INTRODUCTION:

Nevirapine, 11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[3,2-b:2’,3’-e]^1,4 diazepin 6-one¹is a non-nucleoside reverse transcriptase inhibitor (NNRTI) of human immunodeficiency virus type 1 (HIV-1). Treatment with Nevirapine mono-therapy is notorious for rapidly eliciting resistance due to mutations of the amino acids surrounding the NNRTI binding site²Nevirapine is metabolized by cytochrome P450 (CYP3A4) and is a relatively potent inductor of the enzyme; consequently, it has the ability to reduce plasma concentrations of other drugs that are also biotransformed by CYP3A4 as PIs³. Therapeutic drug monitoring of Nevirapine may be warranted to prevent or delay the occurrence of viral resistance, and to ensure optimal therapy for HIV-infected patients⁴.The spectrophotometric^5-7, HPLC^8-12 and HPTLC¹³ methods have been found in literature for its estimation alone or in fixed dose combination with other drugs. But no method has been found for Nevirapine by stability indicating UV spectrophotometric method.

The International Conference on Harmonization (ICH) guideline entitled ‘Stability testing of new drug substances and products’ requires that stress testing be carried out to elucidate the inherent stability characteristics of the active substances¹⁴.

In this paper we describe a very simple yet rapid, selective, and highly sensitive UV spectrophotometric method, not requiring sample treatment, for determination of Nevirapine. We have also laid emphasis on the stability indicating assay method by UV spectrophotometric method.

MATERIAL AND METHODS:

Pure samples of Nevirapine were obtained as gift samples from Hetero Labs Limited (Hyderabad). Tablet Nevimune was purchased from local market. Methanol used was of AR grade and it’s purchased from Merck, India. Spectral runs were made on a shimadzu double beam UV- Visible spectrophotometer, model A-1075 with spectral band width of 2nm with automatic wavelength correction was employed.

Preparation of the standard solution:

About 100mg of Nevirapine hydrochloride was accurately weighed and transferred to a clean dry 100ml calibrated standard flask and dissolved in methanol. It was shaken for few minutes and the solution was diluted to 100ml with same. 1ml of this solution was pipetted to another clean dry calibrated 10ml volumetric flask and the volume was made up with methanol. And again pipette out 1ml of this solution into a 10ml volumetric flask and made up the volume with methanol. The resulting solution was scanned at 283nm against blank. The typical spectrum obtained from a solution is shown in figure no.1.

Figure no.1: Typical spectrum of Nevirapine

Preparation of sample solution:

Twenty tablets were accurately weighed and average weight was taken, weight of sample equivalent to 200mg of Nevirapine was taken 100ml standard flask and the sample is dissolved in methanol and make up to 100ml with the same. The solution was then filtered through whatmann filter paper No.1. 1ml of this solution was pipetted to another clean dry calibrated 10ml volumetric flask and the volume was made up with methanol. And again pipette out 1ml of this solution into a 10ml volumetric flask and made up the volume with methanol. The prepared 10µg/ml was scanned at 283nm against blank. The amount of Nevirapine per tablet was calculated by comparing absorbance value of standard and sample at 283nm.The results were shown in table no.1.

Table no.1: Result of analysis of Nevirapine in tablets (n=3)

Drug

Amount claimed

(mg per tablet)

Amount found

(mg per tablet)

Mean recovery (%)

Nevirapine

200

201.20

100.06%

Stability studies of Nevirapine:

Stability studies were performed by forced degradation study of Nevirapine and it includes the study of effect of temperature, oxidation, acid degradation and base degradation. For acidic hydrolysis 1N HCL and 0.1N HCL, for basic hydrolysis 0.1 N NaOH, for oxidation study 3% H₂O₂ was used and the resultant solution were allowed to stand for one day. And thermal stress was applied by heating the drug at 60⁰C for 24 hrs. The results are shown in the table no.2.and the spectrum are shown in the figure no.2 to 6.

