Development and Validation of HPLC Method for the Estimation of Emtricitabine in Capsule Dosage Form

 

Bhavini N. Patel¹*, Bhanubhai N. Suhagia²and Chaganbhai N. Patel¹

¹Department of Pharm. Chem., Shri Sarvajanik Pharmacy College, Mehsana-384001

²Department of Pharm. Chem., L.M. College of Pharmacy, Navrangpura, Ahmedabad-380009

*Corresponding Author E-mail: bhavi_pharma22783@yahoo.co.in

ABSTRACT:

A simple, precise, rapid and accurate reverse phase HPLC method was developed for the estimation of emtricitabine in capsule dosage form. A Phenomenex (Torrance, CA) C8 column, 250× 4.6 mm id, column, at ambient temperature with mobile phase consisting of 0.03M potassium dihydrogen phosphate (pH 4.86±0.02): Acetonitrile: Methanol (40:20:40 v/v/v) was used. The flow rate was 1 mL/min and the effluent was monitored at 260 nm. The retention time was 2.95 min. The detector response was linear in the concentration of 8-60 µg/mL. The respective linear regression equation being Y=27675x+41556. The limit of detection and limit of quantification was 0.06 and 0.20µg/mL respectively. The percentage assay of emtricitabine was 100.53%. The method was validated by determining its accuracy, precision and system suitability. The results of the study showed that the proposed RP-HPLC method is simple, rapid, precise and accurate, which is useful for the routine determination of emtricitabine in bulk drug and in its pharmaceutical dosage form.

 

 

 

INTRODUCTION:

Emtricitabine^1-2 is chemically known as 4-amino-5-fluoro-1-[(2R, 5S)-2-(hydroxymethyl)-1, 3-oxathiolan-5-yl] pyrimidin-2-one. Emtricitabine is a nucleoside reverse transcriptase inhibitor (NRTI) for the treatment of HIV infection in adults. Literature survey reveals many chromatographic methods^3-8 for the determination of emtricitbine, in biological fluids. So far, no assay procedure has been reported for the estimation of emtricitabine from pharmaceutical dosage form. The aim of the study was to develop a simple, precise and accurate reversed-phase HPLC method for the estimation of emtricitabine in bulk drug samples and in pharmaceutical dosage form.

 

Structure of Emtricitabine

 

MATERIALS AND METHODS:

Materials:

Emtricitabine was obtained as a gift sample from Cipla Ltd, Mumbai. Potassium dihydrogen orthophosphate was of analytical grade, supplied by M/s S. d. Fine Chem Limited, Mumbai. Acetonitrile and water used were of HPLC grade (Finar Lab.). Commercially available Emtricitabine capsule (Emtriva   200mg) were procured from local market.

 

Instrumentation:

A Shimadzu (Columbia, MD) RP-HPLC instrument (LC-10AT vp) equipped with UV detector, manual injector with 20μL loop, Phenomenex (Torrance, CA) C8 column (250× 4.6 mm id, 5 μm particle size), Class-VP software was used.

 

Chromatographic condition:

The chromatographic separation was performed using Phenomenex (Torrance, CA) C8 column (250× 4.6 mm id, column, at ambient temperature. The mobile phase containing KH₂PO₄ (0.03M) pH 4.86±0.02: Acetonitrile: Methanol (40:20:40 v/v/v) was selected because it was found ideally resolves the peak of EMT (RT=2.95), respectively as shown in Fig. 1. Wavelength was selected by scanning standard solutions of drug over 200 nm to 400 nm wavelengths. Measurement was made at the effluent flow rate of 1.0 ml/min with injection volume 20 μl and ultraviolet (UV) detection at 260 nm, as emtricitabine shows reasonable good response at this wavelength.

 

Preparation of standard stock solution:

A standard stock solution of the drug was prepared by dissolving 25 mg of emtricitabine in 25 mL volumetric flask containing 15 mL of methanol, sonicated for about 15 min and then made up to 25 mL with methanol to get 1 mg/mL standard stock solution.

 

Working standard solution:

1 mL of stock solution was taken in 10 mL volumetric flask and thereafter made up to 10 mL with mobile phase to get a concentration of 100 µg/mL.

 

Preparation of sample solution:

Weigh accurately a quantity of the mixed contents of 20 capsules, equivalent to 25 mg of the active ingredient, was mixed with 15 mL of methanol. The solution was sonicated for about 15min, and then filtered through a 0.45 µm membrane filter, followed by adding methanol to obtain a stock solution of 1.0 mg/mL. 1 mL of this solution was transferred to a 10 mL volumetric flask and made up to sufficient volume with mobile phase to give a concentration of 100 µg/mL.

 

Linearity:

Aliquots of standard emtricitabine stock solution were taken in different 10 mL volumetric flasks and diluted up to the mark with the mobile phase such that the final concentrations of emtricitabine are in the range of 8-60 µg/mL. Each of these drug solutions (20 µL) was injected three times into the column, and the peak areas and retention times were recorded. Evaluation was performed with U.V. detector at 260 nm and a calibration graph was obtained by plotting peak area versus concentration of emtricitabine.

