A UPLC method for simultaneous estimation of emtricitabine, tenofovir disoproxil fumarate and efavirenz in pharmaceutical dosage forms

 

Jane Jacob*, Sreekanth Nadig

Nitte University, Dept. of Pharmaceutical Chemistry, NGSM Institute of Pharmaceutical Sciences,  Deralakatte, Mangalore -575018,Karnataka State.

 

ABSTRACT:

A simple, fast, reproducible, reliable and sensitive UPLC method was developed for the simultaneous estimation of anti-retroviral drugs in combination namely, Emtricitabine, Tenofovir disoproxil fumarate and Efavirenz. The chromatographic separation was carried out on a sub 2 micron particle size column Waters Acquity UPLC BEH C18, 100 x 2.1 mm, 1.7 µm to achieve more efficiency and better resolution in a gradient mode elution consisting of mobile phase A : Buffer (0.05% of Trifluro acetic acid in water) and mobile phase B: Methanol. Detection wavelength was set at 262 nm with a flow rate of 0.4ml/min. The retention times of Emtricitibine, Tenofovir disoproxil fumarate and Efavirenz was 0.6, 1.88 and 3.23 minute respectively. Under optimized conditions all three components were well separated from each other and also from main degradation impurities like Monoester impurity of Tenofovir, S-Oxide impurity of Emtricitabine and Amino alcohol and Quinoline impurity of Efavirenz. This was further supported by forced degradation studies. The % RSD was well within the limits and the correlation coefficient was 0.999.The developed method was validated as per ICH guidelines and can be used in routine quality testing of individual dosage forms and in combination.

 

 

 

INTRODUCTION:

Antiretroviral drugs are medications for the treatment of infection caused by retroviruses, primarily HIV. Different class of antiretroviral drugs is used in treating HIV infection by making a single dosage unit containing two or more antiretroviral drugs (Highly Active Antiretroviral therapy, or HAART) ^[1] .  When drugs are given as a fixed dose combination product they have shown to improve therapy in terms of sustained virological suppression and significant reduction in the mortality rates of the HIV/AIDS infected people rather than individual entities ^[2] . 

 

Now complex regimens are being converted into simpler formulas, necessitating development of new analytical methods for estimation of multiple analytes in a single method. Since the uses of these drug products are tremendously increasing, there is a need for controlling the quality of these drug products which are sold in the underdeveloped and developing countries. There are few HPLC methods reported for the estimation of Emtricitabine, Tenofovir disoproxil fumarate and Efavirenz from pharmaceutical dosage form ^[3-5] .

 

However no significant information was available in literature on UPLC method for the estimation of Emtricitabine, Tenofovir disoproxil fumarate and Efavirenz from pharmaceutical dosage forms. With this interest a simple ,economical and fast  stability indicating assay method was developed and validated for the simultaneous estimation of  Emtricitabine, Tenofovir disoproxil fumarate and Efavirenz in formulated tablets in different combination and individually.

Chemically,Emtricitabine is (4-amino-5-fluoro-1-[(2S,5R)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2-dihydropyrimidin-2-one) , Tenofovir disoproxil fumarate is (9-[(R)-2 [[bis[[(isopropoxycarbonyl)oxy]-  methoxy]phosphinyl]methoxy]propyl]adenine fumarate (1:1)) and Efavirenz is ((4S)-6-chloro-4-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3,1- benzoxazin-2-one) belonging to category of reverse transcriptase inhibitors.  This combination of three medications was approved by the U.S. Food and Drug Administration (FDA) in July 2006 under the brand name Atripla, provides HAART in a single tablet taken once a day ^[6]. It results in a simplified drug regimen for many patients.UPLC helps in improving speed, sensitivity, resolution and reduction in solvent consumption. UPLC makes use of stationary phase with particle size less than 2µm.

 

METHODS AND MATERIALS:

Chemicals and reagents: All the reagents and chemicals used are of analytical grade, HPLC grade Methanol, Trifluroacetic acid were obtained from Rankem chemicals and ACROS chemicals respectively. HPLC grade water was obtained from Milli Q water purification system.

 

Equipment: Ultra performance Liquid chromatography system (from Waters) with auto sampler and Photo Diode Array detector were used for the study. Data was acquired and processed by using Waters Empower software.

 

Experimental procedure:  The chromatographic column used was Acquity UPLC BEH C18, 100x 2.1 mm, 1.7µm   (Make:Waters).Separation was achieved through gradient elution .Buffer (0.05% Trifluro acetic acid in water) as mobile phase A and Methanol as mobile phase B was used as binary gradient  with the flow rate of 0.4 ml/min. Detection wavelength was monitored at 262 nm. Column oven temperature was maintained at 40°C with the injection volume of 1 µL. Finalized gradient program is as follows, Time (min)/%Solution B , 0/15,2/70,4/70,4.2/15 and 5.5/15.

