Simultaneous quantification of intracellular natural and antiretroviral nucleosides and nucleotides by liquid chromatography-tandem mass spectrometry

Abstract

Nucleoside reverse transcriptase inhibitors (NRTI) require intracellular phosphorylation, which involves multiple enzymatic steps to inhibit the human immunodeficiency virus type 1 (HIV-1). NRTI-triphosphates (NRTI-TP) compete with endogenous 2'-deoxyribonucleosides-5'-triphosphates (dNTP) for incorporation by the HIV-1 reverse transcriptase (RT). Thus, a highly sensitive analytical methodology capable of quantifying at the low femtomoles/10(6) cells level was necessary to understand the intracellular metabolism and antiviral activity of NRTIs in human peripheral blood mononuclear (PBM) cells and in macrophages. A novel, rapid, and a reproducible ion-pair chromatography-tandem mass spectrometry (MS/MS) method was developed to simultaneously quantify the intracellular phosphorylated metabolites of abacavir, emtricitabine, tenofovir disoproxil fumarate, amdoxovir, and zidovudine, as well as four natural endogenous dNTP. Positive or negative electrospray ionization was chosen with specific MS/MS transitions for improved selectivity on all the compounds studied. The sample preparation, the ion-pair reagent concentration, and buffer composition were optimized, resulting in the simultaneous quantification of 13 different nucleotides in a total run time of 30 min. This novel method demonstrated optimal sensitivity (limit of detection 1-10 nM for various analytes), specificity, and reproducibility to successfully measure NRTI-TP and dNTP in human PBM cells and macrophages.

Figure 1

Chromatography parameters of NRTI-TP represented by the symbols: +, 3TC-TP; ×, DXG-TP; ◆, TFV-DP; □, CBV-TP; △, (−)-FTC-TP; and ○, ZDV-TP: (a) tailing factor (Tf) at five different HMA concentrations; 0, 3, 6, 9, and 12 mM. (b) Effective plate number (Ne) at four different buffer compositions; 1 and 2 mM ammonium phosphate; 10 and 20 mM ammonium hydrogen carbonate. (c) Peak area at pH 7.0, 8.0, 9.2, and 10.0.

Figure 2

Typical chromatograms displaying all the standards used for the partial validation spiked in a PBM cells matrix containing 1 × 10⁶ cells/100 μL of mobile phase. The RIC shows the peaks obtained for DXG, DXG-MP, DXG-TP, TFV, TFV-DP, ZDV-MP, ZDV-TP, (−)-FTC-TP, CBV-TP, [¹³C¹⁵N]dATP, [¹³C¹⁵N]dGTP, and [¹³C¹⁵N]dCTP at 100 nM and [¹³C¹⁵N]TTP at 500 nM. Internal standards, 3TC, 3TC-MP, and 3TC-TP were at 100 nM.

Figure 3

(a) Typical chromatograms for NRTI-TP, obtained from macrophages. Five drugs(ZDV, ABC, TDF, (−)-FTC, and DXG) were incubated separately at 10 μM for 4 h and analyzed in five different LC–MS/MS runs. The left chromatograms represent five background signals obtained from a single injection of untreated macrophage extract; these chromatograms are marked as “blank”. The right chromatograms represent the five signals obtained from five separate injections of treated macrophages extracts. The sample incubated with TDF was diluted 20 times. (b) Typical chromatograms of endogenous dNTP obtained from a single macrophage extract.