 

Figure 1. Typical chromatogram of emtrictabine by HPLC

 

The plot of peak area of each sample against respective concentration of emtricitabine was found to be linear in the range of 8-60 µg/mL with correlation coefficient of 0.9934. Linear regression least square fit data obtained from the measurements are given in Table 1. The respective linear regression equation being Y=27675x+41556. The regression characteristics, such as slope, intercept, and % RSD were calculated for this method and given in Table 1.

 

Table 1. Linear regression data for calibration curves.

Drug	Emtrictabine
Concentration range, µg/mL	8-60
Slope, m	27675
Intercept, b	41556
Correlation coefficient	0.9934
% RSD	0.50

 

Assay:

20 µL of sample solution was injected into the injector of liquid chromatography. The retention time was found to be 2.95 minutes. The amount of drug present per capsule was calculated by comparing the peak area of the sample solution with that of the standard solution. The data are presented in Table 2.

 

Recovery studies:

Accuracy was determined by recovery studies of emtricitabine, known amount of standard was added to the preanalysed sample and subjected to the proposed HPLC analysis. Results of recovery study are shown in Table 2. The study was done at three different concentration levels.

 

RESULTS AND DISCUSSION:

The system suitability tests were carried out on freshly prepared standard stock solution of emtricitabine. Parameters that were studied to evaluate the suitability of the system are given in Table 3.

 

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

The limit of detection (LOD) and limit of quantification (LOQ) for emtrictabine were found to be 0.06 µg/mL and 0.20µg/mL respectively. The signal to noise ratio is 3.3 for LOD and 10 for LOQ. From the typical chromatogram of emtricitabine as shown in Fig 1, it was found that the retention time was 2.95 min. A mixture of 0.03M potassium dihydrogen phosphate (pH 4.86±0.02): Acetonitrile: Methanol (40:20:40 v/v/v) was found to be most suitable to obtain a peak well defined and free from tailing. In the present developed HPLC method, the standard and sample preparation required less time and no tedious extraction were involved. A good linear relationship (r=0.9934) was observed between the concentration range of 8-60 µg/mL. Low values of standard deviation are indicative of the high precision of the method. The assay of emtricitabine capsule was found to be 99.83%. From the recovery studies it was found that about 100.53% of emtricitabine was recovered which indicates high accuracy of the method. The absence of additional peaks in the chromatogram indicates non-interference of the common excipients used in the capsule. This demonstrates that the developed HPLC method is simple, linear, accurate, sensitive and reproducible.

 

Table 2: Recovery Data for the Proposed Method (n=3)

Drug	Level	Amount of sample taken (μg/ml)	Amount of standard Spiked (μg/ml)	% Recovery ± S.D (n=3)
Emtricitabine	1	20	10	100.53 ± 1.05
	2	20	20	99.96± 1.04
	3	20	30	101.10± 1.01

 

Table 3. Validation summary

Validation Parameter	Result
Asymmetry factor	1.26
Tailing factor	1.22
Theoretical plate	4043.44
Retention time in minutes	2.95
Limit of detection (μg/ml)	0.06
Limit of quantification (μg/ml)	0.20

 

Thus, the developed method can be easily used for the routine quality control of bulk and capsule dosage form of emtrictabine within a short analysis time.

 

REFERENCE:

1.       O'Neil MJ, Smith A, Heckelman PE, Budavari S. editors. the Merck Index, 13th ed. Whitehouse Station NJ: Merck and Co Inc; 2001. p. 630.

2.       D Roste J.AH, Burger .D.M. J. of liquid Chromatography and related technologies, 2007, 30(17), 2767.

3.       Nase L.Rerk, Angel D.M, J. Chromatography B, 2005, 822(1-2), 201.

4.       Pai N, Desai AD. Simultaneous reverse phase HPLC estimation of some antiretroviral drugs from tablets. Indian J Pharm Sci 2007; 69:118-20.

5.       Sparidans RW, Crommentuyn KM, Schellens JH, Beijnen JH. Liquid chromatographic assay for the antiviral nucleotide analogue tenofovir in plasma using derivitization with chloroacetaldehyde. J Chromatogr B 2003; 791:227-33.

6.       Sethi P.D., HPTLC-Quantitative Analysis of Pharmaceutical Formulations, 1st ed. New Delhi: CBS publishers; 2001.

7.       ICH Q2A, Text on validation of Analytical Procedures, International Conference on Harmonization, Geneva, October 1994: 1-

8.       Pai N, Desai AD. Simultaneous reverse phase HPLC estimation of some antiretroviral drugs from tablets. Indian J Pharm Sci 2007; 69:118-20.

 

 

Received on 16.02.2010        Modified on 12.03.2010

Accepted on 25.03.2010        © AJRC All right reserved