Bioanalytical Sciences, Research & Development , Bristol-Myers Squibb , Route 206 & Province Line Road , Princeton , New Jersey 08543 , United States.
Anal Chem. 2019 Jul 2;91(13):8652-8659. doi: 10.1021/acs.analchem.9b02136. Epub 2019 Jun 21.
Preparation of multisample external calibration curves and dilution of study samples are critical steps in bioanalytical sample processing for quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) based bioanalysis of small-molecule compounds, biotherapeutics, and biomarkers, but they can be time-consuming and prone to error. It is highly desired to simplify or eliminate these two steps in order to improve the assay throughput and robustness. While multisample external calibration curve preparation using authentic matrices can be eliminated with a previously reported in-sample calibration curve (ISCC) approach using multiple isotopologue reaction monitoring (MIRM) of a stable isotopically labeled (SIL) analyte, dilution of study samples is still inevitable due to limited LC-MS/MS assay ranges. In this work, a one-sample multipoint external calibration curve and isotope sample dilution, both using MIRM of an analyte, for quantitative LC-MS/MS based bioanalysis are proposed and demonstrated. By spiking a known amount of an analyte into one blank authentic matrix sample, a one-sample multipoint external calibration curve in an authentic matrix can be established on the basis of the relationship between the calculated theoretical isotopic abundances (analyte concentration equivalents) and the MS/MS responses in the corresponding MIRM channels. This one-sample multipoint external calibration curve can be used in the same way as the traditional multisample external calibration curve for quantitative LC-MS/MS-based bioanalysis. As isotopic abundance in each MIRM channel can be calculated and measured accurately, isotope sample dilution can be achieved by simply monitoring one or a few of the MIRM channels of the analyte in addition to the most abundant MIRM channel for study samples. While the most abundant MIRM channel (isotopic abundance of 100%) is used for the quantitation of samples having concentrations within the assay calibration curve range, less abundant MIRM channels (isotopic abundance of IA%) can be used for the quantitation of samples having concentrations beyond the assay upper limit of quantitation (ULOQ), resulting in isotope dilution factors (IDF) of 100%/IA%. The approaches of one-sample multipoint external calibration curve and isotope sample dilution were evaluated and demonstrated in this work with an example of the quantitation of daclatasvir in human plasma extracted with liquid-liquid extraction. Using these approaches together with the MIRM-ISCC methodology, accurate and reliable LC-MS/MS bioanalysis can be achieved without the need of preparation of multisample external calibration curve and dilution of study samples.
制备多份样本外部校准曲线和研究样本的稀释是基于液相色谱-串联质谱（LC-MS/MS）的小分子化合物、生物治疗药物和生物标志物定量分析中生物分析样品处理的关键步骤，但这些步骤既耗时又容易出错。为了提高检测通量和稳健性，简化或消除这两个步骤是非常理想的。虽然使用真实基质的多份样本外部校准曲线制备可以通过先前报道的使用稳定同位素标记（SIL）分析物的多重同位素反应监测（MIRM）的内标校准曲线（ISCC）方法来消除，但由于 LC-MS/MS 检测范围有限，研究样本的稀释仍然是不可避免的。在本工作中，提出并证明了一种使用分析物的 MIRM 进行单点多校准曲线和同位素样品稀释的方法，用于基于 LC-MS/MS 的定量生物分析。通过将已知量的分析物加入到一个空白真实基质样品中，可以基于计算的理论同位素丰度（分析物浓度等价物）与相应 MIRM 通道中的 MS/MS 响应之间的关系，建立真实基质中的单点多校准曲线。这种单点多校准曲线可以像传统的多份样本外部校准曲线一样，用于基于 LC-MS/MS 的定量生物分析。由于每个 MIRM 通道中的同位素丰度都可以准确地计算和测量，因此除了研究样本中最丰富的 MIRM 通道之外，还可以通过简单地监测分析物的一个或几个 MIRM 通道来实现同位素样品稀释。当最丰富的 MIRM 通道（同位素丰度为 100%）用于定量浓度在检测校准曲线范围内的样品时，可以使用较少丰度的 MIRM 通道（同位素丰度为 IA%）定量浓度超过检测定量上限（ULOQ）的样品，从而得到同位素稀释因子（IDF）为 100%/IA%。在本工作中，通过使用液液萃取提取人血浆中的达卡他韦的定量分析实例，对单点多校准曲线和同位素样品稀释的方法进行了评估和验证。使用这些方法与 MIRM-ISCC 方法相结合，可以在不需要制备多份样本外部校准曲线和稀释研究样本的情况下，实现准确可靠的 LC-MS/MS 生物分析。
