INTRODUCTION:

Emtricitabine (FTC) is a nucleoside agent that has activity against both human immunodeficiency virus (HIV) and hepatitis B virus. It is very similar to lamivudine (3TC) with respect to its activity, convenience, and safety and resistance profile. Recent treatment guidelines for the treatment of HIV infection by both the International AIDS Society–USA and US Department of Health and Human Services have placed FTC in combination with tenofovir, didanosine, or zidovudine in the preferred category of nucleoside backbone regimens for patients receiving antiretroviral therapy. FTC is a prodrug that must be phosphorylated intracellularly into its active triphosphate form, emtricitabine 5′-triphosphate (E-TP). E-TP is incorporated by reverse transcriptase into the elongating proviral DNA chain leading to termination of DNA synthesis through the inability of the next nucleotide to bind to E-TP at the 3-prime position¹. In adults, FTC has demonstrated linear kinetics over a wide dose range and FTC 200 mg once a day (QD) is the recommended therapeutic dose². Following oral administration, emtricitabine is rapidly absorbed, with C_max occurring at 1 to 2 hours post-dose. The mean absolute bioavailability of emtricitabine was 93%.

Following a single oral dose, the half-life is approximately 10 hours. Approximately 86% of emtricitabine is recovered in the urine and 13% is recovered as metabolites³. The aim of the work was to develop simple and reliable method for the estimation of Emtricitabine from human plasma which can be used routinely for bioequivalence study sample analysis. There are few methods reported in the literature for the analysis of emtricitabine on HPLC using UV detector^4-6and fluorometric detector⁷. LC-MS/MS technique has advantages over HPLC technique in terms of fast, sensitive and specific technique. There are very few LC-MS/MS methods reported for the estimation of Emtricitabine from human plasma^2,8-9. All these LC-MS/MS methods are in combination with other antiretroviral drugs. There was a need to develop simple, fast and specific method for the estimation of Emtricitabine hence we have developed method on LC-MS/MS. Sample preparation was carried out using Water, Oasis, HLB cartridges 1cc/30 mg to get clean samples. The method was validated by evaluating the precision, accuracy and other validation parameters from human plasma mentioned in regulatory guidelines ¹⁰.

MATERIALS AND METHODS:

Apparatus and Chromatographic conditions:

The system used was Shimadzu LC-VP HPLC System consisting of LC-10AD Prominence pump, SIL-HTc autosampler, CTO 10ASvp column oven and DGU-14A degasser was used to setting the reverse-phase LC conditions. The separation of analyte and internal standard was performed on Hypurity Advance, 50 x 2.1 mm, 5µm column at 30°C. The mobile phase consisted of 5 mM ammonium acetate: Acetonitrile (30:70 v/v). Flow rate of mobile phase was kept at 1mL/min. the total chromatographic run time was 1.2 min. The autosampler temperature was maintained at 4°C. Injection volume was 5µL and rinsing solution was water and acetonitrile in the volume ratio of 50:50.

Ionization and detection of emtricitabine and abacavir (internal standard) was carried out on triple quadrupole mass spectrometer, MDS SCIEX API 3200 (Toronto Canada) equipped with ESI operating in positive ion mode. Quantitation was performed using MRM mode to monitor protonated precursor → product ion (m/z) transitions for emtricitabine 248.2→130.1 and for Abacavir 287.2→191.2. The source dependent parameters were GS1- 50 psig, GS2- 40 psig, ion spray voltage- 4000V, Turbo heater temperature-500°C, interface heater-on, Curtain gas -nitrogen 25 psig and CAD gas - 5 psig. The compound dependent parameters like declustering potential, entrance potential, collision energy and cell exit potential were set at 25, 4, 15, 12 V respectively for emtricitabine and 40, 5, 25, 30 V respectively for abacavir. Data processing and chromatographic integration was carried out by using ‘Analyst software version 1.4.2’.

The nitrogen evaporator was procured from Takahe Analytical Instruments. Deep freezers used for storage of plasma samples were procured from SANYO (JAPAN) were used.

Chemicals and Reagents:

Emtricitabine and Abacavir (IS) working standards were obtained from Macleods Pharmaceuticals Ltd, Mumbai, India. Water was deionized and further purified with Milli-Q system (Millipore USA), Acetonitrile (HPLC grade) and Methanol (HPLC grade) was supplied by J. T. Baker (U.S.A.), Ammonium acetate (AR grade) and Formic acid (AR grade) were supplied by Thomas Baker (INDIA). SPE cartridges used for sample preparation were Water Oasis HLB cartridges 1cc/30mg.

