Bevital A/S, Laboratory Building 9th Floor, N-5021 Bergen, Norway.
Rapid Commun Mass Spectrom. 2011 Jul 30;25(14):1942-8. doi: 10.1002/rcm.5073.
We have developed an automated high-throughput assay for the determination of vitamin A (retinol), ergocalciferol (25-OH D2), cholecalciferol (25-OH D3) and vitamin E (α-tocopherol) in a small volume of human plasma. Sample preparation involved mixing 50 μL of plasma with 100 μL of ethanol containing isotope-labelled internal standards, followed by mixing with isooctane/chloroform (3:1, 300 μL). The organic phase was evaporated, and the sample reconstituted in 50 μL methanol. The analysis was performed using reversed-phase liquid chromatography with a gradient mobile phase containing water, methanol and ammonium formate. Chromatographic run-time was 5 min, and positive mode electrospray tandem mass spectrometry (MS/MS) was used for detection. The limits of detection were 0.10 μM for all-trans retinol and 3.3 nM for 25-OH D2 and 25-OH D3. Recoveries were 91.9-105.0%, and within- and between-day coefficients of variance (CVs) 2.4-5.3 and 3.1-8.2, respectively. The assay is presently being used in large-scale studies.
我们开发了一种自动化高通量测定方法，用于在小体积人血浆中测定维生素 A（视黄醇）、麦角钙化醇（25-OH D2）、胆钙化醇（25-OH D3）和维生素 E（α-生育酚）。样品制备包括将 50 μL 血浆与 100 μL 含有同位素标记内标物的乙醇混合，然后与异辛烷/氯仿（3:1，300 μL）混合。将有机相蒸发，并用 50 μL 甲醇重新溶解样品。分析采用反相液相色谱法，梯度流动相包含水、甲醇和甲酸铵。色谱运行时间为 5 分钟，采用正离子电喷雾串联质谱（MS/MS）进行检测。所有反式视黄醇的检测限为 0.10 μM，25-OH D2 和 25-OH D3 的检测限为 3.3 nM。回收率为 91.9-105.0%，日内和日间变异系数（CV）分别为 2.4-5.3%和 3.1-8.2%。该测定方法目前正在大规模研究中使用。
