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.
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 cmx 50 microm id; 192 microm 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.
Bezy
Vincent
V
Institut de Chimie Organique et Analytique (ICOA), Université d'Orléans, Orléans, France.
Chaimbault
Patrick
P
Morin
Philippe
P
Unger
Steve E
SE
Bernard
Marie-Charlotte
MC
Agrofoglio
Luigi A
LA
D019380:Anti-HIV Agents / Q000378:metabolism*; D002457:Cell Extracts / Q000737:chemistry; D003838:Deoxyadenine Nucleotides / Q000032:analysis; D003854:Deoxyribonucleotides / Q000032:analysis*; D016049:Didanosine / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005466:Fluorocarbons / Q000737:chemistry; D006538:Heptanoic Acids / Q000737:chemistry; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry; D009711:Nucleotides / Q000378:metabolism; D010766:Phosphorylation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D018119:Stavudine / Q000378:metabolism*; D013942:Thymine Nucleotides / Q000032:analysis; D014270:Trifluoroethanol / Q000737:chemistry
D019380:Anti-HIV Agents; D002457:Cell Extracts; D003838:Deoxyadenine Nucleotides; D003854:Deoxyribonucleotides; D005466:Fluorocarbons; D006538:Heptanoic Acids; D009711:Nucleotides; D013942:Thymine Nucleotides; C101815:perfluoro-n-heptanoic acid; D014270:Trifluoroethanol; D018119:Stavudine; D016049:Didanosine