Simultaneous determination of endogenous deoxynucleotides and phosphorylated nucleoside reverse transcriptase inhibitors in peripheral blood mononuclear cells using ion‐pair liquid chromatography coupled to mass spectrometry

Abstract

Nucleoside reverse transcriptase inhibitors (NRTIs) are activated intracellularly to their triphosphate (TP) form, which compete with endogenous deoxynucleotide-triphosphates (dNTP) as substrate for HIV reverse transcriptase. The activity of NRTIs is thus described by the NRTI-TP-to-dNTP ratio in relevant cell types. Therefore, we developed an ion-pair (IP) LC-MS method for the simultaneous analysis of the mono-, di-, and TP forms of NRTIs and endogenous deoxynucleosides in peripheral blood mononuclear cells (PBMC). The IP-LC method was applied on an IT mass spectrometer using the MS-mode as well as on a triple quadrupole mass spectrometer using the MS/MS mode. The MS/MS approach on the triple quadrupole mass spectrometer demonstrated the best clinical applicability due to its higher sensitivity. The LOD (minimum amount on column) were 25 fmol for the TP forms of zidovudine, lamivudine, and stavudine, as well as for their endogenous dNTP counterparts. The linearity (R(2) ) of the calibration curves were>0.99. The obtained LOD readily allow for clinical applications using just one million PBMC obtained from HIV-infected patients under therapy.

Liquid chromatographic methods for the determination of endogenous nucleotides and nucleotide analogs used in cancer therapy: A review

“…Since cellular endogenous deoxyribonucleotides concentration are low, their detection could not be performed with an ESI ion trap mass spectrometer in full scan mode [43]. Due to its higher sensitivity a triple quadrupole mass spectrometer in multiple ion monitoring mode was required for this purposes [43]. For exemple, LC-MS/MS device lowered the limit of detection by a factor of 100 compared to the UV detection mode and improved significantly the specificity [4,39,41,43,45,49,51,66,67] ( Table 4).…”

“…Detection was based on either triple quadrupole mass spectrometer or ion trap mass spectrometer. Since cellular endogenous deoxyribonucleotides concentration are low, their detection could not be performed with an ESI ion trap mass spectrometer in full scan mode [43]. Due to its higher sensitivity a triple quadrupole mass spectrometer in multiple ion monitoring mode was required for this purposes [43].…”

“…Protein precipitation was also described using pure methanol [49,60] or a methanol/water mixture with different proportions of both compounds: v/v: 80/20 [4], 70/30 [43], 60/40 [51]. To enhance efficiency of protein precipitation, several authors proposed a supplementary step consisting to a freezing-thawing cycle [4,40,42,56,61] or to a sonication in an ice bath [4,44].…”

“…The analysis of nucleotides have been performed in various matrix such as whole blood [38,39], erythrocytes [40], peripheral blood mononucleous cells (PBMC) [41][42][43], cultured cells [4,42,[44][45][46][47][48][49], cerebrospinal fluid (CSF) [50] or tissue [51][52][53]. These matrixes contain enzymes involved in the purines and pyrimidines metabolism.…”

“…IP [40,42,43,45,46,[49][50][51][52][55][56][57]64,66,67,[72][73][74][75] and anion exchange (AE) chromatography [44,76] are in principle the most suitable methods for the determination of nucleotides. Tomiya et al [44] compared an IP chromatography and an AE chromatography for the determination of nucleotides and sugar nucleotides.…”