TNO, Utrechtseweg 48, 3700 AJ Zeist, The Netherlands.
J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Sep 15;879(26):2772-82. doi: 10.1016/j.jchromb.2011.07.045. Epub 2011 Aug 6.
Nucleoside reverse transcriptase inhibitors (NRTIs) are a key class of drugs for the treatment of HIV infection. NRTIs are intracellularly phosphorylated to their active triphosphate metabolites and compete with endogenous deoxynucleotides (dNTP) for substrate binding. It is therefore important to analyze the intracellular concentrations of these compounds to understand drug efficacy and toxicity. To that purpose an analytical platform was developed that is capable of analyzing 8 NRTIs, 12 phosphorylated NRTIs and 4 dNTPs in small numbers of peripheral blood mononuclear cells, i.e. 1 × 10(6) cells. The platform consists of two liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods: a reversed-phase method for NRTIs using positive electrospray ionization (ESI) and an ion-pair LC-MS/MS method for the phosphorylated compounds using negative ESI. The methods use the same LC-MS system and column and changing from one method to the other only includes changing the mobile phase. The methods were partially validated, focussing on sensitivity, accuracy and precision. Successful transfer of the methods to ultra performance liquid chromatography (UPLC) led to a significant improvement of speed for the analysis of NRTIs and sensitivity for both NRTIs and phosphorylated NRTIs. The latter was demonstrated by the improved separation by UHPLC of dGTP vs. AZT-TP and ATP which made direct analysis of dGTP possible using the optimal MS/MS transition thereby significantly improving the detection limit of dGTP. Typically LLOQs observed for both the NRTIs and phosphorylated NRTIs were 1 nM, while the mean accuracy varied between 82 and 120% and inter- and intra-assay precision was generally <20%.
核苷逆转录酶抑制剂（NRTIs）是治疗 HIV 感染的关键药物类别。NRTIs 在细胞内被磷酸化为其活性三磷酸代谢物，并与内源性脱氧核苷酸（dNTP）竞争底物结合。因此，分析这些化合物的细胞内浓度对于了解药物疗效和毒性非常重要。为此，开发了一种分析平台，能够分析少量外周血单核细胞中的 8 种 NRTIs、12 种磷酸化 NRTIs 和 4 种 dNTPs，即 1×10(6)个细胞。该平台由两种液相色谱-串联质谱（LC-MS/MS）方法组成：一种用于 NRTIs 的反相方法，采用正电喷雾电离（ESI），一种用于磷酸化化合物的离子对 LC-MS/MS 方法，采用负 ESI。这两种方法使用相同的 LC-MS 系统和色谱柱，从一种方法转换到另一种方法只需改变流动相。方法进行了部分验证，重点是灵敏度、准确性和精密度。方法成功转移到超高效液相色谱（UPLC）后，大大提高了 NRTIs 的分析速度和 NRTIs 和磷酸化 NRTIs 的灵敏度。后者通过 UHPLC 对 dGTP 与 AZT-TP 和 ATP 的更好分离得到证明，这使得可以使用最佳 MS/MS 转换直接分析 dGTP，从而大大提高了 dGTP 的检测限。通常，两种 NRTIs 和磷酸化 NRTIs 的LLOQ 观察值均为 1 nM，而平均准确度在 82%至 120%之间变化，批内和批间精密度通常<20%。
