The simultaneous assay of tenofovir and emtricitabine in plasma using LC/MS/MS and isotopically labeled internal standards

Tom Delahunty, Lane Bushman, Brian Robbins, Courtney V. Fletcher

Research output: Contribution to journalArticle

74 Scopus citations

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 (300 mg) and another reverse transcriptase inhibitor, emtricitabine (FTC, 200 mg) 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 13C in the adenine moiety (Iso-TFV) and FTC labeled with 13C and 15N 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 200 μL/min. A Synergi Polar-RP, 2.0 mm × 150 mm, 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 10 ng/mL to 1500 ng/mL for both analytes (250 μL plasma extracted), with a minimum quantifiable limit of 10 ng/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.

Original languageEnglish (US)
Pages (from-to)1907-1914
Number of pages8
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume877
Issue number20-21
DOIs
StatePublished - Jul 1 2009

Keywords

  • Emtricitabine (FTC)
  • Isotopic internal standards (IS)
  • LC/MS/MS
  • Pharmacokinetics
  • Selective reaction monitoring (SRM)
  • Tenofovir (TFV)
  • Tenofovir disoproxil fumarate (TDF)

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry
  • Cell Biology

Fingerprint Dive into the research topics of 'The simultaneous assay of tenofovir and emtricitabine in plasma using LC/MS/MS and isotopically labeled internal standards'. Together they form a unique fingerprint.

  • Cite this