INTRODUCTION:

Chemically, Efavirenz (EFA) is (S)-6-chloro-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one. Its empirical formula is C₁₄H₉ClF₃NO₂. Efavirenz is a white to slightly pink crystalline powder with a molecular mass of 315.68 g/mol. It is practically insoluble in water. EFA is non-nucleoside reverse transcriptase inhibitor (NNRTI) and is used as a part of highly active antiretroviral therapy for the treatment of human immunodeficiency virus. The drug is used in combination with other anti-retroviral agents for the treatment of HIV-1 infection in children and adults. The usual dose of EFA is 600 mg per day and its plasma half-life is ~50 h. (Nikalje, 2009; Rang, 2007). Both nucleoside and non-nucleoside RTIs inhibit the same target, the reverse transcriptase enzyme, an essential viral enzyme which transcribes viral RNA into DNA. Unlike nucleoside RTIs, which bind at the enzyme's active site, NNRTIs act allosterically by binding to a distinct site away from the active site known as the NNRTI pocket. EFA is not effective against HIV-2, as the pocket of the HIV-2 reverse transcriptase has a different structure, which confers intrinsic resistance to the NNRTI class (Ren, 2002).

However, more than 50% of patients starting EFA treatment experience its related neuropsychiatric adverse events (NPAEs), such as dizziness, feeling of drunkeness and sleep disorders and even severe psychiatric symptoms have been reported with EFA (Allcia, 2009). Several analytical methods of EFA were reported including LCMS, HPLC (Gadkari, 2010; Garg, 2009). The information about spectrophotometric method used to analyze the EFA concentration was rather scanty. In the present study an attempt has been made to develop simple, sensitive, and economical method in UV region with greater precision and accuracy for the estimation of EFA in pure drug, tablet formulation.

MATERIALS AND METHODS

EFA sample was supplied by Sandoz Ltd., Mumbai, as a gift sample and used as such. Methanol used was spectra grade purchased from S. D. Fine Chemicals ltd., India. All other reagents used were of analytical reagent grade supplied by Research Lab., India. Spectral and absorbance measurements were made on a UV-Visible spectrophotometer Jasco V 530 model with 10mm matched pair of quartz cell and spectral band width of ±2nm.

EXPERIMENTAL:-

Determination of λ max:-

Accurately weighed 10mg of Efavirenz EFA is transferred into a 100ml volumetric flask and dissolved in 30ml of methanol. It was then sonicated for 10 minutes, and made up to the mark with methanol to give a stock solution having 100 μ g/ml concentrations. This solution was subjected to scanning between 200-400 nm and absorption maxima at 247nm was determined. The effect of dilution maxima was studied by diluting the above solution to 20mcg/ml and scanned from 200-400nm.

Standard solutions:-

Accurately weighed 100mg of Efavirenz is transferred into a 100ml volumetric flask and dissolved in 30ml of methanol. It was then sonicated for 10 minutes, and made up to the mark with methanol to give a stock solution having 1000 μ g/ml concentrations.

Working standard solution:

10 ml of stock solution was further diluted to 100ml with methanol to obtain a working standard solution containing 100 μg/ml.

Linearity and Calibration:-

The aliquots working standard solution was diluted serially with methanol to obtained the range 5-40 μ g/ml. a calibration curve for Efavirenz was obtained by measuring the absorbance at the λ max of 247 nm. Statistical parameters like slope, intercept, coefficient of correlation, standard deviation were determined.

Analysis of marketed tablet formulation :

Determine the content of Efavirenz in conventional tablets (label claim: 600mg EFA per tablet), twenty tablets were weighed, their mean weight was determined and they were finely powdered and powder equivalent to 100 mg of Efavirenz was weighed and transferred into a 10 ml volumetric flask containing 10 ml methanol, sonicated for 10 min and the resulting sample solution was then filtered through Whattman filter paper (No. 41). The filtrate was further diluted to obtain the final concentration of 1000μg/ml. Appropriate dilutions of Efavirenz were scanned over the range of 400-200 nm and the absorbance at wavelength 247nm was measured. From calibration curve the final drug concentration in tablet was calculated.

Recovery studies:

Recovery studies were performed to judge accuracy of the method. 1ml of standard solution (100mg/ml) was taken in three 10 ml volumetric flask and to it 80%, 100% , 120% ( i.e. 0.8ml, 1.0ml, 1.2ml) of working standard solution (100mg/ml) added respectively and made the volume upto the mark. The respective absorbance at 247nm was recorded against the blank. The amount of added concentration was determined from the obtained absorbance values and percent recovery was determined for each formulation.

