Development, Validation and Stress Degradation Studies of Emtricitabine and Tenofovir Disoproxil Fumarate by High Performance Liquid Chromatography

 

Battula Sreenivasa Rao¹*, Siva Nagaraju², B.V. Kiran³

¹Department of Chemistry, GITAM Institute of Technology, GITAM University, Visakhapatnam, A P India

¹Research Scholar, Department of Chemistry, Acharya Nagarjuna University, Guntur A P India

³Research Scholar, Department of Chemistry, GITAM University, Visakhapatnam, A P India

*Corresponding Author E-mail: battula_sr@gitam.edu

 

A simple, selective, rapid, precise and accurate reverse phase high pressure liquid chromatographic method has been developed for simultaneous estimation of Emtricitabine and Tenofovir disoproxil fumerate in pharmaceutical Tablet dosage form. The mobile phase consisted of 65:35 % (v/v) of Methanol and 0.1M of potassium di hydrogen ortho phosphate and pH adjusted to 3.2 with ortho phosphoric acid.The method developed is operated on isocratic mode.The flow rate is 1.0 ml/min. Chromatographic determination of Emtricitabine and Tenofovir disoproxil fumerate was performed on Phenomenex C₁₈ column (150 X 4.6 mm Id, ODS-2, 5µm). The wavelength of detection is 260 nm. The injection volume is 20µL. The retention time of Emtricitabine is 1.92 ± 0.01 minutes while the retention time of Tenofovir disoproxil fumerate is 3.17 ± 0.01minures.The developed method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantitation, solution stability, ruggedness, and robustness. The influence of Acid, Alkaline, Oxidative Stress, Photolytic stress conditions on Emtricitabine and Tenofovir disoproxil fumerate was studied. Results indicated that Emtricitabine and Tenofovir disoproxil fumerate is stable under the experimental conditions. The proposed method has been successfully used for the estimation in tablet dosage forms.

 

 

1. INTRODUCTION:

Emtricitabine [1, 3][Figure-1], with trade name Emtriva (formerly Coviracil), is a nucleoside reverse transcriptase inhibitor (NRTI) for the treatment of HIV infection in adults and children. White to off white powder. Melting Point is 136-140 ⁰C. Molecular Formula: C₈H₁₀FN₃O₃S. Molecular Weight: 247.25. IUPACName: 4-amino-5-fluoro-1-[(2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]pyrimidin-2-one.

 

Emtricitabine is very similar to lamivudine (3TC) and cross-resistance between the two is near-universal. Emtricitabine is an analogue of cytidine. The drug works by inhibiting reverse transcriptase, the enzyme that copies HIV RNA into new viral DNA.

 

By interfering with this process, which is central to the replication of HIV, emtricitabine can help to lower the amount of HIV, or "viral load", in a patient's body and can indirectly increase the number of immune system cells (called T cells or CD4+ T-cells). Both of these changes are associated with healthier immune systems and decreased likelihood of serious illness. Lactic acidosis and severe hepatomegaly with steatosis, including fatalities, have been reported with the use of nucleoside analogs alone or in combination, including emtricitabine and other antiretrovirals. Emtricitabine should always be used in combination with other antiretroviral agents.

 

Emtricitabine was discovered by Dr. Dennis C. Liotta, Dr. Raymond F. Schinazi, and Dr. Woo-Baeg Choi of Emory University. It was approved by the FDA July 2, 2003. Emtricitabine is also marketed in a fixed-dose combination with tenofovir (Viread) under the brand name Truvada. A fixed-dose triple combination of emtricitabine, tenofovir and efavirenz (Sustiva, marketed by Bristol-Myers Squibb) was approved by the U.S. Food and Drug Administration (FDA) on July 12, 2006 under the brand name Atripla™.

 

Tenofovir disoproxil fumarate [2,4] [Figure-2] marketed by Gilead Sciences under the trade name Viread™, belongs to a class of antiretroviral drugs known as nucleotide analogue reverse transcriptase inhibitors (NRTIs), which block reverse transcriptase, a crucial virus enzyme in human immunodeficiency virus 1 (HIV-1) and hepatitis B virus infections.

 

Figure-1: Structure of Emtricitabine

 

Figure-2: Structure of Tenofovir disoproxil fumerate

 

It is white solid, having a melting point between 113-115⁰C. It is molecular formula of C₁₉H₃0N₅O₁₀P and Molecular weight of 633.51.Tenofovir was discovered through a collaborative research effort between Antonín Holý at the Institute of Organic Chemistry and Biochemistry (IOCB), Academy of Sciences of the Czech Republic (AS CR) in Prague, and Erik DeClercq, Rega Institute for Medical Research, Catholic University of Leuven, Belgium.

