Intracellular Nucleotides of (−)-2′,3′-Deoxy-3′-Thiacytidine in Peripheral Blood Mononuclear Cells of a Patient Infected with Human Immunodeficiency Virus

An analytical methodology was developed to quantitate the intracellular nucleotides including mono-, di-, and triphosphates and the diphosphocholine derivative of (−)-2′,3′-deoxy-3′-thiacytidine (3TC) in human peripheral blood mononuclear cells (PBMCs). The procedure includes the resolution of 3TC nucleotides by solid-phase extraction (SPE) on an anion-exchange cartridge, with subsequent enzyme digestion of the resulting phosphates to the parent drug that is ultimately quantitated by high-performance liquid chromatography with UV detection (HPLC-UV). Validation was performed with PBMCs from healthy donors exposed to [(3)H]3TC, leading to the formation of intracellular nucleotides that were quantitated by anion-exchange HPLC with radioactive detection (HPLC-RA). These nucleotide levels served as reference values and were used for cross-validation with data obtained by HPLC-UV. An excellent correlation was established between the results obtained by HPLC-RA and those obtained by HPLC-UV, with a slope of the regression lines close to unity and intercepts near nullity as well as a correlation coefficient close to unity for all 3TC phosphates. The assay was characterized by a limit of quantitation below 1 ng (amount on column) with a precision (percentage of coefficient of variation of repeated measurement) ranging from 0.8 to 18.1% and an accuracy (deviation of the amount determined by HPLC-UV from the nominal reference value) varying from −14.8 to 19.4%. This methodology was successfully applied to determine the quantity of 3TC nucleotides in PBMCs of a patient infected with human immunodeficiency virus after oral administration of 3TC and stavudine.

本研究开发了一种定量分析方法，可实现人外周血单个核细胞（PBMCs）内细胞内核苷酸（涵盖一磷酸、二磷酸与三磷酸形式）以及(−)-2′,3′-二脱氧-3′-硫代胞苷（3TC）的二磷酸胆碱衍生物的定量检测。该流程通过阴离子交换固相萃取（SPE）柱分离3TC核苷酸，随后将所得磷酸酯类产物酶解为原形药物，最终借助紫外检测高效液相色谱法（HPLC-UV）完成定量。本研究采用经[(3)H]3TC处理的健康供者外周血单个核细胞开展方法验证，通过放射性检测高效液相色谱法（HPLC-RA）定量生成的细胞内核苷酸，以所得核苷酸水平作为参考值，并与HPLC-UV获取的实验数据进行交叉验证。HPLC-RA与HPLC-UV的检测结果之间呈现极佳的相关性：所有3TC磷酸酯的回归直线斜率均接近1，截距趋近于零，相关系数亦接近1。该检测方法的定量限低于1 ng（柱上样量），精密度（重复测量的变异系数百分比）介于0.8%至18.1%之间，准确度（HPLC-UV测定值与标称参考值的偏差）范围为−14.8%至19.4%。本方法已成功应用于口服3TC与司他夫定后，人类免疫缺陷病毒感染者外周血单个核细胞中3TC核苷酸的定量检测。