Xia Yuan-Qing, Jemal Mohammed, Zheng Naiyu, Shen Xiaohang
Bioanalytical and Discovery Analytical Sciences, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543, USA.
Rapid Commun Mass Spectrom. 2006;20(12):1831-7. doi: 10.1002/rcm.2517.
A major challenge in selecting an appropriate stationary phase for diastereomeric separation is that it is difficult to predict which of the commercially available stationary phases could achieve the required liquid chromatographic (LC) separation. This work describes the selection and evaluation of a porous graphitic carbon (PGC) column coupled with tandem mass spectrometry (MS/MS) for the simultaneous quantitation of an experimental drug candidate (I), its two diastereomeric metabolites (II and III), and its demethylated metabolite (IV) in rat plasma. In addition, we investigated the PGC column for the separation of another drug candidate (VI), its two diastereomeric metabolites (VII and VIII) and its ketone metabolite (IX). The PGC column showed excellent chromatographic resolution for the two diastereomers II and III, as well as for VII and VIII. In contrast, the required resolution for the diastereomers II and III could not be achieved using silica-bonded C(18), C(30), phenyl, perfluorinated, polar embedded and polar end-capped phases. The PGC column showed ruggedness with excellent reproducibility of retention times, peak symmetry and response over a period of more than 400 injections of a plasma acetonitrile-precipitation extract. Excellent accuracy and precision were achieved, with accuracy of 94-108% and intra- and inter-run precision within 9%. This work indicates that PGC is a valuable addition to the repertoire of LC columns used for quantitative LC/MS/MS bioanalysis, especially where the separation and quantitation of diastereomeric analytes is involved.
为非对映体分离选择合适的固定相面临的一个主要挑战是，很难预测市售的固定相中哪一种能够实现所需的液相色谱（LC）分离。本文描述了一种多孔石墨化碳（PGC）柱与串联质谱（MS/MS）联用，用于同时定量大鼠血浆中一种实验性候选药物（I）、其两种非对映体代谢物（II和III）及其去甲基化代谢物（IV）。此外，我们还研究了PGC柱对另一种候选药物（VI）、其两种非对映体代谢物（VII和VIII）及其酮代谢物（IX）的分离情况。PGC柱对非对映体II和III以及VII和VIII显示出优异的色谱分辨率。相比之下，使用硅胶键合的C(18)、C(30)、苯基、全氟、极性嵌入和极性封端相无法实现非对映体II和III所需的分辨率。在对血浆乙腈沉淀提取物进行400多次进样的过程中，PGC柱表现出良好的耐用性，保留时间、峰对称性和响应具有出色的重现性。实现了优异的准确度和精密度，准确度为94-108%，批内和批间精密度在9%以内。这项工作表明，PGC是用于定量LC/MS/MS生物分析的LC柱系列中的一个有价值的补充，特别是在涉及非对映体分析物的分离和定量时。