 

A total analysis run time of less than 5 min was achieved. The developed method was validated as per ICH Guidelines [15] and used successfully to evaluate 2 brands of Tenofovir and Emtricitabine marketed  drug products.

 

1.1 Review of past work of Analytical methods of Ebastine and Motelukast sodium

From literature survey separate and combination methods have been developed for Emtricitabine and Tenofovir. Several methods have been reported for the quantitative determination of Emtricitabine and Tenofovir in bulk, and pharmaceutical and biological samples .These methods include UV-Visible spectrophotometric and Simultaneous equation method by UV[7-9], HPTLC[5-6] ,HPLC-flurometric-detector[10] HPLC-UV-detector[11-12],and Hyphenated techniques such as HPLC with Mass-spectrophotometer detector[13-14].

 

2. EXPERIMENTAL

2.1. Reagents and chemicals

Potassium di hydrogen ortho phosphate (AR Grade, Merck ltd), Methanol (HPLC grade, Merck ltd), Milli-Q water purchased from Ranbaxy, Emtricitabine and Tenofovir disoproxil fumerate (reference standard is gifted by M/s Corpuscle Research Solutions),Ortho phosphoric acid (GR Grade, SD Fine Chem Ltd).All other chemicals are of the highest grade commercially available unless otherwise specified.

 

2.2. Apparatus and chromatographic conditions

The Chromatographic system consisted of a Shimadzu Class VP Binary pump LC-10ATvp, SIL-10ADvp Auto sampler, CTO-10Avp Column Temperature Oven, SPD-10Avp UV-Visible Detector. All the components of the system are controlled using SCL-10Avp System Controller. Data acquisition was done using LC Solutions software.

 

The mobile phase consisted of 65:35 % (v/v) of Methanol and 0.1M potassium di hydrogen ortho phosphate (pH adjusted to 3.2 with ortho phosphoric acid).The pump is operated on isocratic mode. The flow rate is 1.0ml/min.Chromatographic determination of Emtricitabine and Tenofovir disoproxil fumerate was performed on Phenomenex C₁₈ column (150 X 4.6 mm id, ODS 2, 5µm). The wavelength of detection is 260 nm. The injection volume is 20µL.

 

2.3. Preparation of standard solutions, Calibration Standards and Quality Control Samples

Stock solutions (10 mg/mL) was prepared separately for Emtricitabine and Tenofovir disoproxil fumerate in a volumetric flask and labeled accordingly.Suitable dilutions of Emtricitabine and Tenofovir disoproxil fumerate were prepared using 50:50 %v/v Methanol and Milli-Q water as diluent Solution.A Linear Calibration curve containing 8 non-zero standards were prepared using diluent solution in the concentration range of 2.02 to 50.5µg/mL for Emtricitabine and Tenofovir disoproxil fumerate. The linear standard calibration standard sample is then transferred into the auto sampler for analysis. Samples for Specificity (Sample with Drug; Blank Sample were also prepared accordingly).

 

For the preparation of quality control samples, a separate stock containing approximately the same concentration of the drug substance is prepared and labeled as quality control stock. From this stock, quality control samples were prepared at three concentration levels namely LQC (14.15 μg/mL), MQC (25.25 μg/mL), HQC (37.40 μg/mL). So as to obtain low, median and high concentration quality control samples. The performance of the linear calibration curve is then evaluated using quality control samples.

 

2.4. Assay

The assay of tablets containing Emtricitabine and Tenofovir disoproxil fumerate separately was done using the procedure given in general Pharmacopoeial method for tablets. Briefly, twenty tablets, each containing 30.0mg of Emtricitabine and Tenofovir disoproxil fumerate as labeled claim were weighed separately and finely powdered; a quantity of powder equivalent to 30.0 mg each of Emtricitabine and Tenofovir disoproxil fumerate was weighed and transferred to a 10mL volumetric flask. To this 10mL of methanol was initially added and vortexed thoroughly. The final volume is made up to volume with methanol. Suitable dilution is prepared using diluent solution so as to get a final concentration within the range of the calibration curve. This mixture is then carefully filtered using 0.45 μm membrane filter. The filtrate is then taken and suitably diluted and injected for analysis. The assay content was evaluated using the regression equation of linear calibration curve.

