The simultaneous assay of tenofovir and emtricitabine in plasma using LC/MS/MS and isotopically labeled internal standards. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences [J Chromatogr B Analyt Technol Biomed Life Sci] Journal article | | Title | The simultaneous assay of tenofovir and emtricitabine in plasma using LC/MS/MS and isotopically labeled internal standards. | | Author(s) | Delahunty T, Bushman L, Robbins B, Fletcher CV | | Institution | Antiviral Pharmacology Laboratory, Department of Pharmaceutical Sciences, University of Colorado Denver, P.O. Box 6511, Anschutz Medical Center, Aurora, CO 80045, USA. | | Source | J Chromatogr B Analyt Technol Biomed Life Sci 2009 May 21. | | Abstract | An LC/MS/MS assay we published for tenofovir (TFV) plasma levels is a useful tool for monitoring the pharmacotherapy of HIV-positive individuals [T. Delahunty, L. Bushman, C.V. Fletcher, J. Chromatogr. B 830 (2006) 6-12]. A new combination therapy consisting of the TFV pro-drug (300mg) and another reverse transcriptase inhibitor, emtricitabine (FTC, 200mg) has become available in a convenient once-daily dosage form (Truvada). This widely used medication has prompted us to develop and validate a convenient assay to determine simultaneously TFV and FTC plasma concentrations. In view of their chemical similarity to the analytes, stable isotope internal standards (IS) were chosen. These consisted of TFV labeled uniformly with (13)C in the adenine moiety (Iso-TFV) and FTC labeled with (13)C and (15)N in the cytosine moiety (Iso-FTC). Trifluoroacetic acid was added to the patient's EDTA plasma (containing the IS) to produce a de-proteinated extract after high speed centrifugation. The extracts were directly injected into the mobile phase (3% acetonitrile/1% acetic acid, aq.) stream flowing at 200muL/min. A Synergi Polar-RP, 2.0mmx150mm, reversed-phase analytical column was used to achieve the chromatographic separation. Detection of the analytes was achieved by ESI positive ionization tandem mass spectrometry. The precursor/product transitions (m/z) in the positive ion mode were 288/176 and 293/181 ions for TFV and Iso-TFV, respectively and the precursor/product transitions (m/z) were 248/130 and 251/133 ions for FTC and Iso-FTC, respectively. When the analyte/IS abundance ratios were plotted against the specified concentrations, the linearity of the concentration curves were in the range 10ng/mL to 1500ng/mL for both analytes (250muL plasma extracted), with a minimum quantifiable limit of 10ng/mL for both analytes. The inter- and intra-day accuracy and precision for both TFV and FTC were within+/-20% at the LLOQ and+/-15% at the other QC levels. We have expanded the method originally designed for the assay of TFV alone to incorporate the simultaneous determination of the latter and FTC using stable isotope IS. This assay has been successfully used for the periodic monitoring of 678 HIV-positive patients being treated with the combination therapy. | | Language | ENG | | Pub Type(s) | JOURNAL ARTICLE
| | PubMed ID | 19493710 |
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