一种改良的超高效液相色谱-串联质谱法，用于测定干血样中的异亮氨酸和支链氨基酸。
An improved ultra performance liquid chromatography-tandem mass spectrometry method for the determination of alloisoleucine and branched chain amino acids in dried blood samples.
机构信息
Discipline of Paediatrics and Child Health, University of Sydney, Sydney, Australia.
出版信息
Ann Clin Biochem. 2011 Sep;48(Pt 5):468-70. doi: 10.1258/acb.2011.010283. Epub 2011 Jul 20.
BACKGROUND
Branched chain amino acid (BCAA) analysis is needed for the diagnosis and management of patients with maple syrup urine disease (MSUD). We report an improved ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of BCAAs and Allo-Ile in dried blood spot (DBS) samples.
METHODS
BCAAs were extracted from a 3 mm blood spot into methanol/water containing stable isotope internal standards. Eluents were dried and reconstituted in the mobile phase. Gradient elution was performed on an Acquity™ BEH C(18) (100 × 2.1 mm, 1.7 μm) column. BCAAs were detected and quantified in the multiple reaction monitoring mode in a five-minute analysis.
RESULTS
The assay was calibrated to give best possible alignment with plasma results. Retrospective analysis of newborn DBSs from six classic MSUD patients showed elevated alloisoleucine (Allo-Ile) in all cases. Two of four patients with mild disease had normal values; the other two had significant elevations in Allo-Ile.
CONCLUSIONS
Analysis of BCAA in DBS by UPLC-MS/MS is a useful second tier newborn screening test to identify classical MSUD and for monitoring of remote patients.
背景
支链氨基酸（BCAA）分析对于枫糖尿症（MSUD）患者的诊断和治疗是必需的。我们报告了一种改进的超高效液相色谱-串联质谱（UPLC-MS/MS）法，用于测定干血斑（DBS）样本中的 BCAAs 和别异亮氨酸。
方法
将 3mm 血斑中的 BCAAs 提取到甲醇/水含稳定同位素内标中。洗脱液干燥并在流动相中重新配制。在 Acquity™ BEH C（18）（100×2.1mm，1.7μm）柱上进行梯度洗脱。在五分钟的分析中，以多反应监测模式检测和定量 BCAAs。
结果
该测定法经校准后与血浆结果最佳匹配。对 6 例经典 MSUD 患者的新生儿 DBS 进行回顾性分析显示，所有病例的别异亮氨酸（Allo-Ile）均升高。4 例轻度疾病患者中有 2 例值正常；另外 2 例 Allo-Ile 显著升高。
结论
通过 UPLC-MS/MS 分析 DBS 中的 BCAAs 是一种有用的二线新生儿筛查试验，可用于识别经典 MSUD 并监测远程患者。
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