INTRODUCTION:

Emtricitabine is chemically known as 5-fluoro-1-[(2R, 5S)-2-(hydroxymethyl)-1,3-oxthiolan-5yl]cytosin{4-amino-5-fluoro-1-[(2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2-ihydropyimidin-2-one} is a novel and unique nucleoside reverse transcriptase inhibitor (NRTI) for the treatment of HIV infection in adults. Emtricitabine is an analogue of cytidine. The drug works by inhibiting reverse transcriptase, the enzyme that copies HIV RNA into new viral DNA.^[1,2,3]

Tenofovir Disoproxil Fumarate is chemically known as 9-[(R)-2 [[Bis [(isopropoxycarbonyl)oxy]methoxy]-phosphinyl]methoxy]propyl]adenine fumarate (1:1).^[4,5,6]

No official methods for the Emtricitabine determination is available. Various spectrophotometric methods have been reported for the determination of Emtricitabine and other combination in pharmaceutical tablets using different reagents, UV spectrophotometric ^[7] and HPTC Method method ^[8] were developed. The literature review reveals that few methods for determination of Emtricitabine in biological fluids. Many spectrophotometric methods have been applied for the simultaneous determination of Emtricitabine, Tenofovir Disoproxil Fumarate and Efavirenz in Area Under curve, Dual wavelength^[9], UV spectroscopy^[10] and RP-HPLC^[11].

Various analytical methods have been reported for the assay of Tenofovir Disoproxil Fumarate^[17] in pure form as well as in pharmaceutical formulations. They include RP-HPLC^[12-16].

To the best of our knowledge, no study has been described for the simultaneous determination of this combination in pharmaceutical formulation by RP-HPLC method. Hence the aim of the present investigation was to develop simultaneous method for estimation of Emtricitabine and Tenofovir Disoproxil Fumarate has been developed for its Pharmaceutical Preparation. Therefore it is desirable to develop simple and reproducible analytical methods.

MATERIALS AND METHODS:

Chemicals and Reagents

Emtricitabine (EMTRI) and Tenofovir Disoproxil Fumarate (TENO) reference standard received as gift sample from Lauras Laboratory Ltd., India. The pharmaceutical preparations of combination of Emtricitabine and Tenofovir Disoproxil Fumarate that is Truvada contains 200 mg of Emtricitabine and 300 mg of Tenofovir Disoproxil Fumarate equivalent to Tenofovir Disoproxil was purchased from local market. Methanol of analytical reagent grade was purchased by HPLC grade - Merck Pvt. Ltd (India). Sodium Dihydrogen phosphate monohydrate used for buffer preparation by Merck Pvt. Ltd.. (India). All the solutions were analyzed on the day of preparations.

Apparatus

RP-HPLC was performed with a Waters solvent-delivery system, UV–visible photodiode-array detector, Gradient pump system, degasser and a universal loop injector of injection capacity 10 μL. The monitoring software was Empower Version. The equipment was controlled by a PC workstation. Compounds were separated on a 25 cm × 4.6 mm i.d, 5-μm particle, ACE C18 column under reversed-phase partition chromatographic conditions. Ultrasonicator Model USB 30 was used. The work was carried out in an air-conditioned room maintained at temperature 25±2 °C. The flow rate was 1.5 mL min-1, analytes were monitored at 269 nm and with gradient program run time was 16 min.

Mobile Phase

The Mobile Phase was selected Phosphate buffer pH 5.7 (using dilute Sodium hydroxide solution) : Methanol in the ratio (85:15) as Mobile Phase A and Water: Methanol in the ratio of (15:85) with gradient programme.

Table.1 Gradient Programme

Time (in min.)

Mobile Phase A

Mobile Phase B

0.01

5.0

6.0

9.0

11.0

13.0

15.0

16.0

Selection of Diluent

Based on Solubility Methanol is selected as part of the diluent and as tablets dispersed in water so ratio of Water:Methanol in (20:80) is selected.

Standard Stock preparation

Standard stock solution of EMTRI and TENO each were prepared separately in 100 mL of diluent to get the final concentration of 2000 μg mL-1 and 3000 μg mL-1 respectively.

From the standard stock solution of drugs, different dilutions were prepared, injected and their peak area was measured. After that, calibration curves were drawn between concentration against their respective area for EMTRI and TENO respectively.

