Coldwell R D, Trafford D J, Makin H L, Varley M J
J Chromatogr. 1985 Mar 22;338(2):289-302. doi: 10.1016/0378-4347(85)80100-6.
A specific mass fragmentographic assay for the measurement of 25,26-dihydroxyvitamin D3 [25,26(OH)2D3] in human plasma, using a stable isotope labelled internal standard ([26,27-2H5]25,26(OH)2D3), is described. Plasma samples (5 ml) were extracted with acetonitrile and applied to a C18 Sep-Pak cartridge, from which the vitamin D metabolites were eluted with methanol. The metabolites were then applied to a Sep-Pak SIL cartridge and three fractions were collected. The most polar fraction, containing the polyhydroxylated metabolites, was further purified by high-performance liquid chromatography on Zorbax SIL. The eluent containing 25,26(OH)2D3 was collected, and the 25,26-n-butylboronate cyclic ester 3-trimethylsilyl ether derivative was formed. Gas chromatography-mass spectrometry was carried out, monitoring the intensities of the ions at m/z 449 and m/z 454 (for the internal standard). These ions represent the loss of a methyl group and the 3-silanol group, (M-90-15)+. The minimum limit of detection of the assay was estimated to be approximately 0.05 microgram/l. Inter-assay (3.7%) and intra-assay (8.0%) precision was acceptable and added 25,26(OH)2D3, over the concentration range 0.5-1.5 microgram/l, was recovered quantitatively. The plasma 25,26(OH)2D3 level was estimated in 26 healthy volunteers and ranged from 0.05 to 1.30 microgram/l, with a mean value of 0.54 microgram/l.
本文描述了一种使用稳定同位素标记内标（[26,27-2H5]25,26(OH)2D3）测量人血浆中25,26-二羟基维生素D3 [25,26(OH)2D3] 的特定质量碎片分析法。血浆样本（5 ml）用乙腈萃取并应用于C18 Sep-Pak柱，维生素D代谢产物用甲醇从该柱上洗脱。然后将代谢产物应用于Sep-Pak SIL柱并收集三个馏分。含有多羟基化代谢产物的最极性馏分通过在Zorbax SIL上的高效液相色谱进一步纯化。收集含有25,26(OH)2D3的洗脱液，并形成25,26-正丁基硼酸环酯3-三甲基硅醚衍生物。进行气相色谱-质谱分析，监测m/z 449和m/z 454（内标）处离子的强度。这些离子代表甲基和3-硅醇基团的损失，(M-90-15)+。该分析方法的最低检测限估计约为0.05微克/升。批间精密度（3.7%）和批内精密度（8.0%）可以接受，在0.5 - 1.5微克/升的浓度范围内添加的25,26(OH)2D3定量回收。对26名健康志愿者的血浆25,26(OH)2D3水平进行了估计，范围为0.05至1.30微克/升，平均值为0.54微克/升。
