately and taken in 100 mL volumetric flask containing 50 mL of methanol.The contents were shaken well for 30 minutes and made upto the volume 100 mL with methanol.This solution was further suitably diluted with 1.5% w/v sodium lauryl sulphate solution and determined the efavirenz content by measuring the absorbance at 247 nm.

Recovery experiments
To keep an additional check on the accuracy of the developed method and to study the interference of formulation additives, analytical recovery experiments were performed by adding known amount of pure drug to the previously analyzed pharmaceutical preparation and analyzed by the developed method.The concentration levels used were 10µg/mL.

Method development
To develop accurate, precise and sensitive UV spectrophotometric method for efavirenz, various solvent systems such as water, methanol etc. were tried alone and in combinations or in the presence of surfactants at different propotions.The final decision of using 1% w/v SLS in water was based on sensitivity, minimal interference, ease of preparartion, suitability for drug content estimation, stability, analysis time and cost.The λ max for efavirenz in 1% w/v SLS (Figure 1) showed linear relationship (with correlation coefficient of 0.9988) in the concentration range of 1-5 µg/mL (Table 1& 2).

Sample solution stability studies
Overlay scans obtained at zero time, 12, 24, and 48 h revealed no degradation upto 48 h,in the selected solvent at controlled (25±2 o C; 65±5 %RH) and accelerated (40±2 o C;75±5 %RH).Drug was stable for more than 48 hours, thus there can be time between collection of the sample and analysis of the same.

Recovery studies
The method developed for the estimation of efavirenz in bulk and in its dosage forms was found to be simple, accurate, economical and rapid.Table 3-5 clearly indicates the drug content was uniform ranging from 98 to 99.55% and SD, CV values were found to be satisfactorily low.Recovery studies were also carried out and found to be 98.89 to 98.89% for the both batches of tablets.The method requires only measuring the absorbance of sample solution at the selected wavelength followed by simple calculations.Hence, it was further employed for our study.

Method validation
The developed estimation method proved to be accurate (accuracy varies between 10.2-5.5%) and precise (Intra day precisions were less than 4.5%).The method has been validated for the range 1-5 µg/mL using 1%w/v sodium lauryl sulphate solution.The method is linear over this concentration range as indicated by the F-test for lack of fit.Analyte recovery was better than 90% at all points on the standard curve, Intraday precision was better than 5% CV, while accuracy was between 98-100% of nominal over this range of the estimation.

Conclusions
The developed UV spectrophotometric method for the estimation of efavirenz was found to be simple and useful with high accuracy, precision, repeatability.Sample recoveries in all formulations using the above method was in good agreement with their respective label claim or theoretical drug content, thus suggesting the validity of the method and non interference of formulation excipients in the estimation.In the selected solvent system, drugs were stable for more than 48 hours, thus suggesting that samples need not be estimated immediately after collection.The developed method was found to be stability specific and were validated as per ICH guidelines (2005) and statistical method.

Table 1 .
Calibration curve points of the proposed method for the estimation of efavirenz.

Table 2 .
Results of least square regression analysis of UV methods for the estimation of efavirenz.

Table 3 .
Efavirenz estimation in bulk by proposed method.

Table 4 .
Efavirenz estimation in dosage form by developed method.

Table 5 .
Efavirenz estimation in dosage form in recovery studies by proposed method.