RESULT:-

The UV scan of standard solution between 200-400nm showed the absorption maxima at 247nm, shown in fig. 1. The Beer’s law was verified from the calibration curve by plotting a graph of concentration vs absorbance. The plot shown in fig.2. Regression analysis showed very good correlation. The calibration plot revealed zero intercept which is clear by the regression analysis equation Y=mX+C (where Y is absorbance, m is the slope and X is the conc. ) the results obtained are depicted in Table no. 1,2,3. No significant variations were observed on interday and intraday analysis.

Figure 1: Spectrum of Efavirenz at wavelength 200 to 400 nm

Figure 2: Calibration curve of Efavirenz showing linearity relationship

Table No.1: Optical characteristic

Parameter

Observation

Absorption maxima

Beer’s law limit

Coefficent of correlation

Regression equation

Slope

Intercept (A)

247nm

5-40 μg/ml

0.9993

Y= 0.0504x-0.034

0.0504

-0.0304

Table 2: Results of analysis of laboratory samples

Label claim (mg/tab)	*% Concentration estimated (Mean ± % R.S.D.)**
600 mg	99.74 ± 1.22

* Average of nine determinations; R.S.D., Relative Standard Deviation

Table 3: Recovery data for EFA

Level added (%)	Recovery (%)*	SD
80	98.88	0.7426
100	99.75	0.7823
120	97.77	0.5674

* Mean of three determinations

DISCUSSION:

The spectrum of Efavirenz in methanol showed the absorption maxima at 247nm. No effect of dilution was observed on the maxima which confirmed the maxima at 247nm. The statistical analysis of data obtained for the calibration curve of Efavirenz in the high level of precision for the proposed method. The coefficient of correlation was highly significant. The linearity range was observed between 5-40 mcg/ml. the plot clearly showed a straight line passing through origin. The estimated method was validated by %RSD, accuracy, precision of the methods.

CONCLUSION:

From the above result and discussion the method describe in this paper for the determination of efavirenz from tablet formulation is simple, accurate, sensitive and economical. The proposed method utilizes inexpensive solvent. The proposed method could be applied for routine analysis in quality control laboratories.

REFERENCES:

1. Allcia GV, Pompeyo V, Stepped-dose verses full-dose efavirenz for HIV infection and Neuropsychiatric ad-verse effects. Annals of International Medicine. 2009, 151: 149-156.

2. Beckett AH and Stanlake JB. Practical Pharmaceutical Chemistry, CBS, Delhi. 4^th Edn, 1998.

3. Gadkari T, Chandrachood P, Ruikar A, Tele S, Validated stability indicating LC-PDA-MS method to investigate PH rate profile and degradation on kinetics of efavirenz and identification of hydrolysis product. Inter-national Journal of Pharmacy and Pharmaceutical Sciences. 2010, 2: 169-176.

4. Garg A, Soni LA, Kaskhedikar SG, Kona SS, Singh LR, Gupta KK and Dwivedi D, Development and validation of HPLC method for analysis of some antiretroviral agents in their pharmaceutical dosage forms. Pharmaceutical Chemistry Journal. 2009, 43: 369-374.

5. Joachim Ermer, John H, Mc B Miller; Method Validation in Pharmaceutical analysis, A Guide to Best Practice, 2003.

6. Kappelhoff BS, Hilde RA, Huitema DR, Beijnen JH, Simple and rapid method for the simultaneous determination of the non-nucleoside reverse transcriptase inhibitors Efavirenz and nevirapine in human plasma using liquid chromatography. Journal of Chromatography B. 2003, 792: 353-362.

7. Rang HP, Dale MM. Pharmacology. 5^th Edn, New York: Churchill Livingstone, 2007.

8. Rao BU, Nikalje AP, Stability indicating HPLC method for the determination of efavirenz in bulk drug and in pharmaceutical dosage form. African journal of Pharmacy and Pharmacology. 2009, 3: 643-650.

9. Ren J, Bird LE, Chamberlain PP, Structure of HIV-2 re-verse transcriptase at 2.35-A resolution and the mechanism of resistance to non-nucleoside inhibitors. Proc Natl. Acad Sci USA. 2002, 99: 14410-15.

10. Skoog DA and West DM, In: Fundamentals of analytical Chemistry, 3^rd Edn, 1992.

11. Smith AA, Manvalan R, Kannan K, Rajendiran N, Spectroflurometric determination of bicalutamide in formulation and biological fluids. Asian journal of chemistry. 2009, 21: 459-466.

12. Validation of Analytical Procedures, Methodology Step-3 Consensus Guidelines, ICH Harmonized Tripartite Guidelines, ICH Q2B, 1996.

Received on 28.01.2011 Modified on 25.02.2011

Asian J. Research Chem. 4(5): May, 2011; Page 754-756