Background Urine organic acid (UOA) analysis is essential for the diagnosis of inborn errors of metabolism (IEMs). Traditionally, UOA analysis is performed with gas chromatography-mass spectrometry (GC-MS) and requires time-consuming sample preparation steps including liquid-liquid extraction and derivatization. The rapid development of Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS) in the past few years provides the opportunity to perform UOA analysis with a dilute-and-shoot methodology. We describe the development and validation of a quantitative Ultra-Performance Liquid Chromatography Quadrupole Time-of-Flight (UPLC-QToF) method for UOA analysis. Methods Urine specimens were diluted to normalize creatinine concentrations to 1 mmol/L. 20 µL of urine specimen (diluted), calibrator, or quality control (QC) material was mixed with 400 µL of mobile phase A (0.05% formic acid in water) and a mixture of isotope-labeled internal standards. After centrifugation, 10 µL of the supernatant was analyzed using a Xevo G3 QTOF mass spectrometer (Waters) with a ACQUITYTM Premier HSS T3 1.8 µm VanGuardTM FIT 2.1 x 150 mm column (Waters). Data collection was performed with negative electrospray ionization (ESI) mode using the MSE method to produce fragment ions when applicable. Repeatability, reproducibility, and carryover were assessed using the QC materials. The analytical measuring range (AMR) was assessed using synthetic urine spiked with increasing concentrations of each organic acid. Accuracy was assessed by method comparison with the UOA test performed at Mayo Clinic Laboratory and by spike-recovery study using a pooled urine specimen. Matrix effect was also evaluated with matrix dilution study. Results An optimized LC method was used to enable high-resolution separation of selected UOAs (N = 29) and isomers. Total analytical time was 20 min per injection. Both linear and quadratic regressions were used to build the calibration curves. AMR and correlation coefficients of a few representative UOAs were: orotic acid (3.4 to 214.2 mmol/mol creatinine, R^2 = 0.99, linear regression); 2-methylcitric acid (4 to 189 mmol/mol creatinine, R^2 = 0.99, linear regression); 3-methylcrotonylglycine (0.3 to 18.0 mmol/mol creatinine, R^2 = 0.99, linear regression). Repeatability and reproducibility were mostly <=10% CV and no carryover was observed. Spike-recovery study demonstrated recoveries between 80% and 120%, and method comparison study demonstrated no discrepancies with results from Mayo Clinic Laboratory. Conclusions We have developed and validated a novel UPLC-QTOF method for UOA analysis to support the diagnosis of IEMs with acceptable analytical and clinical performances. Compared with the traditional GC-MS method, the UPLC-QTOF method requires a very small specimen volume and does not require laborious and time-consuming sample preparation steps. Continued optimization of the method will be pursued to measure more UOAs to support the diagnosis of more IEMs.

中文翻译：

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B-168 用于尿液有机酸分析的定量超高效液相色谱四极杆飞行时间 (UPLC-QToF) 方法的开发和验证


背景 尿液有机酸 (UOA) 分析对于先天性代谢缺陷 (IEM) 的诊断至关重要。传统上，UOA 分析是通过气相色谱-质谱 (GC-MS) 进行的，需要耗时的样品制备步骤，包括液液萃取和衍生化。过去几年超高效液相色谱串联质谱 (UPLC-MS/MS) 的快速发展为采用稀释-喷射方法进行 UOA 分析提供了机会。我们描述了用于 UOA 分析的定量超高效液相色谱四极杆飞行时间 (UPLC-QToF) 方法的开发和验证。方法 稀释尿液样本，使肌酐浓度正常至 1 mmol/L。将 20 µL 尿液样本（稀释）、校准品或质量控制 (QC) 材料与 400 µL 流动相 A（0.05% 甲酸水溶液）以及同位素标记内标混合物混合。离心后，使用 Xevo G3 QTOF 质谱仪 (Waters) 和 ACQUITYTM Premier HSS T3 1.8 µm VanGuardTM FIT 2.1 x 150 mm 色谱柱 (Waters) 分析 10 µL 上清液。在适用时，使用 MSE 方法以负电喷雾电离 (ESI) 模式进行数据收集，以产生碎片离子。使用 QC 材料评估重复性、再现性和残留。使用添加了浓度逐渐增加的每种有机酸的合成尿液来评估分析测量范围（AMR）。通过与 Mayo Clinic 实验室进行的 UOA 测试进行方法比较以及使用合并尿液样本的尖峰回收研究来评估准确性。还通过基质稀释研究评估了基质效应。 结果 使用优化的 LC 方法能够对选定的 UOA (N = 29) 和异构体进行高分辨率分离。每次进样的总分析时间为 20 分钟。使用线性回归和二次回归来构建校准曲线。一些代表性 UOA 的 AMR 和相关系数为：乳清酸（3.4 至 214.2 mmol/mol 肌酐，R^2 = 0.99，线性回归）； 2-甲基柠檬酸（4至189 mmol/mol肌酐，R^2 = 0.99，线性回归）； 3-甲基巴豆酰甘氨酸（0.3 至 18.0 mmol/mol 肌酐，R^2 = 0.99，线性回归）。重复性和再现性大多为<=10% CV，并且没有观察到残留。加标回收率研究表明回收率在 80% 到 120% 之间，方法比较研究表明与 Mayo Clinic 实验室的结果没有差异。结论 我们开发并验证了一种用于 UOA 分析的新型 UPLC-QTOF 方法，以支持 IEM 的诊断，具有可接受的分析和临床性能。与传统的GC-MS方法相比，UPLC-QTOF方法需要的样品体积非常小，并且不需要费力且耗时的样品制备步骤。我们将继续优化该方法，以测量更多的 UOA，以支持更多 IEM 的诊断。