Vitamin D status is typically assessed by the measurement of 25-hydroxy vitamin D (25(OH)D). However, in selected patient groups the sole determination of 25(OH)D has been proven insufficient for this purpose. The simultaneous measurement of additional vitamin D metabolites may provide useful information for a better evaluation of the vitamin D status. Therefore, we developed and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of 25(OH)D₃, 25(OH)D₂, 24,25(OH)₂D₃ and additionally 25,26(OH)₂D₃, which was identified with a synthesized pure substance.

Pure and deuterated substances were used to prepare calibrators and internal standards for all target metabolites. Pre-analytical sample preparation comprised protein precipitation followed by liquid-liquid-extraction and derivatization with 4-Phenyl-1,2,4-triazole-3,5-dione (PTAD) using 50 µL sample volume. Samples were analysed on an Agilent HPLC 1260 system equipped with a silica-based Kinetex® 5 µm F5 100Å core-shell column (150 x 4.6 mm) coupled to a Sciex 4500 mass spectrometer.

For all four metabolites, limit of detection (LoD) and limit of quantification (LoQ) ranged from 0.3 to 1.5 nmol/L and 1.0 to 3.1 nmol/L, respectively. Recovery varied between 76.1 % and 84.3 %. Intra- and inter-assay imprecision were <8.6 % and <11.5 %, respectively. The analysis of external and internal quality control samples showed good accuracy for 25(OH)D₃, 25(OH)D₂, 24(R),25(OH)₂D₃ and 25,26(OH)₂D₃. Method comparison studies with human samples that were also analyzed with two other LC-MS/MS methods showed close agreement. Finally, the present method has been shown capable of identifying patients with 24-hydroxylase deficiency, which proves its clinical utility.

维生素D的状态通常通过25-羟基维生素D（25（OH）D）的测量来评估。但是，已证明仅在选定的患者组中测定25（OH）D不足以实现此目的。同时测量其他维生素D代谢物可为更好地评估维生素D状态提供有用的信息。因此，我们开发并验证液相色谱串联质谱法（LC-MS / MS）的25（OH）d同时测定方法₃，25（OH）d ₂，24,25（OH）₂ d ₃，并且另外25,26（OH）₂ D ₃，由合成的纯净物质鉴定。

使用纯净的氘代物质制备所有目标代谢物的校准物和内标。分析前的样品制备包括蛋白质沉淀，然后进行液-液萃取，并使用50 µL样品体积的4-苯基-1,2,4-三唑-3,5-二酮（PTAD）进行衍生化。在配备有基于二氧化硅的Kinetex®5 µm F5100Å核壳色谱柱（150 x 4.6 mm）的Agilent HPLC 1260系统上分析样品，该色谱柱与Sciex 4500质谱仪连接。

对于所有四种代谢物，检出限（LoD）和定量限（LoQ）分别为0.3至1.5 nmol / L和1.0至3.1 nmol / L。回收率在76.1％和84.3％之间。批内和批间不准确性分别为<8.6％和<11.5％。外部和内部质量控制样品的分析显示出良好的精度25（OH）d ₃，25（OH）d ₂，24（R），25（OH）₂ d ₃和25,26（OH）₂ d ₃。用人类样品进行的方法比较研究也用其他两种LC-MS / MS方法进行了分析，结果显示出高度的一致性。最后，已经表明本方法能够鉴定患有24-羟化酶缺乏症的患者，证明了其临床实用性。