Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30332, USA.
Anal Chem. 2010 Mar 1;82(5):1982-9. doi: 10.1021/ac902737j.
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.
核苷逆转录酶抑制剂（NRTI）需要细胞内磷酸化，这涉及多个酶促步骤来抑制人类免疫缺陷病毒 1 型（HIV-1）。NRTI-三磷酸酯（NRTI-TP）与内源性 2'-脱氧核糖核苷酸-5'-三磷酸酯（dNTP）竞争，由 HIV-1 逆转录酶（RT）掺入。因此，需要一种能够在低 femtomoles/10(6)细胞水平定量的高度敏感分析方法，以了解 NRTI 在人外周血单核细胞（PBM）和巨噬细胞中的细胞内代谢和抗病毒活性。开发了一种新的、快速且可重复的离子对色谱-串联质谱（MS/MS）方法，以同时定量阿巴卡韦、恩曲他滨、替诺福韦二吡呋酯、amdoxovir 和齐多夫定的细胞内磷酸化代谢物，以及四种天然内源性 dNTP。选择正或负离子喷雾电离，并针对所有研究化合物进行特定的 MS/MS 转换，以提高选择性。优化了样品制备、离子对试剂浓度和缓冲液组成，使得在 30 分钟的总运行时间内可同时定量 13 种不同的核苷酸。这种新方法表现出最佳的灵敏度（各种分析物的检测限为 1-10 nM）、特异性和重现性，可成功测量人 PBM 细胞和巨噬细胞中的 NRTI-TP 和 dNTP。
