定量 LC–MS/MS。1. 跨峰点对峰面积测量准确度和精密度的影响,Journal of the American Society for Mass Spectrometry

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定量 LC–MS/MS。1. 跨峰点对峰面积测量准确度和精密度的影响
Journal of the American Society for Mass Spectrometry ( IF 3.1 ) Pub Date : 2023-05-04 , DOI: 10.1021/jasms.3c00077
Wei Zeng ₁ , Kevin P Bateman ₁

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长期以来，色谱峰上的点数一直被认为是测量峰面积准确度和精密度的关键决定因素。在药物发现和开发中基于 LC-MS 的定量实验中，“经验法则”一直是使用 15 个或更多点。该“规则”基于描述色谱方法的文献，其目标是在测量中实现尽可能低的不精确性，尤其是在检测未知分析物时。将方法限制为跨峰至少 15 个点的要求可能不利于使用更长的驻留时间和/或转换求和完全优化测定信噪比的开发方法。本研究旨在表明，对于宽度为 9 秒或更短的峰，跨峰的 7 个点可为药物定量研究提供足够的准确度和精密度。使用跨峰 7 点采样间隔的模拟高斯曲线数据使用梯形和黎曼法则计算的峰面积在预期总峰面积的 1% 以内，辛普森法则为 0.6%。低浓度和高浓度样品（n = 5) 在三天的不同时间在两种不同的仪器（API5000 和 API5500）上使用三种不同的 LC 方法进行了分析。峰面积的差异 (%ΔPA) 和峰面积的相对标准偏差 (%RSD) 小于～5%。从不同采样间隔、不同峰宽、不同天数、不同峰大小和不同仪器获得的数据没有观察到显着差异。在三个不同的日子里进行了三个核心分析运行。在每个核心运行中，定量下限（LLOQ，n = 5）、低质量控制（LQC，n = 5）、中等质量控制（MQC，n = 5）和高质量控制样本（HQC，n= 5) 与标准曲线同时处理和运行。7 个数据点的 3 次核心运行的日内和日间准确度和精密度范围分别为 98.0-105% 和 0.9-3.0%，17 个数据点的范围分别为 97.5-105% 和 0.8-4.3%。对于不同的采样间隔没有观察到显着差异。结果表明，对于宽度达 9 秒的峰，7 个点的采样间隔足以准确而精确地定义一个峰，用于药物发现和开发中的药物定量研究。

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Quantitative LC–MS/MS. 1. Impact of Points across a Peak on the Accuracy and Precision of Peak Area Measurements

The number of points across a chromatographic peak has long been recognized as a key determinant of the accuracy and precision of the measured peak area. In LC–MS-based quantitation experiments in drug discovery and development, the “rule-of-thumb” has been to use 15 or more points. This “rule” is based on the literature describing chromatographic methods where the goal was to achieve the lowest possible imprecision in the measurements, especially when unknown analytes are being detected. Restricting methods to the requirement of at least 15 points across a peak can be detrimental to the development methods that fully optimize the signal-to-noise ratio for the assay using longer dwell times and/or transition summing. This study aims to show that 7 points across the peak for peaks that are 9 s or less wide provide more than sufficient accuracy and precision for drug quantitation studies. Data from simulated Gaussian curves using a sampling interval of 7 points across the peak gave peak area calculations within 1% of the expected total peak area using the Trapezoidal and Riemann rules and 0.6% for the Simpson rule. Low and high concentration samples (n = 5) were assayed using three different LC methods on three different days on two different instruments (API5000 and API5500). The difference in peak area (%ΔPA) and relative standard deviation of the peak areas (%RSD) was less than ∼5%. No significant difference was observed from the data that were obtained from different sampling intervals, different peak widths, different days, different peak sizes, and different instruments. Three core analytical runs were performed on three different days. In each core run, the lower limit of quantitation (LLOQ, n = 5), low quality control (LQC, n = 5), middle quality control (MQC, n = 5), and high-quality control samples (HQC, n = 5) were processed and run simultaneously with a standard curve. The range of the intra- and interday accuracy and precision for 3 core runs was 98.0–105% and 0.9–3.0% for 7 data points and 97.5–105% and 0.8–4.3% for 17 data points, respectively. No significant difference was observed for the different sampling intervals. The results show a sampling interval of 7 points for peaks up to 9 s wide is sufficient to define a peak accurately and precisely for drug quantitation studies in drug discovery and development.

更新日期：2023-05-04

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