Department of Clinical Laboratory, Peking Union Medical College Hospital, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100730, China.
Beijing Hospital National Center for Clinical Laboratories, Ministry of health, Beijing 100730, China.
J Steroid Biochem Mol Biol. 2019 Mar;187:146-151. doi: 10.1016/j.jsbmb.2018.11.012. Epub 2018 Nov 23.
This study establishes and validates a rapid method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization steps to simultaneously measure of 24,25(OH)D, 24,25(OH)D, 25OHD, and 25OHD, while efficiently separating the 3-epi analogs. Samples were prepared by precipitation and liquid-liquid extraction. The linearity, precision, accuracy, recovery and matrix effect of the method were thoroughly evaluated according to the Clinical & Laboratory Standards Institute guidelines. Additionally, the four vitamin D metabolites in the serum of 38 apparently healthy Chinese volunteers were evaluated. The total analysis time was 8.0 min, with efficient separation of 3-epi 24,25(OH)D and 3-epi 25OHD, without interference from isomers such as 23,25(OH)D or 1,25(OH)D, 1,25(OH)D. Good reproducibility was obtained for all four metabolites with within-run coefficient variations (CVs) of 4.07%-6.55%, 4.26%-7.84%, 2.46%-7.21%, and 4.90%-6.87% for 25OHD, 25OHD, 24,25(OH)D, and 24,25(OH)D, respectively, and the total CVs were 4.29%-6.64%, 6.14%-7.84%, 4.33%-7.21%, 5.82%-9.90%, respectively. The limit of quantification was 0.625 ng/mL for 25OHD and 25OHD, and 0.5 ng/mL for 24,25(OH)D and 24,25(OH)D. The relative bias of the LC-MS/MS method compared to the certified results of SRM 972a for 25OHD, 25OHD and 24,25(OH)D was -2.21% to 1.01%, 3.38% to 6.73%, and -7.72% to -3.9%, respectively. The mean±SD values for 25OHD, 24,25(OH)D and 25OHD/24,25(OH)D in the volunteers were 13.5±4.4 ng/mL(range:7.6-27.5 ng/mL), 0.84±0.42 ng/mL (range:0.26-2.1 ng/mL), and 18±7(range:8-37), respectively. Thus, a simple, precise LC-MS/MS method for appropriate retention and separation of vitamin D metabolites and their epi analogs was developed.
本研究建立并验证了一种无需衍生化步骤的基于液相色谱-串联质谱（LC-MS/MS）的快速方法，可同时测量 24,25（OH）D、24,25（OH）D、25OHD 和 25OHD，并有效分离 3-差向异构体。样品通过沉淀和液液萃取进行制备。根据临床和实验室标准协会（CLSI）指南，对方法的线性、精密度、准确度、回收率和基质效应进行了全面评估。此外，还评估了 38 名中国健康志愿者血清中的四种维生素 D 代谢物。总分析时间为 8.0 分钟，3-差向 24,25（OH）D 和 3-差向 25OHD 得到有效分离，没有异构体如 23,25（OH）D 或 1,25（OH）D、1,25（OH）D 的干扰。所有四种代谢物均获得良好的重现性，25OHD、25OHD、24,25（OH）D 和 24,25（OH）D 的批内变异系数（CV）分别为 4.07%-6.55%、4.26%-7.84%、2.46%-7.21%和 4.90%-6.87%，总 CV 分别为 4.29%-6.64%、6.14%-7.84%、4.33%-7.21%和 5.82%-9.90%。25OHD 和 25OHD 的定量限为 0.625ng/mL，24,25（OH）D 和 24,25（OH）D 的定量限为 0.5ng/mL。与 SRM 972a 的认证结果相比，LC-MS/MS 方法对 25OHD、25OHD 和 24,25（OH）D 的相对偏差分别为-2.21%至 1.01%、3.38%至 6.73%和-7.72%至-3.9%。志愿者中 25OHD、24,25（OH）D 和 25OHD/24,25（OH）D 的平均±SD 值分别为 13.5±4.4ng/mL（范围：7.6-27.5ng/mL）、0.84±0.42ng/mL（范围：0.26-2.1ng/mL）和 18±7（范围：8-37）。因此，开发了一种简单、精确的 LC-MS/MS 方法，可适当保留和分离维生素 D 代谢物及其差向异构体。