 

2.5 Method Validation

2.5.1 System Suitability

The system suitability was assessed by six replicate analysis of the drug at a concentration of 30.30μg/ml. The acceptance criterion is ± 2 % for the percent coefficient of the variation for the retention time for the drug.

 

2.5.2 Detection and Quantitation Limits (Sensitivity)

Limits of detection (LOD) and quantification (LOQ) (Figure-3) were estimated from both linearity calibration curve method and signal to noise ratio method. The detection limit was defined as the lowest concentration level resulting in a peak area of three times the baseline noise. The quantification limit was defined as the lowest concentration level that provided a peak area with signal to noise ratio higher than 10, with precision (%CV) and accuracy with (±) 20%.

 

2.5.3 Linearity (Calibration Curve)

The calibration curve was constructed with eight concentrations ranging from 2.0-50.5 μg/mL. The linearity was evaluated by linear regression analysis, which was calculated by least square method. It is depicted in         (Figure-4).

 

Figure-3: Chromatograms shown indicate limit of Detection (LOD) above and Limit of Quantitation (LOQ) below

 

Figure-4:Linear calibration curve of Emtricitabine(above) and Tenofovir (below)respectively.

 

2.5.4 Accuracy and Precision

Accuracy of assay method was determined for both intra-day and inter-day variations using triplicate analysis of the QC samples. Precision of the assay was determined by repeatability (intra-day) and intermediate precision (inter-day). Repeatability refers to the use of the analytical procedure within the laboratory over the shorter period of the time that was evaluated by assaying the QC samples during the same day. Intermediate precision was assessed by comparing the assays on different days (3 days).

 

2.5.5 Specificity

Specificity of the method was determined by comparing the Blank sample with that of the sample containing Emtricitabine and Tenofovir disoproxil fumerate.(Figure-5). A less than 20% interference of the peak area at the retention time of the drug in the blank sample is taken as acceptance criteria for the analyte. Sample Specificity is also observed in the degradation study of the drug. None of the degraded products must interfere with the quantification of the drug.

 

2.5.6 Stability

The stability of the drug is determined by placing the MQC samples for the short term stability by keeping at room temperature up to 12 hours and then comparing the obtained peak area with that of the similarly prepared fresh sample. Further, auto-sampler stability for up to 24 hrs was studied and established.

 

Figure-5: Comparison of Chromatograms of Blank(above) and that of Emtricitabine and   Tenofovir respectively.

 

2.5.7 Stress Degradation Studies

For Stress Degradation Analysis, 1 mL aliquots (in duplicate) of samples containing MQC level concentration are treated separately with 100 μL of 0.1N HCl (Acid stress), 0.1N NaOH (Alkaline stress), 5% v/v Hydrogen Peroxide (Oxidative Stress), for 24 Hrs. Samples for Photolytic stress are placed in a transparent glass vial and placed in a UV chamber for 24 Hrs. Samples are then injected for analysis.(Figure-6) The results of analysis are then compared with similarly prepared fresh samples.

 

Acidic degradation

 

Oxidative degradation

 

Photolytic degradation

 

Alkaline degradation

 

Acidic degradation

 

Oxidative degradation

 

Photolytic degradation

 

Alkaline degradation

Figure-6: Chromatograms showing the influence of various stress conditions on Emtricitabine; Data 1 – Freshly prepared Sample; Data 2 – Oxidative Stress; Data 3 – Photolytic Stress; Data 4 – Acid Stress; Data 5 – Alkaline Stress. Data 5 clearly indicates the spectral degradation of Pregabalin due to alkaline instability.

 

