Validation and clinical application of a high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of 10 anti-retrovirals in human peripheral blood mononuclear cells.

Elens, Laure;Veriter, Sophie;Yombi, Jean Cyr;Di Fazio, V.;Haufroid, Vincent;et.al.
(2009) Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences — Vol. 877, n° 20-21, p. 1805-1814 (2009)

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Authors
  • Elens, LaureUCLouvain
    Author
  • Veriter, SophieUCLouvain
    Author
  • Yombi, Jean CyrUCLouvain
    Author
  • Di Fazio, V.
    Author
  • Lison, Dominiqueorcid-logoUCLouvain
    Author
  • Wallemacq, PierreUCLouvain
    Author
  • Vandercam, BernardUCLouvain
    Author
  • Haufroid, Vincentorcid-logoUCLouvain
    Author
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Abstract
This paper reports the validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method that allows the quantification of 10 antiretroviral (ARV) drugs in peripheral blood mononuclear cells (PBMCs) using 6 different isotopic internal standards (IS) and its clinical application. PBMCs are isolated from blood by density gradient centrifugation and drugs are extracted with a 60% methanol (MeOH) solution containing the 6 IS. The cell extract is then injected in the HPLC system and analytes are separated on a Symmetry Shield RP18 2.1mmx50mm column. The different molecules are then detected by MS/MS in electrospray positive or negative ionisation modes and data are recorded using the multiple reaction monitoring (MRM) mode. Calibration curves are constructed in the range of 0.25-125ng/ml of cell extract by a 1/x(2) weighted quadratic regression. The regression coefficients obtained are always greater than 0.99 and back calculated values always comprised in the range of +/-15% from their nominal concentration. Mean extraction recoveries are greater than 80% for all analytes and the method is accurate and precise with CV and bias lower than 9.4%. The lower limits of quantification (LLOQ) of the different drugs range from 0.0125 to 0.2ng/ml of cell extract. This method was successfully applied to a cohort of 98 HIV-infected patients treated with Kaletra((R)) (400/100mg of lopinavir/ritonavir (LPV/RTV) twice a day, n=48) or with Stocrin((R)) (600mg once a day, n=50) and has been tested for cellular quantification of tipranavir (TPV) in 2 patients treated with Aptivus((R)) (500mg twice a day). The patients treated by Kaletra((R)) showed mean cell-associated concentrations (CC) of 1819.0 and 917.2ng/ml, for LPV and RTV, respectively. Patients treated with Stocrin((R)) showed mean CC of 2388.11ng/ml while both patients under Aptivus((R)) showed TPV CC of 4322.7 and 1078.0ng/ml, respectively. This method can be used to analyze ARV drug concentrations within the target tissue.
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Elens, L., Veriter, S., Yombi, J. C., Di Fazio, V., Vanbinst, R., Lison, D., Wallemacq, P., Vandercam, B., & Haufroid, V. (2009). Validation and clinical application of a high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of 10 anti-retrovirals in human peripheral blood mononuclear cells. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, 877(20-21), 1805-1814. https://doi.org/10.1016/j.jchromb.2009.04.046 (Original work published 2009)