Sun Li, Chavez-Eng Cynthia, Fillgrove Kerry L, Lu Bing, Xie Iris, Rudd Deanne Jackson, Breidinger Sheila, Anderson Melanie, Yeh Suzie, Zhang Rena, Woolf Eric J
Department of Pharmacokinetics, Pharmacodynamics & Drug Metabolism, Merck & Co., Inc., West Point, PA, USA.
Bioanalysis. 2019 Feb;11(4):233-250. doi: 10.4155/bio-2018-0101. Epub 2019 Feb 15.
MK-8591 (EFdA), a novel anti-HIV nucleoside analog, is converted to mono-, di- and tri-phosphates (MK-8591-MP, MK-8591-DP and MK-8591-TP) intracellularly, among which MK-8591-TP is the active pharmacological form. An ultrasensitive LC-MS/MS assay was required to measure MK-8591-DP and MK-8591-TP levels in human peripheral blood mononuclear cells (PBMCs). Sensitivity and reproducibility were major bottlenecks in these analyses. Human PBMCs were isolated from blood and lysed with 70/30 methanol/RPMI-1640. An LC-MS/MS method was developed to simultaneously quantify MK-8591-DP and MK-8581-TP in PBMC lysates. Low flow LC and dimethyl sulfoxide mediated signal enhancement enabled an extreme sensitivity with limit of quantitation at 0.1 ng/ml. Assay accuracy was 92.5-106% and precision was 0.7-12.1% for a linear curve range of 0.1-40 ng/ml. Matrix variability and interference liability were comprehensively evaluated. Our study findings and steps taken in addressing clinical sample issues help understand and overcome the challenges facing intracellular nucleotide analog analysis.
新型抗HIV核苷类似物MK-8591（EFdA）在细胞内转化为一磷酸、二磷酸和三磷酸形式（MK-8591-MP、MK-8591-DP和MK-8591-TP），其中MK-8591-TP是活性药理形式。需要一种超灵敏的液相色谱-串联质谱（LC-MS/MS）测定法来测量人外周血单个核细胞（PBMC）中的MK-8591-DP和MK-8591-TP水平。灵敏度和重现性是这些分析中的主要瓶颈。从血液中分离出人PBMC，并用70/30的甲醇/RPMI-1640进行裂解。开发了一种LC-MS/MS方法来同时定量PBMC裂解物中的MK-8591-DP和MK-8581-TP。低流速液相色谱和二甲亚砜介导的信号增强实现了极高的灵敏度，定量限为0.1 ng/ml。对于0.1-40 ng/ml的线性曲线范围，测定准确度为92.5-106%，精密度为0.7-12.1%。全面评估了基质变异性和干扰可能性。我们的研究结果以及在解决临床样本问题时采取的步骤有助于理解和克服细胞内核苷酸类似物分析面临的挑战。
