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Analysis and validation of the phosphorylated metabolites of two anti‐human immunodeficiency virus nucleotides (stavudine and didanosine) by pressure‐assisted CE‐ESI‐MS/MS in cell extracts: Sensitivity enhancement by the use of perfluorinated acids and alcohols as coaxial sheath‐liquid make‐up constituents
Author(s) -
Bezy Vincent,
Chaimbault Patrick,
Morin Philippe,
Unger Steve E.,
Bernard MarieCharlotte,
Agrofoglio Luigi A.
Publication year - 2006
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200500850
Subject(s) - didanosine , stavudine , chromatography , chemistry , lamivudine , electrospray , abacavir , analytical chemistry (journal) , mass spectrometry , virus , hepatitis b virus , virology , biology
A CE method utilizing triple quadrupole electrospray (ES) MS (MS/MS) detection was developed and validated for the simultaneous measurement of nucleoside 5'‐triphosphate and 5'‐monophosphate anabolites of the anti‐HIV (human immunodeficiency virus) didanosine (ddAMP, ddATP) and stavudine (d4TMP, d4TTP), among a pool of 14 endogenous 5'‐mono‐, di‐, and triphosphate nucleosides. These compounds were spiked and extracted from peripheral blood mononuclear cells (PBMCs) which are the sites of HIV replication and drug action. An acetic acid/ammonia buffer (pH 10, ionic strength of 40 mM) was selected as running electrolyte, and the separation was performed by the simultaneous application of a CE voltage of +30 kV and an overimposed pressure of 28 mbar (0.4 psi). The application of pressure assistance was needed to provide stable ES conditions for successful coupling. The coupling was carried out with a modified sheath‐flow interface, with one uninterrupted capillary (80 cm× 50 µm id; 192 µm od) in a dimension that fits into the ESI needle to get a stable ion spray. Some CE‐MS parameters such as overimposed pressure, sheath‐liquid composition, sheath‐liquid and sheath‐gas flow rates, ES voltage, and the CE capillary position were optimized in order to obtain an optimal sensitivity. The use of perfluorinated alcohols and acids in the coaxial sheath‐liquid make‐up (2,2,2‐trifluoroethanol + 0.2 mM tridecafluoroheptanoic acid) appeared to provide the best MS sensitivity and improve the stability of spray. The linearity of the CE‐MS and CE‐MS/MS methods was checked under these conditions. Validation parameters such as accuracy, intraday and interday precision, and LOQs were determined in CE‐MS/MS mode. Finally, the quantitation of d4T‐TP and ddA‐TP was validated in this CE‐MS/MS system.