Matuszewski B K, Constanzer M L, Chavez-Eng C M
Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
Anal Chem. 1998 Mar 1;70(5):882-9. doi: 10.1021/ac971078+.
Contrary to common perceptions, the reliability of quantitative assays for the determination of drugs in biological fluids using high-performance liquid chromatography with tandem mass spectrometric (LC/MS/MS) detection methods and the integrity of resulting pharmacokinetic data may not be absolute. Results may be adversely affected by lack of specificity and selectivity due to ion suppression caused by the sample matrix, interferences from metabolites, and "cross-talk" effects. In this paper, an example of the effect of the sample matrix on the determination of finasteride (I) in human plasma is presented. The ion suppression effect was studied by analyzing standards of I injected directly in mobile phase and comparing the response (peak areas) of I and an internal standard (II) with the peak areas of the same analytes spiked before extraction into five different plasma pools and standards spiked into the plasma extracts after extraction. The LC/MS/MS analyses were performed using a turbo ion spray interface (TISP) under chromatographic conditions, characterized by minimal (total run time of 2 min, capacity factors, k' of 1.50 and 1.75 for I and II, respectively) and high retention of the analytes (total run time 6 min, k' of 3.25 and 13.25 for I and II, respectively). The absolute peak areas for I and II in different plasmas were calculated, and the slopes and peak area ratios at all concentrations within the standard curve ranges were compared. When analyses were performed under conditions of minimal HPLC retention, the slope of the standard line for one set of plasma samples was substantially different (about 50% higher) from that from other plasma sources. The precision of the assay, expressed as coefficient of variation (CV, %) was also inadequate and varied from 15 to 30% at all concentrations within the standard curve range. When the same experiments were repeated using high HPLC retention, the slopes from different plasma sources were practically the same, and the CV was improved to 6-14%. By increasing k' and providing more chromatographic retention of analytes, the "unseen" interferences from plasma matrix were mostly separated from analytes, practically eliminating the ion suppression. In addition, by eliminating from plasma extracts a number of endogenous components through more selective extraction, the ion suppression was also minimized. The detailed data and the design of these experiments are presented. In addition, development of a highly sensitive assay for I in human plasma at low picogram per milliliter concentrations using LC/MS/MS with a heated nebulizer (HN) interface, instead of a TISP interface, is described. In this case, the effects of sample matrixes were not observed.
与普遍认知相反，使用高效液相色谱-串联质谱（LC/MS/MS）检测方法测定生物流体中药物的定量分析的可靠性以及所得药代动力学数据的完整性并非绝对。由于样品基质引起的离子抑制、代谢物的干扰以及“串扰”效应，结果可能会受到缺乏特异性和选择性的不利影响。本文给出了一个样品基质对人血浆中非那雄胺（I）测定影响的例子。通过分析直接注入流动相中的I标准品，并将I和内标（II）的响应（峰面积）与在萃取前加入到五个不同血浆池中的相同分析物的峰面积以及萃取后加入到血浆提取物中的标准品的峰面积进行比较，研究了离子抑制效应。LC/MS/MS分析使用涡轮离子喷雾接口（TISP）在色谱条件下进行，其特点是分析物的保留时间最短（总运行时间为2分钟，I和II的容量因子k'分别为1.50和1.75）且保留率高（总运行时间6分钟，I和II的k'分别为3.25和13.25）。计算了不同血浆中I和II的绝对峰面积，并比较了标准曲线范围内所有浓度下的斜率和峰面积比。当在HPLC保留时间最短的条件下进行分析时，一组血浆样品的标准曲线斜率与其他血浆来源的斜率有很大差异（高出约50%）。该分析方法的精密度以变异系数（CV，%）表示，在标准曲线范围内的所有浓度下也不充分，从15%到30%不等。当使用高HPLC保留时间重复相同实验时，不同血浆来源的斜率实际上相同，CV提高到6 - 14%。通过增加k'并提供更多分析物的色谱保留，血浆基质的“不可见”干扰大多与分析物分离，实际上消除了离子抑制。此外，通过更具选择性的萃取从血浆提取物中去除一些内源性成分，离子抑制也降至最低。本文给出了这些实验的详细数据和设计。此外，还描述了使用带加热雾化器（HN）接口而非TISP接口的LC/MS/MS开发一种用于测定人血浆中低至皮克每毫升浓度的I的高灵敏度分析方法。在这种情况下，未观察到样品基质的影响。
