Specificity enhancement with LC‐positive ESI‐MS/MS for the measurement of nucleotides: application to the quantitative determination of carbovir triphosphate, lamivudine triphosphate and tenofovir diphosphate in human peripheral blood mononuclear cells

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Abstract
Our previous negative ESI‐LC‐MS/MS method developed for nucleoside reverse transcriptase inhibitor (NRTI) triphosphate (‐TP) measurements in human peripheral blood mononuclear cells (PBMC) encountered some specificity problems for several NRTI‐TP and simultaneous endogenous nucleotide triphosphates analysis. As LC‐MS/MS offers several possibilities to circumvent such problems, we have investigated the contribution of the positive electrospray ionization mode in enhancing the specificity of the intracellular analyses of triphosphate metabolites of lamivudine, abacavir, and tenofovir. For intracellular NRTI‐TP analysis, after disruption of PBMCs, concentrated supernatants were directly injected into the LC‐MS/MS system, dimethylhexylamine being used as ion‐pairing agent to resolve NRTI‐TP. MS/MS detection was performed after positive electrospray ionization. Total run time was 12 min instead of 26 min for NRTI‐TP analysis. The validation parameters of the method met the international requirements, and endogenous chromatographic interferences were eliminated. The use of positive ESI, offering a better specificity and a slightly better sensitivity than the negative ESI mode for these compounds, resulted in specificity enhancement and more robust assay methods. Copyright © 2007 John Wiley & Sons, Ltd.

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