Visible Spectrophotometric Determination of Lamivudine in Tablet Dosage Form

Lakshmi Aswini G.¹*, Dhachinamoorthi D.² and Prasada Rao CH.²

¹Dept. of Pharmaceutical Analysis, Vagdevi College of Pharmacy, Gurazala -522415, India.

²Dept. of Pharmaceutical Analysis, QIS College of Pharmacy, Ongole -523272, India.

*Corresponding Author E-mail: aswinipharmagl@gmail.com

ABSTRACT:

A simple visible spectrophotometric method has been developed for the estimation of Lamivudine in bulk and tablet dosage form. This method is based on the diazotization of Lamivudine with nitrous acid to form diazotized Lamivudine, followed by its coupling with β-naphthol to form a red coloured chromogen which shows maximum absorption at 553.0 nm and obeys Beer’s law in the concentration range of 5-20 mcg/ml. This method was validated for accuracy, precision and ruggedness. Statistical analysis proves that the method is reproducible and selective for the estimation of said drug.

KEYWORDS: Visible spectrophotometry; Lamivudine; β-naphthol; Validation.

INTRODUCTION:

Lamivudine is a nucleoside reverse transcriptase inhibitor used in the treatment of AIDS. Chemically Lamivudine is (1R, cis)-4-amino-1-(1-hydroxy methyl-1, 3-oxathiolan-5-yl)- (1H)-pyrimidine-1-one¹. A very few analytical methods appeared in the literature for the determination of Lamivudine includes its estimation with other drugs^2-9. There is no reported method on spectrophotometry and therefore, require much more investigation. The proposed method is based on the visible spectrophotometric technique.

MATERIAL AND METHODS:

A Systronics UV-Vis double beam spectrophotometer (model 2201) with 1 cm matched quartz cells was used for all spectral measurements. All the chemicals used in the investigation were of analytical grade. Authentic drug sample of Lamivudine was given as a gift sample by Hetero drugs limited, Hyderabad. Tablets of Lamivudine are procured from local market.

Working Standard Solution of Lamivudine:

Standard stock solution was prepared by dissolving accurately weighed, 100mg of Lamivudine in distilled water and the volume was made upto 100ml with distilled water (stock solution-I, 1000 mcg/ml).From this, a working standard solution containing 100 mcg/ml was prepared with distilled water.

Sample Preparation of Lamivudine:

20 tablets of two different brands of Lamivudine were weighed and powdered in glass mortar and the powder equivalent to 25 mg of Lamivudine was weighed accurately and transferred into a 25 ml standard volumetric flask. The contents were dissolved in distilled water and sonicated for five minutes. This solution was filtered through 0.45 µWhatmann filter paper. 5 ml of the filtrate was diluted to 50 ml with distilled water to get the solution of 100 mcg/ml.

Development of colour and study of spectra in visible region:

Aliquots of standard solution of Lamivudine ranging from 0.5 to 2.0 ml (1 ml = 100 µg) were transferred into a series of 10 ml volumetric flasks. To each flask, 1.0 ml of hydrochloric acid (2 N) and 1.0 ml of sodium nitrite (0.1% w/v) was added and a reaction time of 10 min at 0-5°C was given for the completion of reaction. Then 1.0 ml of alkaline β-naphthol solution (0.1% w/v in 2% aqueous NaOH) was added to each flask with gently shaking and after 10 min, the volume in each flask was made up to 10 ml with distilled water. The absorbances of red coloured chromogen were measured at 553.0 nm against the reagent blank. The coloured chromogen was stable for 3 h. The amount of Lamivudine present in the sample solution was computed from the respective calibration curve.

Fig. 01- Absorption Spectrum of Lamivudine with β-naphthol.

RESULTS AND DISCUSSION:

In the present investigation the λmax of the Lamivudine was found to be 553.0 nm. Lamivudine follows linearity in the concentration range of 5- 20 mcg/ml. Two brands of tablets were analyzed and amount of drug were determined by proposed method; it was in good agreement with the label claim. The proposed method was validated as per the ICH guidelines. The recovery studies were carried out by adding a known amount of drug to pre analysed sample at two different levels and the % recoveries were ranges from 99.74-99.95%, which shows the accuracy of method. Intra-day and Inter-day precision of the assay was determined by analyzing the drug sample using same concentration. The intra-day and inter-day % RSD values were calculated and lying in the range of 1.121-1.261%. Ruggedness of proposed method was studied with the help of two different analysts and results were evaluated by calculating the % RSD values; lying within the range of 1.193-1.235%.

RESULTS OF ANALYSIS:

Table 01: Optimum conditions, optical characteristics and statistical data of the regression equation in the proposed method

Parameter	Values
l_max (nm)	553
Beer’s law limits (mcg/ml)	5-20
Molar extinction coefficient (mol^-1cm^-1)	0.0465 X10⁴
Sandell’s sensitivity (mcg/cm²-0.001 absorbance units)	0.021
Regression equation (Y*)	Y=0.0092C + 0.0006
Slope (b)	0.0092
Intercept (a)	0.0006
Correlation coefficient(r²)	0.9995
% RSD**	0.648
Limit of detection (mcg/ml)	0.130
Limit of quantitation (mcg/ml)	0.422

*Y= bC + a where C is the concentration of Lamivudine in mcg/ml and

Y is the absorbance at the respective l_max.

**Average of five determinations.

Table 02: Results of analysis

Brand used	Label claimed(mg)	Amount found by proposed method(mg)	% label claim	% RSD*
Tab-a	100	99.77	99.77	0.86
Tab-b	100	99.56	99.56	1.18