Fresh frozen plasma containing di-potassium EDTA as an anticoagulant was used during validation and study sample analysis was collected in Macleods Pharmaceuticals Ltd, Mumbai India. Plasma was stored at -20°C before sample preparation and analysis.

Standards and Working Solutions:

Standard stock solutions of Emtricitabine containing 1000 µg/mL was prepared by in methanol and stock solution of internal standard (IS) 1000 µg/mL was prepared by in 10 % formic acid in methanol. Intermediate dilutions and IS spiking dilutions were prepared from respective stock solutions by dilution with 50% methanol in water. Calibration standards for plasma were prepared in the range of 50.07 ng/mL to 5006.59 ng/mL using eight concentration levels. Quality control standards were prepared at three different levels low (135.37 ng/mL), medium (2506.89 ng/mL) and high (4220.35 ng/mL).

Sample Treatment:

50µL of IS dilution was added to 250 µL of plasma sample. These samples were further diluted with 250 µL of water. The plasma samples were extracted on Water’s OASIS HLB SPE cartridges. Conditioning of cartridges was carried out with 1 mL methanol followed by 1 mL water. The washing of the sample loaded cartridges was carried out twice with 1 mL of water. The samples were eluted with 1 mL of methanol and evaporated to dryness at 50°C under nitrogen. Reconstitution of the samples was carried with 2 mL mobile phase.

Results:

Method Validation:

Carryover effect:

Carry over effect of the autosampler was evaluated by sequentially injecting solutions of mobile phase and blank after extracted high concentration sample containing Emtricitabine and IS (concentration equivalent to 1.7 times of ULOQ) and its aqueous recovery comparison sample. No significant carry over was observed when rinsing cycle before and after with 500 µL of rinsing solution was applied.

Specificity and Selectivity:

Ten different lots of plain plasma were analysed to ensure selectivity of the method. There was no significant interference observed at the retention time of Emtricitabine and IS. Representative chromatogram of blank sample is given in Fig. 1.

Fig. 1 Representative chromatogram of blank plasma sample

Lower Limit of Quantification (LLOQ):

Set of six LLOQ sample prepared in plasma and processed along with one set of calibration curve. The lowest limit of quantification was set at the concentration of 50.07 ng/mL from plasma (Fig 2.). The precision and accuracy at LLOQ was found to be 2.85 % and 100.70 %.

Linearity:

The linearity of the method was determined by analysis of eight point calibration standards. Three linearity curves were analyzed. A regression equation with a weighting factor of 1/x and 1/x² of concentration ratio of drug to IS were checked for better results in terms of accuracy. Finally 1/x²was used to produce the best fit for the concentration-detector response relationship. Correlation coefficients (r²) were greater than 0.98 in the concentration range of 50.07 ng/mL to 5006.59 ng/mL (ULOQ Fig. 3). Accuracy of all calibration standards was within 85-115% except LLOQ where it was 80-120%.

Fig. 2 Representative chromatogram of LLOQ sample in plasma

Fig. 3 Representative chromatogram of highest concentration sample in plasma

Matrix effect:

Matrix effect may arise due to co-elution of some unintended components present in biological samples or which are added as part of analysis. These components result in ion suppression / enhancement, decrease / increase in sensitivity of analytes over a period of time, increase in baseline, imprecision of data, drift in retention time and distortion or tailing of chromatographic peak. Thus assessment of matrix effect constitutes an important and integral part of validation for quantitative LC-MS/MS method. Matrix factor was found to be 1.07 with precision of 2.47 % CV.

Accuracy and Precision:

The precision of the assay was measured as the percent coefficient of variation over the concentration range of LLOQ QC, LQC, MQC and HQC samples during the course of validation. The accuracy of the assay was defined as the absolute value of the ratio of the calculated mean values of LLOQ QC, LQC, MQC and HQC samples to their respective nominal values, expressed in percentage. Six replicates of each QC sample were analyzed together with a set of calibration standard. For determining the intra-day accuracy and precision, replicate analysis of plasma samples was performed on the same day. The inter-day accuracy and precision were assessed by analysis of three precision and accuracy batches on two different days. The obtained precision and accuracy (inter and intra-day) are presented in Table 1.

Table 1: Inter and intra-day accuracy and precision of emtricitabine from plasma

QC levels	Mean Accuracy (%)	Precision (% RSD)
Intra day (n=12)
LLOQ QC	104.17	5.33
LQC	93.12	4.21
MQC	96.42	2.60
HQC	99.25	2.89
Inter day (n=18)
LLOQ QC	103.88	4.59
LQC	93.02	3.58
MQC	95.99	2.43
HQC	98.62	2.76

Recovery Study:

The recovery study was performed by comparing processed QC samples of three different concentrations with aqueous recovery comparison samples representing 100 % extraction. The recovery was found to be 90.47 % and for IS 83.92 %.