Standard Mixture Solution

Mixed standard analysis was performed to validate the procedure. From the standard stock solutions of the drugs, Final concentration that achieved 2000 μg mL-1 for EMTRI and 3000 μg mL-1 TENO respectively were prepared and analyzed, statistical results were within the range of acceptance i.e. % RSD<2.0.

Sample Preparation

Twenty tablets were weighed accurately and their average weight was determined. Then take 10 intact tablets weighed and transferred to 1000 ml volumetric flask. 600 ml diluent was added to it and solution was sonicate for 30 min. The volume was adjusted up to the mark with diluent. The solution was then filtered through 0.45µ membrane Filter. Filtrate contained mixture of 2000 μg/ml Emtricitabine and 3000 μg/ml Tenofovir Disoproxil Fumarate. From these Stock solution 5.0 ml was taken in 50 ml volumetric flask and volume was made up with diluents and mix. (200 μg/ml of Emtricitabine and 300 μg/ml of Tenofovir Disoproxil Fumarate).

Table.2 Assay of Tablet Formulation

Sr.No. Drug Label Claim %Assay RSD

(mg tab^-1)(n=10)

1. Emtricitabine 200 99.0 0.11

2. Tenofovir 300 99.4 0.12

Disoproxil Fumarate

RSD: Relative Standard Deviation

RESULTS AND DISCUSSION:

HPLC method development and optimization

Column chemistry, solvent type, solvent strength, detection wavelength and flow rate were varied to determine the chromatographic conditions giving the best separation. The mobile phase conditions were optimized so that the components were not interfered from the solvent and excipients.

After trying column C8 and C18, the final choice of stationary phase giving satisfactory resolution and run time was the reversed phase column ACE C18. Mobile phase and flow rate selection was based on peak parameters (height, area, tailing, theoretical plates and resolution) and run time. The best result was obtained by use of 85:15 ratio of Sodium dihydrogen phosphate (pH 5.7 ± 0.05 adjusted with Diluted Sodium Hydroxide solution) : Methanol as Mobile Phase A and Water: Methanol in ratio of (15:85) as Mobile Phase B with gradient programme with 1.5 mL min.^-1. From the overlain UV spectra (Shimadzu-1700), suitable wavelength considered for monitoring the drugs was 269 nm (Fig 1). Solutions of EMTRI and TENO in diluent were also injected directly for HPLC analysis and the responses (peak area) were recorded. It was observed that there was no interference from the diluent or baseline disturbances and both the analytes absorbed well at 269 nm. The chromatogram of Blank and standard mixture is shown in Fig 2 and 3 respectively.

Under the optimum chromatographic conditions, the retention time obtained for EMTRI and TENO were 3.02 and 9.54 min. respectively for sample preparation shown in Fig 6. The result of tailing factor, theoretical plate number and resolution are reported in Table 3.

The values obtained for these shows that, the chromatographic conditions are appropriate for separation and determination of compounds.

Fig 1. Overlain Spectra of EMRI and TENO

Table 3.System Suitability parameters

Property EMTRI TENO

Rt 3.02 9.54

T f 1.10 1.08

N 5428 214575

R s 25.5 --

Rt-retention time; Tf - tailing factor; N- number of theoretical plates; RS- resolution

Fig 2. Chromatogram of Blanks

Validation of the developed method

The method was validated for linearity, accuracy, precision, repeatability, selectivity and specificity study as per ICH norms ^[19]. All the validation studies were carried out by replicate injection of the sample and standard solutions.

Linearity

Linearity was found to be 50-320 μgmL-1 for EMTRI and 75-480 μgmL-1 for TENO. The linear regression equations for EMTRI and TENO were-

For EMTRI: y = 2995.4328x+198618.0728

For TENO: y = 10737.2411x-1125.8605

Where y is response (peak area) and x is the concentration.

Accuracy

Accuracy of developed method was confirmed by doing recovery study as per ICH norms at three different concentration levels 50%, 100% and 150% by replicate analysis (n=3). The result of accuracy study was reported in Table 4. From the recovery study it was clear that the method is very accurate for quantitative estimation of EMTRI and TENO in tablet dosage form as all the statistical results were within the range of acceptance i.e. %RSD<2.0.

Precision, Limit of Detection, and Limit of Quantitation

The concentrations of both the drugs were measured three times on the same day with different six preparation and with different day, different system and another six preparation for interday study. The limits of detection and quantitation, LOD and LOQ, were calculated by use of the equations LOD = 3.3σ/S and LOQ = 10σ/S, where σ is the standard deviation of the blank and S is the slope of the calibration curve. The results are reported in Table 5.

