INTRODUCTION:

Efavirenz belongs to the class of non-nucleoside reverse transcriptase inhibitors and is indicated in the treatment of HIV infection, Class: Nonnucleoside Reverse Transcriptase Inhibitor(NNRTI) ,VA Class: AM800,Chemical Name: (±) - 6 - Chloro - 4 - (cyclopropylethynyl) - 1,4 - dihydro - 4 - (trifluoromethyl) - 2H - 3,1 - benzoxazin - 2 – one, Molecular Formula: C₁₄H₉ClF₃NO₂, CAS Number: 154635-17-3, Brands: Sustiva, structural formula(fig. 1) and Ball and stick model of Efavirenz (fig. 2)¹. It is white to slightly pink powder, practically insoluble in water and freely soluble in methanol. It should be kept in a well closed container, protected from light. Literature survey reveals that only HPLC methods are available for the estimation of Efavirenz alone, in combination with other drugs and in its dosage form, no UV Spectrophotometric method using sodium hydroxide as solvent was found in literature⁷. The present investigation has been undertaken to develop simple UV Spectrophotometric method for the estimation of Efavirenz in pure form and its formulations⁵.

MATERIAL:

Efavirenz pure drug was obtained as a gift sample from Ranbaxy lab ltd (Dewas, India). SUSTIVA® (600mg) tablet were purchased from Civil Hospital (A.R.T center) Ahmednagar. All the reagents in this assay along with sodium hydroxide were of analytical grade and these agent solution were prepared using preanalysed double distilled water.

APPARATUS:

Spectral analysis were made on a Jasco Spectrophotometer, Model- V-630 (Japan), was employed with spectral bandwidth of 1 nm and wavelength accuracy of ±0.3 nm with automatic wavelength correction with a pair of 10 mm quartz cells. Glass wares used in each procedure were soaked overnight in a mixture of chromic acid and sulphuric acid rinsed thoroughly with double distilled water and dried in hot air oven.

EXPERIMENTAL:

Standard Stock Solution:

Accurately weighed Efavirenz (10mg) was transferred to a 100ml volumetric flask, dissolved in 10ml with 0.1 N sodium hydroxide solution and made up the volume up to the mark with 0.1 N sodium hydroxide solution. A stock solution contained 100µg/ml of Efavirenz⁴.

Determination of absorbance maximum:

Weighed an accurate amount of 10mg Efavirenz was dissolved in 10ml with 0.1 N sodium hydroxide solutions and diluted up to100ml by same to obtain a 100µg/ml conc. of Efavirenz in solution. This solution was subjected to scanning between 200-600 nm. The effect of dilution on absorption maxima was studied by diluting the above solution to 30µg/ml and scanned from 200-600nm⁸.

Preparation of calibration curve for Efavirenz:

Stock solutions of Efavirenz (0.1ml to 1.0ml) were pipette out in to a series of six volumetric of 10ml. The volume in each volumetric flask was made up to the mark with 0.1N sodium hydroxide solution. It produced the concentration range of 1-10 µg/ml. The absorbance of the solution was measured at 247 nm. against 0.1N sodium hydroxide as a blank in table no. 1. The calibration curve was given in figure 3, Spectra of 5μg/ml solution of Efavirenz in 0.1 N sodium hydroxide solution (figure no.4) and statistical parameters are summarized in table no.2.

Figure 1. Structural formula of Efavirenz

Figure 2. Ball and Stick model of Efavirenz

Figure no.3 Calibration Curve of Efavirenz at 247 nm.

Table no.1 Results of least square regression analysis and absorbance of UV methods for the estimation

Sr. no.	Concentration, μg/ml	Absorbance Mean ±SD(n=6)	CV (%)
1	1	0.055 ±0.00634	1.20
2	2	0.107 ±0.00624	1.43
3	3	0.157 ±0.00610	1.58
4	4	0.214 ±0.00650	1.62
5	5	0.266 ±0.00818	1.71
6	6	0.319±0.00981	2.05

Figure no.4 Spectra of 5μg/ml solution of Efavirenz in 0.1 N sodium hydroxide solution.

