Schwartz Michael S, Desai Rajesh B, Bi Sheng, Miller Alisha R, Matuszewski Bogdan K
Department of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2006 Jun 6;837(1-2):116-24. doi: 10.1016/j.jchromb.2006.04.022. Epub 2006 May 22.
A method for the determination of a prostaglandin D(2) receptor antagonist (I, a compound being evaluated for the prevention of niacin induced flushing) and its acyl glucuronide metabolite (II) in human plasma is presented. The method utilized high performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection using an atmospheric pressure chemical ionization (APCI) interface operated in the positive ionization mode. The product ion was a radical cation generated via a homolytic bond cleavage. A chemical analog of the drug was used as internal standard (III). The acyl glucuronide metabolite (II) was detected using the same precursor-to-product ion transition used for the parent compound after chromatographic separation of I and II. Drug and metabolite were extracted using semi-automated, 96-well format solid phase extraction (SPE), and chromatography was performed using a reverse phase analytical column with an isocratic mobile phase. The chromatographic retention factor (k') of II was found to be highly sensitive to mobile phase formic acid concentration. An adjustment in mobile phase formic acid concentration improved the chromatographic separation between II and a mono-hydroxylated metabolite after an unexpected lack of MS/MS selectivity between the two molecules was observed. The dependence of retention factor on formic acid concentration (k' increased as formic acid concentration decreased) was thought to indicate polar interactions between II and the stationary phase. The stability of II in spiked human plasma was determined. The rate of hydrolysis back to parent compound was relatively low (approximately 0.1 and 0.5% per hour at room temperature and 4 degrees C, respectively) indicating that significant changes in analyte concentrations did not occur during sample processing. The concentration range of the assay was 10-2500 ng/mL for both drug and glucuronide metabolite.
本文介绍了一种测定人血浆中前列腺素D₂受体拮抗剂（化合物I，正被评估用于预防烟酸引起的潮红）及其酰基葡萄糖醛酸代谢物（化合物II）的方法。该方法采用高效液相色谱（HPLC）与串联质谱（MS/MS）检测联用，使用在正离子模式下运行的大气压化学电离（APCI）接口。产物离子是通过均裂键裂解产生的自由基阳离子。该药物的一种化学类似物用作内标（化合物III）。在对化合物I和II进行色谱分离后，使用与母体化合物相同的前体-产物离子转换来检测酰基葡萄糖醛酸代谢物（化合物II）。药物和代谢物采用半自动96孔板形式的固相萃取（SPE）进行提取，并使用具有等度流动相的反相分析柱进行色谱分析。发现化合物II的色谱保留因子（k'）对流动相甲酸浓度高度敏感。在观察到这两个分子之间意外缺乏MS/MS选择性后，调整流动相甲酸浓度改善了化合物II与单羟基化代谢物之间的色谱分离。保留因子对甲酸浓度的依赖性（k'随着甲酸浓度降低而增加）被认为表明化合物II与固定相之间存在极性相互作用。测定了化合物II在加标人血浆中的稳定性。水解回母体化合物的速率相对较低（在室温下和4℃时分别约为每小时0.1%和0.5%），表明在样品处理过程中分析物浓度没有发生显著变化。该测定方法对药物和葡萄糖醛酸代谢物的浓度范围均为10 - 2500 ng/mL。
