尿儿茶酚胺 LC－MS/MS 检测方法的建立

摘要

Objective To establish a liquid chromatography-tandem mass spectrometry ( LC-MS/MS) for the determination of urinary catecholamines, including epinephrine ( E), norepinephrine ( NE) and dopamine ( DA). Methods E, NE and DA were separated by WATERS ACQUITY UPLC® HSS T3 (2.1 mm ×100 mm,1.8 μm column) using E-d3, NE-d6 and DA-d4 as internal standards.The mobile phase consisted of 2‰ formic acid aqueous solution ( A1) and methyl alcohol ( B1, purity =99.9%).The flow rate of 0.50 mL/min was applied to the chromatographic column.Flow starting was performed with A1 ∶B1 =95 ∶5.Electrospray ionization ( ESI) was operated in positive ion mode.The LC-MS/MS was used, with linearity, accuracy (recovery rate), imprecision and lower limit of determination being evaluated.A total of 65 healthy subjects were enrolled, and the reference ranges for E, NE and DA were established.Results The retention times of E, NE and DA were 0.58, 0.89, 0.63 min, respectively.The determination range was from 0.50 ng/mL to 800.00 ng/mL.The mean recovery ranges were 97.95%, deter mination range 97.78% and 101.03%.The lower limits of determination for E, NE and DA were 0.25, 2.50 and 2.50 ng/mL.The reference ranges were 5.4-25.8 μg/24 h for E, 28.5-73.1 μg/24 h for NE and 269.1-420.5 μg/24 h for DA.Conclusions The LC-MS/MS for the simultaneous determination of E, NE and DA has been established and is suitable for clinical application.It can provide reliable information for the diagnosis of phaeochromocytoma.%目的：建立尿液儿茶酚胺[包括肾上腺素（E）、去甲肾上腺素（NE）和多巴胺（DA）]的液相色谱－串联质谱（LC－MS／MS）检测方法。方法以 E－d3、NE－d6和 DA－d4盐酸盐为内标，采用 WATERS ACQUITY UPLC® HSS T3色谱柱（2．1 mm ×100 mm，1．8μm）进行分离。流动相 A1为2‰甲酸水溶液，流动相 B1为甲醇（纯度为99．9％），流速为0．5 mL／min，按 A1∶B1＝95∶5等度洗脱。采用电喷雾（ESI）正离子模式进行质谱分析。对方法的线性、准确度（回收率）、不精密度及最低检出限等进行性能验证。选取表面健康人群65名，建立 E、NE 和 DA 的参考区间。结果采用建立的 LC－MS／MS 检测尿液 E、NE 和 DA，保留时间分别为0．58、0．89和0．63 min，检测范围为0．50～800．00 ng／mL。 E、NE 和 DA 的加标平均回收率分别为97．95％、97．78％和101．03％，最低检出限分别为0．25、2．50和2．50 ng／mL。 E、NE 和 DA 的参考区间分别为5．4～25．8、28．5～73．1和269．1～420．5μg／24 h。结论建立的LC－MS／MS 方法能同时检测尿液中的 E、NE 和 DA，可为嗜铬细胞瘤的诊断提供可靠的信息。