INTRODUCTION:

It is a 4-amino-5-fluoro-1-[(2R,5S)-2 (hydroxyl -methyl)-1,3-oxathiolan-5-yl]-1,2-dihydro- pyrimidin-2-one. Emtricitabine is a nucleoside reverse transcriptase inhibitor (NRTI) for the treatment of HIV infection in adults. EMT is an analogue of cytidine. The drug works by inhibiting reverse transcriptase, the enzyme that copies HIV RNA into new viral DNA. According to the literature review several methods has been developed for drug, like spectroscopy methods^1-6. HPLC^7-22, and HPTLC^{23, 24}.

These spectroscopy methods can be used for the routine analysis. In the proposed methods optimization and validation of this method are reported. The proposed methods involve formation of ion pair complexes of emtricitabine with reactive dyes like Congo red, eriochrome black T, methyl orange in acidic medium.

Structure of Emtricitabine:

MATERIALS AND METHODS:

A Shimadzu-160 A double beam UV-Visible recording spectrophotometer with pair of 10mm matched quartz cell was used to measure absorbance of solutions. A Shimadzu analytical balance was used.

Congo red, Eriochrome black T, Methyl orange, Hydrochloric acid, Potassium hydrogen phthalate and Chloroform of A.R. grade were used in the study.

Preparation of Standard Solution and Reagents:

Stock solution of Emtricitabine (100μg/ml) was prepared in distilled water. From this stock solution working standard (10μg/ml) was prepared by diluting 10ml stock solution to 100ml with distilled water. A 0.05% w/v Congo red, 0.25% eriochrome black T and 0.02% methyl orange solutions were prepared in distilled water respectively.

Potassium hydrogen phthalate buffer solution of pH 4.01was prepared in distilled water. Dilute hydrochloric acid was used to adjust desired pH of buffer solution.

EXPERIMENTAL:

Method 1 (with Congo Red):

Into a series of separating funnels appropriate amount of the working standard drug solutions were pipetted out. To each funnel 1.0ml of buffer (pH= 3.7) and 5.0ml of 0.05% w/v congo red were added. 10 ml of chloroform was added to each funnel. The solutions were shaken for thorough mixing of the two phases and were allowed to stand for clear separation of the layers. The absorbance values of the chloroform layers were measured against their respective reagent blank at the wavelength of the maximum absorbance (λ max =490nm.)

Method 2(with Eriochrome Black T):

Into a series of separating funnels appropriate amount of the working standard drug solutions were pipetted out. To each funnel 4.0ml of buffer (pH = 3.7) and 3.5ml of 0.25% w/v eriochrome black T were added. 10ml of chloroform was added to each funnel. The solutions were shaken for thorough mixing of the two phases and were allowed to stand for clear separation of the layers. The absorbance values of the chloroform layers were measured against their respective reagent blank at the wavelength of the maximum absorbance (λ max= 500 nm).

Method 3(with Methyl Orange):

Into a series of separating funnels appropriate amount of the working standard drug solutions were pipetted out. To each funnel 1.0ml of buffer (pH= 3.7) and 5.5ml of 0.02% w/v methyl orange were added. 10ml of chloroform was added to each funnel. The solutions were shaken for thorough mixing of the two phases and were allowed to stand for clear separation of the layers. The absorbance values of the chloroform layers were measured against their respective reagent blank at the wavelength of the maximum absorbance (λ max =430 nm).

Estimation from Tablets:

Twenty tablets were weighed accurately and average weight of each tablet was determined. Powder equivalent to 10 mg of Emtricitabine was weighed and transferred in 100 ml of volumetric flask. A 30 ml of distilled water was added and sonicated for 15 minutes and filtered. The filtrate and washing were diluted up to the mark with distilled water to give concentration as 100 μg /ml. Such solution was used for analysis.

Appropriate aliquots of drug solution were taken and the individual assay procedures were followed for the estimation of drug contents in tablets. The concentration of the drug in the tablets was calculated using calibration curve. The recovery experiment was carried out by standard addition method. Results of analysis of optical and regression of drug are given in table 1.