Table no.2: Forced degradation data of Nevirapine

S. No	Type of degradation	% Recovery
1	Alkali (0.1N NaOH, 27˚C,24 hours)	125.16% (degraded)
2	Acid (0.1 N HCL, 27˚C 24hrs)	96.02%
3	Acid ( 1N HCL 27˚C, 24hrs)	12.037 (degraded)
4	Oxidation (3% H₂O₂,27˚C,24hrs)	137.94%(degraded)
5.	Thermal stress( 60˚C, 24hrs)	107.47%(degraded)

Figure no.2: Degradation of Nevirapine by 0.1N NaOH

Figure no.3: Degradation of Nevirapine by 0.1N HCL

Figure no. 4: Degradation of Nevirapine by 1N HCL

Figure no.5: Degradation of Nevirapine by 3% H₂O₂

Figure no.6: Degradation of Nevirapine by thermal stress at 60⁰c

RESULT AND DISCUSSION:

Accuracy:

Accuracy of the method was demonstrated at three different concentration levels (80‐120%) by spiking a known quantity of standard drugs into an analyzed sample in triplicate. The results of accuracy table no.3 revealed that the method was more accurate.

Table no.3: Recovery results of Nevirapine

S. No	Target concentration %	Known amount ( µg/ml)	Amount added (µg/ml)	Amount found (µg/ml)	% Recovery
1	50	10	5	15.6	100.4
2	100	10	10	20.33	101.65
3	150	10	15	25.24	100.96

Linearity:

At absorption maxima 283nm, the absorbance were measured for the standard solutions of varying concentrations like 10, 12, 14, 16, 18, 20, 22 and 24μg/ml. Linearity of Nevirapine was found to exist between the concentration ranges of 10 to 24 μg/ml. The linear regression parameters like correlation coefficient, slope and intercept are calculated and tabulated in table no.4.

Table no.4: Optical characteristics and precision of the developed method

Parameters	Optical characteristics
Lambda max	283nm
Beer′s law limit	10 to 24µg/ml
Slope	0.0301
Intercept	0.0036
Correlation coefficient	0.9998
%RSD	0.0431

Precision:

Precision was evaluated by carrying out six replicates of concentration (10 μg/ml of Nevirapine). The intra‐ and inter‐day variation of the method was carried out for one concentration level. Percentage relative standard deviation (%RSD) was found to be less than 2% for within a day and day to day variations, which proves that method is precise. Results are shown in table no.5.

Limit of detection (LOD) and limit of quantization (LOQ):

The limit of detection and limit of quantification for Nevirapine were calculated from the linearity data using relative standard deviation of the response and slope of the calibration curve. The limit of detection of a compound is defined as the lowest concentration of analyte that can be detected. LOD value of Nevirapine was found to be 0.0059 μg/ml. The limit of quantification is the lowest concentration of a compound that can be quantified with acceptable precision and accuracy. LOQ value of Nevirapine was found to be 0.0178μg/ml.

Table no.5: Precision results of Nevirapine

S. No	Concentration in µg/ml	Absorbance at 283nm
1	10	0.2310
2	10	0.2314
3	10	0.2324
4	10	0.2303
5	10	0.2330
6	10	0.2309
Mean		0.2315
SD		0.0001
%RSD		0.0431

CONCLUSION:

During the study it was observed that upon treatment of Nevirapine with 0.1N NaOH, 0.1N HCl, 1 N HCL and 3% hydrogen peroxide was kept aside at room temperature for 24 hrs and the solution in thermal stress at 60⁰c temperature was kept aside for 24 hrs. This developed UV method for estimation of Nevirapine is accurate, precise and stability indicating from the above results, it was concluded that the drug degraded more in 0.1N NaOH, 1N HCL, 3% H₂O₂ compared to 0.1N HCL and thermal stress at 60⁰c.

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Received on 03.07.2011 Modified on 09.07.2011

Research J. Pharm. and Tech. 4(9): Sept. 2011; Page 1386-1388