Stability Studies:

The stability of Emtricitabine and IS was investigated in the stock and working solutions, in plasma during storage, during processing, after three freeze-thaw cycles and in the final extract. The stability samples were compared with freshly prepared calibration curve and quality control samples. All stability exercises were carried out on six sets of LQC and HQC samples. Analyte and IS were considered stable when the change of concentration was ± 15 % of nominal value.

Freeze Thaw (FT) Stability:

For freeze thaw stability, first FT cycle was carried out after 24 hours of freezing the plasma samples, then five more FT cycles were carried out after at least 12 hours of freezing for each cycle. The samples were found to be stable after six FT cycles. Summary of stability data is presented in Table 2.

Post preparative Stability:

The post preparative stability of extracted plasma samples was evaluated after final reconstitution step. The samples were stored at room temperature for 4 hours and results showed that samples were found to be stable for 4 hours. Summary of stability data is presented in Table 2.

Bench Top Stability:

Bench top stability, using six sets each of LQC and HQC was evaluated by placing quality control samples at room temperature for 16 hours. The plasma samples were found to be stable for 16 hours at room temperature. Summary of stability data is presented in Table 2.

Auto Sampler Stability:

In assessing the autosampler stability, QC samples placed in the autosampler, were injected after 54 hours. The samples were found to be stable for 54 hours at 4°C. Summary of stability data is presented in Table 2.

Long Term Stability in Plasma:

For long-term stability, concentrations obtained are compared with the results of 1^st day of analysis of bulk-spiked samples. QC samples at low, medium and high concentration were processed after 55 days of storage in deep freezer maintained at below -50°C and checked for the stability. The samples were found to be stable for 55 days. Summary of this stability data is presented in Table 2.

Stock Solution Stability:

Bench top stock solutions stability and refrigerator stock solutions stability were evaluated by injecting six replicates of stock dilutions of both stability and comparison stock solution of Emtricitabine and Abacavir. The stock solution of Emtricitabine was found to be stable for 23 hours and Abacavir stock solution was found to be stable for 16 hours at room temperature and in refrigerator stock solutions were found to be stable for 5 days 17 hours.

Table 2: Summary of stability data for emtricitabine in plasma

Stability	QC level	Mean Accuracy (%)	Precision (%RSD)	Stability Duration
Bench Top	LQC	89.47	1.89	16 hours
Bench Top	HQC	98.16	4.44	16 hours
Freeze Thaw	LQC	89.07	4.41	6 Cycles
Freeze Thaw	HQC	98.26	3.62	6 Cycles
Post Preparative	LQC	101.13	1.51	4 hours
Post Preparative	HQC	110.29	3.01	4 hours
Autosampler	LQC	91.30	2.60	54 hours
Autosampler	HQC	100.92	3.56	54 hours
Long Term	LQC	100.19	4.09	55 days
	MQC	94.79	1.23
	HQC	90.84	2.77

Each mean accuracy, % CV of each stability represents six observations (n=6) of corresponding QC levels.

Bioequivalence Study:

The validated method was applied to study sample analysis. Design of the study was an open label, balanced, analyst blind, randomized, two-treatment, two-period, two sequences, single dose, crossover bioequivalence study. The study was conducted as per study protocol approved by Independent Ethics Committee. The study was conducted as per ICH GCP guidelines. The study was performed on 12 healthy, adult human subjects under fasting conditions. The concentrations (ng/mL) of test and reference formulations were calculated. The comparative linear plots of emtricitabine test and reference mean plasma concentration Vs time were plotted.

Fig. 4 Comparative Linear Plot of Emtricitabine Mean Plasma Concentration (ng/mL) Vs Time (Hour)

DISCUSSION:

Optimization of Chromatographic conditions and sample Clean-up:

The successful analysis of the analyte in biological fluids using LC-MS/MS method relies on the optimization of sample preparation, chromatographic separation and post column detection etc. The method development was initiated to achieve adequate selectivity, sensitivity and minimize overall analysis time and using small plasma volume for processing. Optimum mass acquisition parameters were obtained by direct infusion of 500 ng/mL solution for Emtricitabine at flow rate of 10µL/min. This was done by maintaining optimized declustering potential and ion spray voltage at 26V and 4000V respectively. The present study was conducted using positive ESI as the analyte is basic in nature. The most stable and consistent product ion for Emtricitabine was observed at m/z 130.1 and for Abacavir the most abundant ion seen was m/z 191.2 in the product ion mass spectra. All the state file parameters were optimized to obtain a consistent and adequate response for the Emtricitabine and IS.

