建立并验证一种 LC-MS/MS 方法，用于定量检测 19 种潜在的哮喘/COPD 尿生物标志物。
Development of a validated LC- MS/MS method for the quantification of 19 endogenous asthma/COPD potential urinary biomarkers.
机构信息
College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK, Canada.
Department of Pediatrics, College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada.
出版信息
Anal Chim Acta. 2017 Oct 9;989:45-58. doi: 10.1016/j.aca.2017.08.007. Epub 2017 Aug 16.
Obstructive airways inflammatory diseases sometimes show overlapping symptoms that hinder their early and correct diagnosis. Current clinical tests are tedious and are of inadequate specificity in special population such as the elderly and children. Therefore, we are developing tandem mass spectrometric (MS/MS) methods for targeted analysis of urine biomarkers. Recently, proton-nuclear magnetic resonance (H-NMR) analysis proposed 50 urinary metabolites as potential diagnostic biomarkers among asthma and chronic obstructive pulmonary disease (COPD) patients. Metabolites are divided into 3 groups based on chemical nature. For group 1 (amines and phenols, 19 urinary metabolites), we developed and validated a high performance liquid chromatographic (HPLC)-MS/MS method using differential isotope labeling (DIL) with dansyl chloride. Method development included the optimization of the derivatization reaction, the MS/MS conditions, and the chromatographic separation. Linearity varied from 2 to 4800 ng/mL and the use of C-labeled derivatives allowed for the correction of matrix effects as well as the unambiguous confirmation of the identity of each metabolite in the presence of interfering isomers in urine. Despite the challenges associated with method validation, the method was fully validated as per the food and drug administration (FDA) and the European medicines agency (EMA) recommendations. Validation criteria included linearity, precision, accuracy, dilution integrity, selectivity, carryover, and stability. Challenges in selectivity experiments included the isotopic contributions of the analyte towards its internal standard (IS), that was addressed via the optimization of the IS concentration. In addition, incurred sample analysis was performed to ensure that results from patient samples are accurate and reliable. The method was robust and reproducible and is currently being applied in a cohort of asthma and COPD patient urine samples for biomarker discovery purposes.
阻塞性气道炎症性疾病有时表现出重叠的症状，这阻碍了它们的早期和正确诊断。目前的临床检测既繁琐，在特殊人群（如老年人和儿童）中的特异性又不足。因此，我们正在开发串联质谱（MS/MS）方法，用于靶向分析尿液生物标志物。最近，质子核磁共振（H-NMR）分析提出了 50 种尿液代谢物，作为哮喘和慢性阻塞性肺疾病（COPD）患者的潜在诊断生物标志物。代谢物根据化学性质分为 3 组。对于第 1 组（胺和酚类，19 种尿液代谢物），我们使用丹磺酰氯的差分同位素标记（DIL）开发并验证了一种高效液相色谱-MS/MS 方法。方法开发包括衍生化反应、MS/MS 条件和色谱分离的优化。线性范围为 2 至 4800ng/mL，使用 C 标记的衍生物可以校正基质效应，并在尿液中存在干扰异构体的情况下，明确确认每种代谢物的身份。尽管方法验证存在挑战，但该方法完全按照食品和药物管理局（FDA）和欧洲药品管理局（EMA）的建议进行了验证。验证标准包括线性、精密度、准确度、稀释完整性、选择性、交叉污染和稳定性。选择性实验中的挑战包括分析物对其内标（IS）的同位素贡献，这是通过优化 IS 浓度来解决的。此外，还进行了实际样品分析，以确保患者样品的结果准确可靠。该方法稳健且重现性良好，目前正在哮喘和 COPD 患者尿液样本的队列中用于生物标志物发现。
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