3. RESULTS AND DISCUSSION:

3.1 Method Development and Validation

The HPLC procedure was optimized with a view to develop a stability indicating assay method. Reverse phase HPLC separations usually require optimization of the mobile phase pH particularly for the highly ionic drugs. Therefore we evaluated the chromatographic behavior at different pH values ranging from pH 3.0 to pH 6.5 using various columns like Hypersil-BDS-C18, Symmetry C18, Ymc-pack C18, Ymc-pack pro, Spherisorb C18, Phenomenex C18 have been tried with different buffer salts such ammonium Formate, ortho phosphoric acid, di-potassium hydrogen orthophosphate, in combination with acetonitrile, methanol and tetrahydrofuran. However less tailing and high theoretical plates are obtained with Phenomenex ODS 2 column C18 150 X 4.6 cm 5μm column. Mobile phase composition consisted of 65:35 % (v/v) of Methanol and 0.1M of potassium di hydrogen ortho phosphate (pH adjusted to 3.2 ± 0.1 with ortho phosphoric acid) on isocratic mode. The flow rate of the method is 1.0 ml/min. Calibration standards were prepared in diluents solution containing 50:50 % v/v of methanol and Milli-Q water. The wavelength of detection is 210nm. The column temperature is maintained at 25 ^OC. At the reported flow rate, peak shape was excellent; however increasing or decreasing the flow rate resulted in unacceptable tailing factor and poor peak shape. Hence 0.7 ml/min was optimized flow rate decreasing the consumption of the mobile phase, which in turn proves to be cost effective for long term routine quality control analysis.

 

3.2 Method Validation

3.2.1 System Suitability

The % RSD of the peak area and the retention time for both drug and internal standard are within the acceptable range (Table-1). The efficiency of the column was expressed as the number of theoretical plates for the six replicate injections was around 5095 ±32 and the USP tailing factor was 1.10 ± 0.01 for Emtricitabine and around 3975 ± 26 and the USP tailing factor was 1.10 ± 0.01.

 

3.2.2 Determination and Quantification Limits (Sensitivity)

Figure-3 represents the chromatogram of limit of detection and limit of quantification. The method is found to be sensitive which can be determined from the data obtained from the (Table-2).

 

3.3.3 Linearity

The linearity was demonstrated in triplicate. The results of the best fit line (y = mx + c) for the triplicate analysis is given in (Table 3). The accuracy of the calibration standards was evaluated from the back calculated concentrations (Table 4). All the standards were found to be within the range of 98 – 102 %.

 

Table 1.

System Suitability test for Emtricitabine
SR NO	Retention Time	Peak Area	Theoretical Plates	Tailing Factor
1	1.9	926224	5012	1.14
2	1.94	950286	5089	1.09
3	1.91	949426	5109	1.10
4	1.92	939729	5090	1.12
5	1.91	938761	5098	1.15
6	1.91	933188	5100	1.10
MEAN	1.915	939602.33	5095	1.10
ST DEV	0.014	9289.70	31.92	0.01
% CV	0.72	0.99	0.80	0.80
System Suitability test for Tenofovir disoproxil fumerate
SR NO	Retention Time	Peak Area	Theoretical Plates	Tailing Factor
1	3.15	926610	4017	1.09
2	3.19	917328	4004	1.10
3	3.16	910097	3971	1.12
4	3.17	890537	3979	1.08
5	3.16	900646	3942	1.09
6	3.16	899422	3938	1.10
MEAN	3.165	907440.00	3975.2	1.10
ST DEV	0.0138	13179.37	26.89	0.01
% CV	0.44	1.45	0.25	0.06

 

 

Table 2. Sensitivity of Emtricitabine and Tenofovir by HPLC

 

 

Table 3. Results of Regression Analysis

Emtricitabine

                                    

Tenofovir

 

 

Table 4. Linearity and Range for Emtricitabine demonstrating accuracy, carryover effect and specificity of the method (Data represented for 1^st calibration curve).

SR NO	SAMPLE ID	CONCENTRATION (µg/mL)	DRUG (Emtricitabine)			Calculated Conc. (µg/mL)	Accuracy (%)
			RETENTION TIME		PEAK AREA
1	BLANK	0.000	0.00		NO PEAK	Not Applicable	Not Applicable
2	CC 01	2.02	1.9		109234	2.019	99.96
3	CC 02	5.05	1.91		211137	5.003	99.09
4	CC 03	10.10	1.91		381951	10.006	99.08
5	CC 04	20.20	1.91		739143	20.467	101.33
6	CC 05	30.30	1.89		1069668	30.147	99.50
7	CC 06	40.40	1.89		1404827	39.963	98.93
8	CC 07	44.44	1.89		1570332	44.810	100.84
9	CC-08	50.50	1.89		1752237	50.137	99.29
SR NO	SAMPLE ID	CONCENTRATION (µg/mL)	DRUG (Tenofovir)			Calculated Conc. (µg/mL)	Accuracy (%)
			RETENTION TIME	PEAK AREA
1	BLANK	0.000	0.00	NO PEAK		Not Applicable	Not Applicable
2	CC 01	2.02	3.15	78390		2.032	100.54
3	CC 02	5.05	3.16	189243		5.066	100.26
4	CC 03	10.10	3.16	371390		10.051	99.46
5	CC 04	20.20	3.16	733940		19.973	98.82
6	CC 05	30.30	3.16	1113670		30.365	100.16
7	CC 06	40.40	3.18	1490640		40.682	100.64
8	CC 07	44.44	3.17	1633914		44.604	100.31
9	CC-08	50.50	3.17	1840916		50.269	99.49