Fig.3 Standard Mixture Chromatogram

Fig.4 Chromatogram of EMTRI and TENO in Sample solution with their retention time

Table 4. Recovery Studies

Drug	Level	Amount added % (μgmL-1)*	% Recovery	%RSD
EMTRI	50%	300	101.5	0.8
	100%	400	100.8	0.8
	150%	500	100.2	0.2
TENO	50%	450	101.1	0.1
	100%	600	101.2	0.1
	150%	750	101.5	0.1

*Recovery performed by standard addition method; %RSD: Relative Standard Deviation

Table 5. Precision and Inter Day Precision, LOD and LOQ Studies

Drug	Precision (%RSD) (n=6)	Inter day Precision (%RSD) (n=6)	LOD (μgmL-1)	LOQ (μgmL-1)
EMTRI	0.8	0.60	4.01	12.14
TENO	1.06	0.64	8.20	24.86

Selectivity and Specificity

The selectivity was checked by injecting the solution of both the drugs into the system and it was observed that two sharp peaks of EMTRI and TENO having resolution of 25.5 were obtained at retention time of 3.2 and 9.5 min respectively in reference to blank solution. Specificity of the method was assessed by comparing the chromatograms obtained from standard drugs, with the chromatogram obtained from tablet solutions. As the retention time of standard drugs and the retention time of the drugs in sample solution was same, so the method was specific.

Also Specificity covers the degradation of the drug substances and drug product in Acid degradation, Base degradation, Peroxide degradation, Thermal degradation and Photo degradation. All degradation conditions with % degradation and peak purity were mention in table no. 5.

Fig.5 Chromatogram of Blank

Fig. 6 Chromatogram of standard mixture of Emtricitabine (200 µg/ml)and Tenofovir Disoproxil Fumarate Std (300 µg/ml)

Fig. 7 Chromatogram of Emtricitabine Std 200 µg/ml

Fig. 8 Chromatogram of Tenofovir Disoproxil Fumarate Std 300 µg/ml

Fig.9 Chromatogram of Tablet Formulation (200µg/ml Emtricitabine, 300µg/ml Tenofovir Disoproxil Fumarate)

Fig.10 Chromatogram of Blank Acid degradation (0.1 N HCl 40°C at 30min.)

Fig.11 Chromatogram of Tablet Formulation-Acid degradation (0.1 N HCl 40°C at 30min.)

Fig.12 Chromatogram of Blank - Base degradation (0.1 N NaOH 15 min. at Room Temperature)

Fig.13 Chromatogram of Tablet Formulation - Base degradation (0.1 N NaOH 15 min. at Room Temperature)

Fig.14 Chromatogram of Blank Oxidation degradation (30% H₂O₂ 15 min. at Room Temperature)

Fig.15 Chromatogram of Tablet formulation - Oxidation degradation (30% H₂O₂ 15 min. at Room Temperature)

Fig.16 Chromatogram of Tablet formulation - Thermal degradation (105ºC for 48 Hrs.)

Fig.17 Chromatogram of Tablet formulation - Photo degradation

Fig.18 Chromatogram of Emtricitabine standard preparation-200mg/mL

Fig.19 Chromatogram of Tenofovir Disoproxil Fumarate standard preparation-300 mg/mL

The developed method was found specific and selective, as there was no interference of blank and individual drug. Also method was found stability indicating based on degradation study.

CONCLUSION:

A new, reversed-phase HPLC method has been developed for simultaneous analysis EMTRI and TENO in a tablet formulation. It was shown above that, the method was linear, accurate, reproducible, repeatable, precise, selective, specific and stability indicating proving the reliability of the method. The run time is relatively short, as method is stability indicating which enable rapid determination of many samples in routine and quality control analysis of tablet formulations. The same solvent was used throughout the experimental work and no interference from blank and in degradation study was observed. Hence, the proposed method was successfully applied to analyze preparation containing EMTRI and TENO.

ACKNOWLEDGEMENT:

The authors are thankful to Lauras Lab Pvt. Ltd, Ahmadabad for providing the gift sample of Emtricitabine and Tenofovir Disoproxil Fumarate.

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Received on 03.04.2016 Modified on 20.04.2016

Research J. Pharm. and Tech. 9(4): April, 2016; Page 463-468

DOI: 10.5958/0974-360X.2016.00086.X