Chromatographic conditions of the Emtricitabine and IS was initiated under isocratic conditions to obtain adequate response, sharp peak shape and a short run time. Various mobile phase containing combination of volatile acids and buffers like formic acid, acetic acid, ammonium acetate with acetonitrile and methanol were tried to get good, stable and reproducible response. Finally 5mM ammonium acetate: acetonitrile combination in proportion of 30: 70 was selected for plasma analysis. Various C8 and C18 columns were evaluated for suitable peak shape, response and retention of the analyte and IS. Best results were observed with Hypurity advance, 50 x 4.6 mm, 5µm.

Different techniques like protein precipitation, liquid-liquid extraction and solid phase extraction were evaluated for sample clean-up but solid phase extraction technique was optimized to get maximum recovery and clean samples. The extraction procedure was finally optimized using solid phase extraction HLB cartridges 1cc / 30 mg. During sample preparation washing step was optimized using 1 mL water two times to get cleaner samples and to reduce matrix effect. Elution of the sample was carried out with 1mL of methanol. Abacavir was used as an IS in the present study as it showed similar chromatographic behavior and both the drugs were quantitatively extracted via solid phase extraction.

The results of method validation show that the method is highly accurate and precise. Further application of the method was checked by bioequivalence study samples analysis.

CONCLUSION:

A simple sensitive, selective, precise and accurate LC-MS/MS method for the determination of emtricitabine in human plasma was developed and validated. The method was found to be precise and accurate. The plasma samples containing emtricitabine were found to be stable under experimental conditions. The method was further applied for the estimation of concentrations from plasma samples of emtricitabine study which ensures the applicability of the method.

ACKNOWLEDGEMENTS:

The authors thank Macleods Pharmaceuticals Ltd, Mumbai, for providing all the working standards, chemicals, Laboratory instruments and facility to carry out this work.

REFERENCES:

1. Louis D. Saravolatz, Section Editor and Michael S. Saag, Emtricitabine, a New Antiretroviral Agent with Activity against HIV and Hepatitis B Virus, Clin Infect Dis. 42 (1), 2006: 126-131.

2. Wang L.H., Wiznia A.A., Rathore M.H., Chittick G.E., Bakshi S.S., Emmanuel P.J. and Flynn P.M., Pharmacokinetics and Safety of Single Oral Doses of Emtricitabine in Human Immunodeficiency Virus–Infected Children, Antimicrob. Agents Chemother. 48(1), 2004: 183-191.

3. http://www.rxlist.com/truvada-drug.htm, accessed on June 15, 2011.

4. Peepliwal A.K., Bonde C.G., Determination of emtricitabine in human plasma by RP-HPLC with UV- detection, J. Pharm. Research, 3(8), 2010: 1712-1715.

5. Darque A., Valette G., Rousseau F., Wang L.H., Sommadossi J.P. and Zhou X.J., Quantitation of Intracellular Triphosphate of Emtricitabine in Peripheral Blood Mononuclear Cells from Human Immunodeficiency Virus-Infected Patients, Antimicrob. Agents Chemother., 43(9), 1999, 2245-2250.

6. Rezk N.L., Crutchley R.D. and Kashuba A.D.M., Simultaneous quantification of emtricitabine and Tenofovir in human plasma using high performance liquid chromatography after solid phase extraction, J. Chromtogr. B. 822(1-2), 2005, 201-208.

7. Droste J.A.H., Aarnoutse R.E. and Burger D.M., J. Liq. Chromatogr. Relat. Technol., 30(17-20), 2007, 2769-2778.

8. Yadav M., Singhal P., Goswami S., Pande U.C., Sanyal M. and Shrivastav P.S., Selective Determination of antiretroviral Agents Tenofovir, Emtricitabine, and Lamivudine in Human Plasma by a LC-MS-MS method for a Bioequivalence study in Healthy Indian Subjects, J Chromatogr Sci., 48, 2010,704-713.

9. Delahunty T., Bushman L., Robbins B., Fletcher C., The Simultaneous assay of tenofovir and emtricitabine in plasma using LC-MS/MS and isotopically labeled internal standard, J. Chromatography B., Analytical Technologies in the Biomedical and Life Sciences, 877(20-21), 2009, 1907-14.

10. Guidance for Industry, Bioanalytical Method Validation, US Department of Health and Human Services, Food and Drug Administration, Centre for Drug and Research (CDER), May 2001.

Received on 11.11.2011 Modified on 02.12.2011

Research J. Pharm. and Tech. 5(1): Jan. 2012; Page 133-137