Direct and indirect quantification of phosphate metabolites of nucleoside analogs in biological samples.

Nucleoside reverse transcriptase inhibitors (NRTIs) are prodrugs that require intracellular phosphorylation to active triphosphate nucleotide metabolites (NMs) for their pharmacological activity. However, monitoring these pharmacologically active NMs is challenging due to their instability, high hydrophilicity, and their low concentrations in blood and tissues. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the gold standard technique for the quantification of NRTIs and their phosphorylated NMs. In this review, an overview of the publications describing the quantitative analysis of intracellular and total tissue concentration of NMs is presented. The focus of this review is the comparison of the different approaches and challenges associated with sample collection, tissue homogenization, cell lysis, cell counting, analyte extraction, sample storage conditions, and LC-MS analysis. Quantification methods of NMs via LC-MS can be categorized into direct and indirect methods. In the direct LC-MS methods, chromatographic retention of the NMs is accomplished by ion-exchange (IEX), ion-pairing (IP), hydrophilic interaction (HILIC), porous graphitic carbon (PGC) chromatography, or capillary electrophoresis (CE). In indirect methods, parent nucleosides are 1st generated from the dephosphorylation of NMs during sample preparation and are then quantified by reverse phase LC-MS as surrogates for their corresponding NMs. Both approaches have advantages and disadvantages associated with them, which are discussed in this review.