 

Sample Preparation: Methanolic HCl (0.1N) was used as extraction diluent and further dilution was done with water and methanol in the ratio of 65:35 respectively.

 

Standard Preparation: Stock solution was prepared by accurately weighing 120 mg of Efavirenz,
40 mg of Emtricitabine and 60 mg of Tenofovir in 100 mL volumetric flask, 70 mL diluent was added then sonicated to dissolve and diluted to volume with diluent. Further 5 mL of this stock solution was diluted to 50 mL and diluted to volume with mixture of water and methanol to get concentration of 120μg/ml for Efavirenz, 40μg/ml for Emtricitabine and 60μg/ml for Tenofovir disoproxil fumarate respectively.

 

Test Preparation: Test solution was prepared by taking homogenous mixture of tablet powder equivalent to 300 mg of Efavirenz or 100 mg Emtricitabine or 150 mg of Tenofovir in to a 250 mL volumetric flask, added about 180 mL of methanolic HCl, sonicated for about 30 minutes. Volume was made up to 250 mL with methanolic HCl and mixed well. Further 5 mL of this solution was diluted to 50 mL with mixture of water and methanol (65:35 v/v) and filtered through 0.45 µm filters to get final concentration of 120μg/ml for Efavirenz, 40μg/ml for Emtricitabine and 60μg/ml for Tenofovir disoproxil fumarate respectively.

 

Optimized Conditions: Column used (Acquity UPLC BEH C18 , 100x 2.1 mm ,1.7µm  ) is a most commonly used UPLC column. All chemicals are also easily available. A gradient elution method was developed based on the polarity of individual molecule. Diluent for extraction was selected based on the solubility of drug substances.

 

Under above optimized conditions all three components were well separated from each other and also from main degradation impurities like Monoester impurity of Tenofovir, S-Oxide impurity of Emtricitabine and Amino alcohol impurity, Quinoline impurity of Efavirenz.

 

Method Validation:Above optimized analytical method was subjected for validation to check its Specificity, Precision, Accuracy and Linearity.Validation activity was planned as per the International Conference on Harmonization (ICH) guidelines[7]. Procedures, experimental design and acceptance criteria were followed as per the general industrial practices.

 

RESULTS AND DISCUSSIONS:

System suitability: As integral part of chromatographic method system suitability parameters like USP Tailing and Relative standard deviation (RSD) for replicate injections were evaluated and found to be satisfactory as per general chromatographic practices. Results are shown in Table No 1.

 

Fig 1.Typical Chromatogram of Std solution Spiked with Known Degradent impurities.

Table 1: Results of System Suitability Test

System suitability parameters	Emtricitabine	Tenofovir disoproxil	Efavirenz	Acceptance criteria
%RSD for area response of replicate standard injections.	1.3	1.3	1.3	NMT 2%
Tailing factor	1.3	1.1	1.0	NMT 2.0

 

Specificity: To demonstrate the stability indicating power of analytical method used, specificity was proved by injecting the known impurities, placebo and by performing forced degradation studies under different stress conditions like Acid hydrolysis, Alkali hydrolysis, Oxidation by Peroxide and thermal degradation studies. From this it was proved that possible degradents are well separated from the analytes peak, which was supported by peak purity results of each analyte peak. Results are shown in Table 2 and 3.

 

Table 2: Results of specificity-impurity interference experiment

S. No	Name of Impurity/Analyte	Retention time (min)
1	Emtricitabine	0.560
2	Tenofovir	1.885
3	Efavirenz	3.229
4	S-Oxide Impurity of Emtricitabine	0.377
5	Des amino Impurity of Emtricitabine	0.670
6	Monoester impurity of Tenofovir	1.015
7	Amino alcohol impurity of Efavirenz	2.553
8	Qinoline impurity of Efavirenz	Not detected

 

 

 

Table 3: Results of specificity forced degradation experiment

Stress Conditions	% Degradation	Peak Purity		Purity Flag
		Purity    Angle	Purity    Threshold
Treated with 0.1N HCl solution at room temperature for about 10 minutes
Emtricitabine	5.9	0.182	1.455	No
Tenofovir		0.913	2.530	No
Efavirenz		0.221	1.165	No

 

Stress Conditions	% Degradation	Peak Purity		Purity Flag
		Purity    Angle	Purity    Threshold
Treated with 0.1N NaOH solution at room temperature for about 10 minutes
Emtricitabine	27.8	0.156	1.210	No
Tenofovir		3.739	4.550	No
Efavirenz		0.171	0.918	No

 

 

 

Stress Conditions	% Degradation	Peak Purity		Purity Flag
		Purity    Angle	Purity    Threshold
Treated with 3% H2O2 solution for about 10 minutes at room temperature
Emtricitabine	9.3	1.019	7.857	No
Tenofovir		8.673	24.898	No
Efavirenz		1.822	11.629	No

