Lankes Ulrich, Elder Peter A, Lewis John G, George Peter
Steroid and Immunobiochemistry Unit, Canterbury Health Laboratories, Christchurch, New Zealand
Steroid and Immunobiochemistry Unit, Canterbury Health Laboratories, Christchurch, New Zealand.
Ann Clin Biochem. 2015 Jan;52(Pt 1):151-60. doi: 10.1177/0004563214533316. Epub 2014 Apr 8.
Extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis is the method of choice when it comes to the accurate quantification of 25-OH-vitamin D in blood samples. It is generally assumed that the addition of exogenous internal standard allows for the determination of the endogenous analyte concentration. In this study we investigated the extraction properties of endogenous and exogenous 25-OH-vitamin D.
Eight samples were used for the evaluation of the extraction procedure and 59 patients' samples for a method comparison. The methanol-to-sample ratio (v/v) and the sample-to-hexane ratio (v/v) were varied and the LC-MS/MS signals of endogenous 25-OH-vitamin D3, spiked 25-OH-vitamin D2 and internal standard of the extracts recorded. The optimized 'in-house' LC-MS/MS assay was compared to two automated chemiluminescence immunoassays from DiaSorin and Abbott.
Mathematical analysis of the data revealed a differential extraction of endogenous 25-OH-vitamin D3, spiked 25-OH-vitamin D2 and non-equilibrated internal standard. Exogenous 25-OH-vitamin D can be measured accurately if a definite methanol-to-sample ratio is used. Endogenous 25-OH-vitamin D is affected by critical quantification issues due to a differential slope in the extraction profile. The actual 25-OH-vitamin D concentration can be one-third above the measured extractable concentration. Results confirm that the 'in-house' LC-MS/MS assay provides reproducible 25-OH-vitamin D results.
Discordant concentrations of 25-OH-vitamin D from LC-MS/MS assays can be caused by selection of suboptimal extraction conditions. Furthermore, a different sample pretreatment or solvent extraction system may result in a different dissociation and extraction yield of endogenous 25-OH-vitamin D and therefore contribute to variations of LC-MS/MS results.
对于血液样本中25-羟基维生素D的准确定量，采用萃取后进行液相色谱-串联质谱（LC-MS/MS）分析是首选方法。通常认为添加外源性内标可用于测定内源性分析物浓度。在本研究中，我们对内源性和外源性25-羟基维生素D的萃取特性进行了研究。
使用8个样本评估萃取程序，并使用59例患者样本进行方法比较。改变甲醇与样本的比例（v/v）以及样本与己烷的比例（v/v），记录萃取物中内源性25-羟基维生素D3、添加的25-羟基维生素D2和内标的LC-MS/MS信号。将优化后的“内部”LC-MS/MS测定法与DiaSorin和Abbott的两种自动化化学发光免疫测定法进行比较。
数据的数学分析显示内源性25-羟基维生素D3、添加的25-羟基维生素D2和未平衡内标的萃取存在差异。如果使用确定的甲醇与样本比例，则可准确测量外源性25-羟基维生素D。由于萃取曲线斜率不同，内源性25-羟基维生素D受到关键定量问题的影响。实际的25-羟基维生素D浓度可能比测得的可萃取浓度高1/3。结果证实“内部”LC-MS/MS测定法可提供可重复的25-羟基维生素D结果。
LC-MS/MS测定中25-羟基维生素D浓度不一致可能是由于选择了次优的萃取条件所致。此外，不同的样本预处理或溶剂萃取系统可能导致内源性25-羟基维生素D的解离和萃取产率不同，从而导致LC-MS/MS结果出现差异。
