Coldwell R D, Trafford D J, Varley M J, Makin H L, Kirk D N
Department of Chemical Pathology, London Hospital Medical College, UK.
Biomed Environ Mass Spectrom. 1988 Oct;16(1-12):81-5. doi: 10.1002/bms.1200160116.
Selected ion monitoring of vitamin D metabolites has previously been described but there has been only one detailed description of the measurement by gas chromatography/mass spectrometry (GC/MS) of a number of metabolites in a single plasma sample. We describe here a GC/MS method, using stable isotope labelled internal standards, which allows the estimation of vitamins D2 and D3, and their 25-hydroxy, 24,25-dihydroxy and 25,26-dihydroxy metabolites in a single 2 ml sample of plasma, although more is needed for the measurement of 1,25-dihydroxyvitamin D3. Plasma was extracted on Bond Elut C18 cartridges and initial fractionation carried out on Sep-Pak SIL. Straight-phase high-performance liquid chromatography was required for separation of polyhydroxylated metabolites prior to GC/MS using an LKB 2091 mass spectrometer with conventional packed columns. n-Butylboronate esters were formed across vicinal hydroxyls, followed by formation of trimethylsilyl ethers using trimethylsilylimidazole. The [M - 90 - 15]+ ion for each compound was monitored. Deuterated internal standards were not available for all metabolites and it was necessary to use (2H6)D3 and (2H6)25OHD3 as standards for the measurement of D2 and D3, and 25OHD3 and 25OHD2, respectively, and (2H6)24,25(OH)2D3 as a standard for 24,25(OH)2D3 and 25,26(OH)2D2. Although the [M - 90 - 15]+ ion of 24,25(OH)2D and 25,26(OH)2D has the same mass: charge ratio, derivatives of these compounds are completely separated in the GC system used. The intra-assay precision for all these assays is usually less than 5%.
先前已有关于维生素D代谢物的选择离子监测的描述，但仅有一篇详细描述通过气相色谱/质谱法（GC/MS）对单个血浆样本中的多种代谢物进行测定的文章。我们在此描述一种GC/MS方法，该方法使用稳定同位素标记的内标物，可在2ml单个血浆样本中估算维生素D2和D3及其25-羟基、24,25-二羟基和25,26-二羟基代谢物，不过测定1,25-二羟基维生素D3需要更多样本。血浆在Bond Elut C18柱上进行萃取，并在Sep-Pak SIL上进行初步分离。在使用配备常规填充柱的LKB 2091质谱仪进行GC/MS分析之前，需要采用正相高效液相色谱法分离多羟基化代谢物。邻位羟基之间形成正丁基硼酸酯，随后使用三甲基硅咪唑形成三甲基硅醚。监测每种化合物的[M - 90 - 15]+离子。并非所有代谢物都有氘代内标物，因此有必要分别使用（2H6）D3和（2H6）25OHD3作为测定D2和D3以及25OHD3和25OHD2的标准，使用（2H6）24,25(OH)2D3作为24,25(OH)2D3和25,26(OH)2D2的标准。尽管24,25(OH)2D和25,26(OH)2D的[M - 90 - 15]+离子具有相同的质荷比，但这些化合物的衍生物在所用的GC系统中能完全分离。所有这些测定的批内精密度通常小于5%。
