Mass spectrometry in the quantitative analysis of therapeutic intracellular nucleotide analogs

Abstract

Nucleoside analogs are widely used in anti-cancer, anti-(retro)viral, and immunosuppressive therapy. Nucleosides are prodrugs that require intracellular activation to mono-, di-, and finally triphosphates. Monitoring of these intracellular nucleotides is important to understand their pharmacology. The relatively involatile salts and ion-pairing agents traditionally used for the separation of these ionic analytes limit the applicability of mass spectrometry (MS) for detection. Both indirect and direct methods have been developed to circumvent this apparent incompatibility. Indirect methods consist of de-phosphorylation of the nucleotides into nucleosides before the actual analysis. Various direct approaches have been developed, ranging from the use of relatively volatile or very low levels of regular ion-pairing agents, hydrophilic interaction chromatography (HILIC), weak anion-exchange, or porous graphitic carbon columns to capillary electrophoresis and matrix-assisted light desorption--time of flight (MALDI-TOF) MS. In this review we present an overview of the publications describing the quantitative analysis of therapeutic intracellular nucleotide analogs using MS. The focus is on the different approaches for their direct analysis. We conclude that despite the technical hurdles, several useful MS-compatible chromatographic approaches have been developed, enabling the use of the excellent selectivity and sensitivity of MS for the quantitative analysis of intracellular nucleotides.

Fully validated assay for the quantification of endogenous nucleoside mono- and triphosphates using online extraction coupled with liquid chromatography–tandem mass spectrometry

“…The reduction of peak tailing by DEA was previously observed for nucleoside di-and triphosphates [9]. According to the literature, peak tailing results from interactions between phosphate compounds and stainless steel [22][23][24]. Therefore, triphosphate compounds are more affected than diphosphates and monophosphates [9].…”

Decitabine triphosphate levels in peripheral blood mononuclear cells from patients receiving prolonged low-dose decitabine administration: a pilot study

“…High-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) currently allows the sensitive and speciWc detection of nucleotide analogs [17]. The primary aim of this pilot study was to develop a sensitive method for the quantitative determination of non-radioactive aza-dC nucleotides, making studies in patients feasible.…”

Validation of a method for the determination of the anticancer agent Combretastatin A1 phosphate (CA1P, OXi4503) in human plasma by HPLC with post-column photolysis and fluorescence detection

“…TBAHSO 4 is well known to cause ion suppression in MS and the high concentration required for extraction was one of the reasons for rejecting this as a detection technique. We were unable to achieve satisfactory reproducibility using these samples in eluents compatible with mass spectrometric detection, although we have not investigated alternative ion pairing agents which are reported to be suitable for use in MS systems [7]. Notably, this requirement for the ion pairing agent to enhance extraction was species and even strain specific, with some mouse strains giving high recovery using just methanol as extractant.…”