 

3.3.4 Accuracy and Precision

Accuracy and precision calculated for the QC samples during the intra- and inter –day run are given the (Table-5). The intra-day (day-1) and inter-day accuracy ranged from 98.00 to 102.00 %. The results obtained from intermediate precision (inter-day) also indicated a good method precision. All the data were within the acceptance criteria.

 

Table 5. Results of inter and intra-day accuracy and precision for  Emtricitabine and Tenofovir by HPLC

	Nominal Concentration (µg/mL) for Emtricitabine
DAY 1	14.14	25.25	37.37
MEAN ACCURACY S.DSD	100.54	100.74	99.17
SD % CV	0.74	0.37	0.21
% CV	0.73	0.37	0.21
DAY 2
MEAN ACCURACY S.DSD	99.94	100.51	98.73
SD  % CV	0.37	0.20	0.23
% CV	0.37	0.20	0.24
DAY 3
MEAN ACCURACY S.DSD	99.60	100.43	99.77
SD % CV	0.30	0.10	0.55
% CV	0.31	0.10	0.55
	Nominal Concentration (µg/mL) for Tenofovir
DAY 1	14.14	25.25	37.37
MEAN ACCURACY S.DSD	99.40	99.47	99.65
SD % CV	0.20	0.54	0.28
% CV	0.20	0.54	0.29
DAY 2
MEAN ACCURACY S.DSD	99.47	99.65	98.91
SD % CV	0.54	0.28	1.33
% CV	0.54	0.29	1.34
DAY 3
MEAN ACCURACY S.DSD	99.34	99.15	98.64
SD % CV	0.52	0.13	0.95
% CV	0.52	0.13	0.96

 

3.3.5 Specificity 

Specificity was determined by comparison of the Blank chromatogram with that of the Standard chromatogram (Figure-4).

 

3.3.6 Room Temperature Stability

Stability studies were done for short term stability up to 12 hrs on the bench top for the MQC levels conditions. Stability is calculated as the ratio of the mean peak area of the stability sample to the mean peak area of the fresh sample and expressed as the percentage (n=6). The room temperature stability was found to be 96.43 %. The results are tabulated in Table-6.

 

3.3.7 Stress Degradation

The stress studies involving acid, light (UV) and oxidation revealed that Pregabalin was stable under the stress conditions (Figure- 5). However in alkaline conditions (0.1N NaOH), the baseline resulted in high noise without affecting the peak shape. For all stress conditions studied, the drug content was within 97 – 99 % (Table-7)indicating the stability and specificity of the analytical method to differentiate the degradation peaks.

 

3.3.8 Robustness study  

Robustness is the measure of method capacity to remain unaffected by deliberate small changes in the chromatographic conditions. The experimental conditions were deliberately altered to evaluate the robustness of the method. The impact of flow-rate (1.0 ± 0.1 ml/min), and effect of mobile-phase composition (± 5%) on chromatographic parameters such as retention time, theoretical plates, and tailing factor, were studied. There was no significant variation due to the variation of mobile phase composition or flow rate variation.

 

 

Table 6. Room Temperature Stability of Pregabalin (n = 6).

 

Table-7 Results of Emtricitabine and Tenofovir exposed to different pathways

	Average area	Average Recovery	% Degradation
Oxidation	859389.5	101.00	No degradation
Alkaline	----	88.52	Degradation
Acidic	850719.5	99.98	No degradation
U.V	852836.5	100.23	No degradation
Untreated	850870	Not applicable	Not applicable
Tenofovir	Average area	Average Recovery	% Degradation
Oxidation	806185	101.94	No degradation
Alkaline	-----	No good Recovery	Complete degradation
Acidic	802740	101.51	No degradation
U.V	805059.5	101.80	No degradation
Untreated	790816.5	Not applicable	Not applicable

Each value is a result of triplicate analysis.

