Annesley T M, Kurzyniec S, Nordblom G D, Buchanan N, Pool W, Reily M, Talaat R, Roberts W L
University of Michigan Medical Center, Ann Arbor, MI 48109-0054, USA.
Clin Chem. 2001 May;47(5):910-8.
This investigation was undertaken to identify the structure of a novel immunoreactive metabolite derived from fosphenytoin that has been hypothesized previously as present in sera from renally impaired patients receiving this prodrug.
The metabolite was isolated from uremic sera using solid-phase extraction and HPLC. Structural analysis was performed using HPLC-tandem mass spectrometry, nuclear magnetic resonance (NMR), deuterium exchange, and chemical derivatization. Immunoreactivity was evaluated using a fluorescence polarization immunoassay.
The metabolite had a parent ion at m/z 457 in the negative-ion mode and fragmented to yield the m/z 251 of phenytoin, as well as other mass fragments of phenytoin. Mass fragments associated with glucuronic acid were also present. The chromatographic peak corresponding to this metabolite demonstrated immunoreactivity sufficient to lead to falsely increased reported values for phenytoin immunoassays. The observed immunoreactivity was also proportional to the relative concentration of the metabolite in collected fractions. Analysis by NMR indicated the presence of phenyl groups with chemical shifts identical to those of phenytoin, as well as the presence of a methylene bridge, which was consistent with the same methylene bridge present on the phosphate ester of fosphenytoin. Comparative analysis of serum samples from renally impaired patients receiving phenytoin vs fosphenytoin using multiple reaction monitoring quantification demonstrated that this metabolite was associated with fosphenytoin administration.
A unique immunoreactive oxymethylglucuronide metabolite derived from fosphenytoin has been isolated from sera from uremic patients receiving this prodrug.
本研究旨在鉴定一种源自磷苯妥英的新型免疫反应性代谢物的结构，此前曾推测该代谢物存在于接受这种前体药物的肾功能受损患者的血清中。
使用固相萃取和高效液相色谱法从尿毒症血清中分离该代谢物。采用高效液相色谱 - 串联质谱、核磁共振（NMR）、氘交换和化学衍生化进行结构分析。使用荧光偏振免疫分析法评估免疫反应性。
该代谢物在负离子模式下的母离子为m/z 457，裂解后产生苯妥英的m/z 251以及苯妥英的其他质量碎片。还存在与葡萄糖醛酸相关的质量碎片。对应于该代谢物的色谱峰显示出足以导致苯妥英免疫测定报告值假性升高的免疫反应性。观察到的免疫反应性也与收集馏分中代谢物的相对浓度成正比。核磁共振分析表明存在化学位移与苯妥英相同的苯基，以及一个亚甲基桥，这与磷苯妥英磷酸酯上存在的相同亚甲基桥一致。使用多反应监测定量法对接受苯妥英与磷苯妥英的肾功能受损患者的血清样本进行比较分析表明，这种代谢物与磷苯妥英的给药有关。
已从接受这种前体药物的尿毒症患者血清中分离出一种源自磷苯妥英的独特免疫反应性氧甲基葡萄糖醛酸代谢物。
