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

Abstract

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, mononucleotides, 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 approximately 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.

Mass spectrometry in the quantitative analysis of therapeutic intracellular nucleotide analogs

“…The nucleotides could be separated using an ammonium acetate buffer on bare fused silica. Similar methods were used to study the metabolism of 3TC in HepG2 cells (Liu et al, 2005) and d4T-and ddA-MP and -TP in PBMCs (Bezy et al, 2006). Bezy et al, however, applied a coaxial sheath liquid composed of decafluoroheptanoic acid in 2,2,2,-trifluoroethanol to improve sensitivity and spray stability.…”

Recent advances in the application of capillary electrophoresis with mass spectrometric detection

“…An interesting example for the possibilities of CE-MS in modern bioanalytical chemistry is the work of Liu et al [101]. They presented a study on the phosphorylation of antihuman immunodeficiency virus nucleosides in a human hepatoma cell line.…”

Sheath Liquids in CE-MS: Role, Parameters, and Optimization

“…Several papers describe the use of pure methanol as SL [16,[33][34][35][36]. Methanol as a protic solvent is well compatible with the ESI process.…”