 

 

3.4 Application of the method to dosage forms

The HPLC method developed is sensitive and specific for the quantitative determination of Pregabalin. Also the method is validated for different parameters, hence has been applied for the estimation of drug in pharmaceutical dosage forms. Individual content tablets of Emtricitabine and Tenofovir from two different manufacturers were evaluated. The amount of Emtricitabine in tablet 1 is 99.05 ± 0.46 and tablet 2 is 99.09 ± 0.29.The amount of Tenofovir in tablet 1 is 98.95 ± 0.76 and tablet 2 is 100.05 ± 0.29. None of the tablets expedients interfered with the analyte peak.

 

4. CONCLUSIONS:

The method gave accurate and precise results in the concentration range of 2.0 to 50.50 μg/mL. The mobile phase consisted of 65:35 % (v/v) of Methanol and 0.1M potassium di hydrogen ortho phosphate (pH adjusted to 3.2 with ortho phosphoric acid).The pump is operated on isocratic mode. The flow rate is 1.0ml/min. Chromatographic determination of Emtricitabine and Tenofovir disoproxil fumerate was performed on Phenomenex C₁₈ column (150 X 4.6 mm id, ODS 2, 5µm). The wavelength  of detection is 260 nm. The injection volume is 20µL.The complete validation is done based on ICH Quality guidelines [15].

 

5. REFERENCES:

1.        www.pubchem/Emtricitabine

2.        www.pubchem/Tenofovir disoproxil fumerate

3.        www.wikepedia/Emtricitabine

4.        www.Wikepedia/Tenofovir disoproxil fumerate

5.        HPTLC method Development, Validation for Simultaneous Determination of Efavirenz, Emtricitabine and Tenofovir in combined tablet formulation and Forced Degradation Studies. Nikalje Anna Pratima, Maniyar Ajhar, Obaid Shaikh, AJPTR, volume-3,February 2013.

6.        Joshi M, Nikalje A P, Shahed M, Dehghan M. HPTLC method for the simultaneous estimation of emtricitabine and tenofovir in tablet dosage form. Indian J Pharm Sci 2009;71:95-7.

7.        Choudhari VP, Ingale S, Gite SR, Tajane DD, Modak VG, Ambekar A. Spectrophotometric simultaneous determination of Tenofovir disoproxil fumarate and Emtricitabine in combined tablet dosage form by ratio derivative, first order derivative and absorbance corrected methods and its application to dissolution study. Pharm Methods 2011;2:47-52.

8.        Choudhari VP, Ingale KD, Barhate A, Kale AN, Bobade CD, Kuchekar BS. Development and validation of Simultaneous and Isoabsorptive UV -Spectrophotometric methods for Tenofovir and Emtricitabine in Pharmaceutical Formulations. J Pharm Res. 2010;9:11–13.

9.        Ghorpade SA, Sali MS, Kategaonkar AH, Patel DM, Choudhari VP, Kuchekar BS. Simultaneous determination of emtricitabine and tenofovir by area under curve and dual wavelength spectrophotometric method. J Chil Chem. Soc. 2010;54:331–33.

10.     Jullien V, Treéluyer J, Pons G, Rey E. Determination of tenofovir in human plasma by high-performance liquid chromatography with spectrofluorimetric detection. J Chromatogr B.2003;785: 377–81. 

11.     Notari S, Bocedi A, Ippolito G, Narciso P, Pucillo LP, Tossini G, et al. Simultaneous determination of 16 anti-HIV drugs in human plasma by high-performance liquid chromatography. J Chromatogr B.2006;831:258–66.

12.     Rezk NL, Crutchley RD, Kashuba AD. Simultaneous quantification of emtricitabine and tenofovir in human plasma using high-performance liquid chromatography after solid phase extraction. J Chromatogr B. 2005;822:201–08. 

13.     Gomes NA, Vaidya VV, Pudage A, Joshi SS, Parekh SA. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of tenofovir and emtricitabine in human plasma and its application to a bioequivalence study. J Pharm Biomed Anal. 2008;48:918–26.

14.     Bezy V, Morin P, Couerbe P, Leleu G, Agrofoglio L. Simultaneous analysis of several antiretroviral nucleosides in rat-plasma by high-performance liquid chromatography with UV using acetic acid/hydroxylamine buffer Test of this new volatile medium-pH for HPLC-ESI-MS/MS. J Chromatogr B. 2005; 82:132–43.

15.     ICH-Q2B Validation of Analytical Procedures: Methodology International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use.

 

 

Received on 19.07.2013          Modified on 01.08.2013

Accepted on 05.08.2013         © AJRC All right reserved