Development and validation of a liquid chromatographic/tandem mass spectrometric assay for the quantitation of nucleoside HIV reverse transcriptase inhibitors in biological matrices

Abstract

Besides liquid chromatographic (LC)/UV methods adapted to therapeutic drug monitoring, there is still a need for more powerful techniques that can be used for pharmacological research and clinical purposes. We developed an LC method coupled with tandem mass spectrometry (MS/MS) to separate, detect and quantify with high sensitivity the nucleoside analogues used in multitherapies (zidovudine, stavudine, zalcitabine, didanosine, lamivudine and abacavir) in plasma and in the intracellular medium. We worked on two essential issues: (i) the need to use two ionization modes in order to achieve the best sensitivity, which leads to the optimization of the chromatographic separation of drugs detected in the positive ionization mode and drugs detected in the negative ionization mode, and (ii) the need to optimize the extraction step in order to enhance sample recovery. The peripheral blood mononuclear cells were lysed in Tris buffer-MeOH. A clean-up procedure was performed by solid-phase extraction only for plasma samples. The LC separation was carried out on a Zorbax Stable Bond C(18) column followed by MS/MS analysis after electrospray ionization in either the negative or positive mode. The positive ionization mode was applied at the beginning of the run to detect zalcitabine and lamivudine, then the ionization mode was changed to negative for the detection of didanosine, stavudine, internal standard and zidovudine. The calibration range for all the analytes was 0.5-200 ng ml(-1). The recoveries were between 64 and 90%, with coefficients of variation (CVs) lower than 15%. The inaccuracy (bias) was +/-15% with CVs always lower than 12%. The analytes were stable at room temperature and in the extraction solvent for at least 24 h, after storage at -80 degrees C for 3 months, after three freeze-thaw cycles and in the injection solvent after 48 h at 4 degrees C. Together with the measurement of intracellular triphosphorylated metabolites thanks to the powerful plasma and intracellular assay method for intact drugs, it is possible to describe the behaviour of nucleoside analogues against HIV through plasma pharmacokinetics, cell membrane diffusion including drug transport involvement, and also the intracellular metabolism.

High Levels of Zidovudine (AZT) and Its Intracellular Phosphate Metabolites in AZT- and AZT-Lamivudine-Treated Newborns of Human Immunodeficiency Virus-Infected Mothers

“…The resulting solutions were filtered and analyzed using standard LC-MS/MS conditions (calibration range, 0.5 to 200 ng/ml). Intra-and interday precisions and accuracies tallied with previously reported recommendations (48) and previously reported values (15). No interlaboratory tests have been performed, since this kind of quality control exists only for protease inhibitors and nonnucleoside reverse transcriptase inhibitors (19,33) but not for NRTI.…”

“…Extraction and LC-MS/MS analysis of NRTI from plasma samples. The procedure for extraction and LC-MS/MS analysis of NRTI from plasma samples was an improved version of a previously reported assay (15), with minor modifications concerning the working volume of samples. Only 250 l of plasma was taken, spiked with 50 l of internal standard (2-chloroadenosine and zalcitabine), but the preparation process remained unchanged.…”

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

“…The boost given by the technological developments of novel mass analyzers [13], has produced several LC coupled with MS/MS methods [14][15][16]. Recently, Benech and co-workers [17][18][19][20][21] used LC-MS/ MS with selected reaction monitoring (SRM) to directly quantitate the intracellular triphosphorylated metabolites of anti-HIV nucleosides, alone or in combination, in human PBMCs of HIV-positive patients. As part of our on going program to develop new bioanalytical methods, our group has previously reported that CE coupled with ESI-MS/MS was an excellent technique for the separation and quantitation of various anti-HIV nucleosides [22,23].…”

Rapid and simple liquid chromatography tandem mass spectrometry method for the quantification of zidovudine in rat plasma

“…More recently, LC coupled with tandem mass spectrometry (MS/MS) was applied to measure zidovudine concentrations in plasma Volosov et al, 2002;Font et al, 1998;Compain et al, 2005Compain et al, , 2007King et al, 2006;Pereira et al, 2000). This technique combines the advantages of LC separation and the high sensitivity and specificity of MS/MS, but sometimes it is not sufficiently exploited with respect to the chromatographic step.…”