Capillary electrophoresis-electrospray-mass spectrometry of nucleosides and nucleotides: application to phosphorylation studies of anti-human immunodeficiency virus nucleosides in a human hepatoma cell line.

We report the use of capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) for the determination of antiretroviral dideoxynucleosides (ddNs), their nucleotides, and a set of ribonucleosides and ribonucleotides. A CE system for separation of most commonly used antiretroviral ddNs has been developed based on a basic buffer with a volatile electrolyte suitable for ESI-MS detection in an untreated capillary column. Positive and negative ionization modes are investigated and compared for sensitive and stable electrospray performance. A 14-compound mixture of nucleosides and nucleotides is profiled in a single capillary zone electrophoresis separation with a distinct elution order: electroosmotic flow, ddNs, mono-nucleotides, dinucleotides, and trinucleotides in less than 18 min. The fragmentation pathways of the nucleosides and nucleotides in ESI-MS have been interpreted. Concentration limits of detection are 100 to 200 nm with an injection volume of similar to 10 nL. This technique has been used to detect naturally occurring nucleotides and to study the metabolism of lamivudine (3TC) in the human hepatoma cell line Hep G2. 3TC and its metabolites 3TC-monophosphate, 3TC-diphosphate, and 3TC-triphosphate were detected after 10 h of incubation of 3TC with the cells.