Table no. 2 Calibration curve points of the proposed method for the estimation of Efavirenz

Sr. no.	Parameters	Values
1	Absorption maxima, nm	247
2	Beer’s law limit, μg/ml	1-10
3	Molar absorptivity, 1 mole-1/cm-1	2.21×10-4
4	Coefficient of correlation	0.9998
5	Regression equation	Y=0.053x+0.00
6	Slope(b)	0.05306 ± 0.0004014
7	Intercept(a)	0.0005238 ± 0.001215
8	1/slope	-18.85
9	Correlation coefficient(f)	0.9999
10	LOD and LOQ	0.4ppm and 1ppm

Analysis of Marketed Tablet Formulation:

Accurately weighed the 20 tablets of SUSTIVA® (600mg) and fine powdered. The powder equivalent to 100mg of Efavirenz was transferred to 100ml volumetric flask and 20ml 0.1N sodium hydroxide is added and sonicated for 15 minutes to dissolve the Efavirenz in it and made the volume to mark with same. The solution was filtered through Whatmann filter paper No.40. 10 ml of this was diluted with 0.1N sodium hydroxide diluted with same as blank. The concentration of Efavirenz present in marketed tablet formulation were determined, table no.3².

METHOD VALIDATION:

Accuracy (Recovery studies):³

Recovery studies were performed to judge the accuracy of the method. 1ml of standard formulation (100mcg/ml) was taken in three 10 ml volumetric flask and to it 80%,100% and 120% (i.e. 0.8ml,1.0ml and 1.2ml) of working standard solution (100mcg/ml) added respectively and made the volume up to the mark. The respective absorbance at 247 nm was recorded against the blank. The amount of added concentration was determined from the obtained absorbance values and percent recovery was determined for formulation Table no.4

Table no.3 Result of Analysis of Efavirenz in tablet SUSTIVA® (600mg)

Tablet

sample

Label Claim,

mg/tablet

Actual content

found, mg±S.D

Percent Actual

content found, ±S.D

Efavirenz

Tab.(n=3)

600

590±2.04

98.33±0.74

0.679

Table no.4 Efavirenz estimation in dosage form in recovery studies by proposed method.

Sr. no.	Concentration of added amount of drug in the final dilution, ( μg/ml )	Recovery, (μg/ml )	Percent Recovery (%)	Mean recovery ±SD	CV
1	8	7.91	79.1
2	10	9.89	98.9	98.87± 0.975	0.958
3	12	11.86	118.6

Precision:⁶

The Precision of the proposed method was ascertained by actual determination of ten replicates of fixed concentration of the drugs within the Beer’s range and finding out the absorbance by the proposed method. From this absorbance, mean, SD, %RSD was calculated. The readings were shown in table no.5.

Table no.5 Precision reading of Efavirenz

Sr. no.	Concentration (µg/ml)	Absorbance	Statistical analysis
1	20	1.064	Mean 1.060
2	20	1.051	S.D. 0.975
3	20	1.061	%RSD.91.98.
4	20	1.063
5	20	1.060
6	20	1.064
7	20	1.065
8	20	1.056
9	20	1.066
10	20	1.046

RESULT AND DISCUSSION:

From the optical characteristics of the proposed method, it was found that Efavirenz obeys linearity within the concentration ranges 1-100µg/ml. 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 0.1 N sodium hydroxide 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.

CONCLUSION:

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 (0.1 N sodium hydroxide solution), 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.

ACKNOWLEDGEMENT:

Authors are sincerely thankful to P.D.V.V.P.F’s College of

Pharmacy Ahmednagar for providing necessary facilities for work.

REFERENCES:

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Received on 21.09.2011 Modified on 29.09.2011

Research J. Pharm. and Tech. 4(12): Dec. 2011; Page 1816-1818