Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology and University of Tübingen, Stuttgart, Germany.
Anal Bioanal Chem. 2011 Jul;400(10):3429-41. doi: 10.1007/s00216-011-5045-9. Epub 2011 May 1.
Since the 1960s, clomiphene citrate is used for ovulation induction. Since nonresponse to clomiphene therapy is still not well understood, interindividual variability of clomiphene metabolism has been considered to be a plausible explanation. Therefore, a comprehensive, rapid, sensitive, and specific analytical method for the quantification of (E)- and (Z)-isomers of clomiphene and their putative N-desethyl, N,N-didesethyl, 4-hydroxy, and 4-hydroxy-N-desethyl metabolites, and the N-oxides in human plasma has been newly developed, using HPLC-tandem mass spectrometry and stable isotope-labeled internal standards. All standards other than the parent drug were synthesized in our laboratory. Following protein precipitation analytes were separated on a ZORBAX Eclipse plus C18 1.8 μm column with a gradient of 0.1% formic acid in water and 0.1% formic acid in acetonitrile and detected on a triple quadrupole mass spectrometer with positive electrospray ionization in the multiple reaction monitoring mode. Lower limit of quantification for metabolites ranged from 0.06 ng/mL for clomiphene-N-oxides to 0.3 ng/mL for (E)-N-desethylclomiphene. The assay was validated according to FDA guidelines. Plasma levels of clomiphene and its metabolites were measured in two women after single-dose treatment with clomiphene.
自 20 世纪 60 年代以来，枸橼酸氯米酚已被用于诱导排卵。由于对枸橼酸氯米酚治疗无反应的机制仍未得到充分理解，因此个体间枸橼酸氯米酚代谢的差异被认为是一个合理的解释。因此，我们新开发了一种用于定量测定人血浆中（E）-和（Z）-异构体及其可能的 N-去乙基、N，N-二去乙基、4-羟基和 4-羟基-N-去乙基代谢物以及 N-氧化物的综合、快速、灵敏和特异的分析方法，采用高效液相色谱-串联质谱法和稳定同位素标记的内标法。除母体药物外，所有标准品均在我们的实验室中合成。在 ZORBAX Eclipse plus C18 1.8 μm 柱上进行分析，采用水相中的 0.1%甲酸和乙腈中的 0.1%甲酸进行梯度洗脱，在三重四极杆质谱仪上采用正电喷雾电离，以多反应监测模式进行检测。代谢物的定量下限范围为 0.06 ng/mL 至 0.3 ng/mL（从氯米酚 N-氧化物到（E）-N-去乙基氯米酚）。该测定方法按照 FDA 指南进行了验证。在两名女性单次服用枸橼酸氯米酚后，测量了她们血浆中枸橼酸氯米酚及其代谢物的水平。