 

 

 

 

Stress Conditions	% Degradation	Peak Purity		Purity Flag
		Purity    Angle	Purity    Threshold
Exposed to heat at 90°C temperature for about 24 hours
Emtricitabine	6.3	0.145	1.190	No
Tenofovir		0.583	1.662	No
Efavirenz		0.140	0.820	No

 

 

Acceptance criteria : "Purity angle should be less than purity threshold " and purity flag should be "No"

 

Linearity: Linearity of the analytical procedure was demonstrated by preparing and analyzing the standard preparations at six different levels. Linearity solutions were prepared at 25, 50, 75, 100, 125 and 150% of standard concentration for all the three analytes. From this experiment co efficient of correlation, Intercept and slope were calculated by plotting a graph between peak responses and concentrations. Correlation coefficient was >0.999 and the components showed good correlation between concentration and peak responses. Results are shown in Table 4.

 

 

 

 

Table 4:  Results of Linearity Studies (Response Vs Concentration)

 

Precision: Method precision was performed by preparing the six test preparation from same lot of homogenous tablet powder and they were injected in to chromatography. From this experiment relative standard deviation (RSD) was determined for % Assay results for each analyte. The results indicate that the method is precise. Results are shown in Table 5.

 

Accuracy: Accuracy of the method was tested by preparing the test preparation at three different levels 50,100 and 150% and analyzing by chromatography. Test preparations were prepared by mixing placebo and drug substances at above said levels. Test preparations were done in triplicate at each level. Amount of drug added, amount drug found, % recovery and Relative standard deviation was determined for %recovery results for each analyte. % Recovery results were found to be satisfactory . This indicates the capabability of method to recover the analytes from placebo matrix. Results are shown in fig 5.

 

Table 5: Results of Method precision (Repeatability studies)

Sample No	% Assay  of Emtricitabine	% Assay  of Tenofovir	% Assay  of  Efavirenz
Sample 1	99.0	100.7	99.6
Sample 2	97.0	99.0	97.9
Sample 3	98.3	99.9	99.0
Sample 4	96.9	98.6	97.9
Sample 5	96.4	98.1	97.1
Sample 6	97.2	99.0	98.1
Mean	97.5	99.2	98.3
%RSD	1.0	0.9	0.9
Accepatance criteria : %RSD should be NMT 2%

 

 

 

 

 

Table 6: Results of Recovery Study at Different Levels

  Note: Number of samples analyzed at each level is in triplicate

 

CONCLUSION:

A simple, faster and economical UPLC method could be developed for the estimation of Emtricitibine,Tenofovir Disoproxil fumarate and Efavirenz in formulated products in combination or individually. Method requires all commonly available materials for analysis. Analytical method was validated as per ICH guidelines and general industrial practices and proved that it is suitable for its intended purpose.

 

The above validated chromatographic method can be used by government agencies, government laboratories, research institutions and manufacturing companies to analyze the drug product to check the quality of it.

 

ACKNOWLEDGEMENT:

The authors are grateful to Nitte University and  NGSM Institute of Pharmaceutical Sciences, Mangalore, India for their support.

 

REFERENCES:

1.     Bartlett JG. Ten years of HAART: Foundation for the future. In 13th Conference on Retro. 2006, February.

2.     Katlama C, Ingrand D, Loveday C, et al. Safety and efficacy of lamivudine-zidovudine combination therapy in antiretroviral-naive patients. JAMA: the journal of the American Medical Association. 1996, 276:118-125.

3.     Montgomery ER, Edmanson AL, Cook SC, et al. Development and validation of a reverse-phase HPLC method for analysis of efavirenz and its related substances in the drug substance and in a capsule formulation. J Pharm Biomed Anal. 2001, 25:267-284.

4.     Matthews CZ, Woolf EJ, Mazenko RS, et al. Determination of efavirenz, a selective non-nucleoside reverse transcriptase inhibitor, in human plasma using HPLC with post-column photochemical derivatization and fluorescence detection. J Pharm Biomed Anal. 2002, 28:925-934.

5.     Bhargavi YG and Reddy KT. Method development and validation of Lamivudine, Tenofovir and Efavirenz in a combined dosage form by RP-HPLC. Journal of Pharmacy Research. 2012, 5:711-714.

6.      www.fda.gov, HIV/AIDS Historical Time Line 2000- 2010.

7.     Guideline, I. H. T. (2005). Validation of analytical procedures: text and methodology Q2 (R1). IFPMA: Geneva

 

 

 

 

Accepted on 15.07.2017        © RJPT All right reserved

Research J. Pharm. and Tech 2017; 10(12): 4463-4466.