Section for Forensic Chemistry, Department of Forensic Medicine, Aarhus University, Brendstrupgaardsvej 100, 8200 Aarhus N, Denmark.
Clin Biochem. 2013 Dec;46(18):1877-83. doi: 10.1016/j.clinbiochem.2013.08.011. Epub 2013 Aug 29.
For the quantification of β-hydroxybutyrate (BHB) and β-hydroxy-β-methylbutyrate (HMB) in human whole blood, a method using hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) was developed, which does not require chemical modification of the analytes.
Samples were deproteinised by a mixture of methanol and acetonitrile, and the extracts were cleaned-up using both polymeric strong cation exchange and strong anion exchange sorbents. The analytes and their structural isomers were separated using a column with a zwitterionic stationary phase. Isotope dilution of both analytes was used for quantitative analysis.
Separation of BHB from isobaric interferences was achieved through chromatography. The relative intra-laboratory reproducibility standard deviations were better than 10% for blood samples at concentration levels of 10-20μM BHB and 1μM HMB and better than 5% at concentration levels 10 times higher. The mean true extraction recoveries were close to 100%. The trueness expressed as the relative bias of test results was within ±5% at concentration levels of 10-1000μM BHB and 1-20μM HMB. The lower limits of quantification were estimated to be 3μM for BHB and 0.4μM for HMB.
A simple and highly sensitive and selective HILIC-MS/MS method was developed that is suitable for the quantification of BHB and HMB in whole blood.
为了定量检测人全血中的β-羟基丁酸（BHB）和β-羟基-β-甲基丁酸（HMB），开发了一种亲水相互作用液相色谱串联质谱（HILIC-MS/MS）方法，该方法不需要对分析物进行化学修饰。
样品用甲醇和乙腈的混合物进行蛋白沉淀，提取液用聚合物强阳离子交换和强阴离子交换吸附剂进行净化。采用带有两性离子固定相的色谱柱分离分析物及其结构异构体。采用同位素稀释法对两种分析物进行定量分析。
通过色谱分离实现了 BHB 与等摩尔干扰物的分离。在 BHB 浓度为 10-20μM 和 HMB 浓度为 1μM 的血液样本中，实验室内部重现性标准偏差优于 10%，在 BHB 浓度 10 倍以上的样本中，实验室内部重现性标准偏差优于 5%。真实提取回收率接近 100%。以测试结果的相对偏差表示的准确度在 BHB 浓度为 10-1000μM 和 HMB 浓度为 1-20μM 时，偏差在±5%范围内。BHB 的定量下限估计为 3μM，HMB 的定量下限估计为 0.4μM。
开发了一种简单、灵敏、选择性高的亲水相互作用液相色谱串联质谱法，适用于全血中 BHB 和 HMB 的定量检测。