Table 1: Values of results of optical and regression of drug

Parameter	Congo Red	Eriochrome black T	Methyl orange
Detection Wavelength (nm)	490	500	430
Beer Law Limits (µg/ml)	5-30	2-14	2-18
Correlation coefficient (r²)	0.9999	0.9999	0.9999
Regression equation (y=b+ac)
Slope (a)	0.01	0.0154	0.0203
Intercept (b)	0.0023	0.0023	0.0005

RESULTS:

The extractive spectrophotometric methods are popular due to their sensitivity in assay of the drug and hence ion pair extractive spectrophotometric methods have gain considerable attention for quantitative determination of many pharmaceutical preparations. These proposed methods are extractive spectrophotometric methods for the determination of emtricitabine, by using chloroform as solvent from its formulations. The colour ion pair complexes formed are very stable. The working conditions of these methods were established by varying one parameter at time and keeping the other parameters fixed by observing the effect produced on the absorbance of the colour species. The various parameters involved for maximum colour development for these methods were optimized. The proposed methods were validated statistically and by recovery studies. The molar absorptivity values showed the sensitivity of methods while the precision was confirmed by %RSD (relative standard deviation). The optical characteristics such as absorption maxima (nm), co-relation coefficient (r) were calculated and are also summarized in table 1. Assay results of recovery studies are given in table 2 A, B, C.

Table no 2: A (Congo red)

Amount of Sample Added in (µg/ml)	Amount of Standard Added in (µg/ml)	Total amount recovered	Percentage recovery (%)	Standard deviation	Percentage of relative standard deviation (C.O.V.)
5	0	5.014257	100.2851	0.011316	0.225683
5	5	10.0941	100.941	0.068869	0.682268
5	10	15.08412	100.5608	0.089794	0.595288
5	15	20.17821	100.8911	0.013425	0.066532
				Mean= 0.045851	Mean= 0.392443

Table no 2: B (Eriochrome black T)

Amount of Sample Added in (µg/ml)	Amount of Standard Added in (µg/ml)	Total amount recovered	Percentage recovery (%)	Standard deviation	Percentage of relative standard deviation (C.O.V.)
2	0	2.015179	100.7589	0.010739	0.532909
2	2	3.996429	99.91071	0.054469	1.362946
2	4	6.014286	100.2381	0.00935	0.155466
2	6	8.014286	100.1786	0.022452	0.28015
				Mean= 0.024253	Mean= 0.582868

Table no 2:C (Methyl orange)

Amount of Sample Added in (µg/ml)	Amount of Standard Added in (µg/ml)	Total amount recovered	Percentage recovery (%)	Standard deviation	Percentage of relative standard deviation (C.O.V.)
2	0	2.013415	100.6707	0.012437	0.617689
2	2	4.031707	100.7927	0.051028	1.265676
2	4	6.042683	100.7114	0.054926	0.908974
2	6	8.012195	100.0762	0.050493	0.630197
				Mean= 0.042221	Mean= 0.855634

Results are in good in agreement with labelled value.

DISCUSSION:

The percent recovery obtained indicates noninterference from the common excipients used in the formulation. The reproducibility, repeatability and accuracy of these methods were found to be good, which is evidenced by low standard deviation. The proposed methods are simple, sensitive, accurate, precise and reproducible. They are directly applied to drug to form chromogen. Hence, they can be successfully applied for the routine estimation of emtricitabine, in bulk and pharmaceutical dosage form even at very low concentration and determination of stability of drug in formulation. The strong recommendation is made here for the proposed methods for determination of emtricitabine, from its formulation.

ACKNOWLEDGMENTS:

Authors express sincere thanks to the Principal, of D.G. Ruparel College, for providing necessary facility for research work.

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Received on 22.01.2025 Revised on 12.02.2025

Accepted on 19.02.2025 Published on 24.02.2025

Available online from February 27, 2025

Asian J. Research Chem.2025; 18(1):1-4.

DOI: 10.52711/0974-4150.2025.00001

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