Jin Zhicheng, Bertholf Roger L, Yi Xin
Department of Pathology and Genomic Medicine, Houston Methodist Hospital, Houston, TX, USA.
Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY, USA.
J Appl Lab Med. 2022 May 4;7(3):737-746. doi: 10.1093/jalm/jfab112.
The circulating concentration of 1α,25-dihydroxyvitamin D [1α,25(OH)2D] is very low, and the presence of multiple isomers may lead to inaccurate quantitation if not separated prior to analysis. Antibody-based immunoextraction procedures are sometimes used to remove structurally related isomers of 1α,25(OH)2D prior to an LC-MS/MS analysis. However, immunoextraction increases sample preparation time and cost. In addition, some dihydroxyvitamin D metabolites are not completely removed by immunoextraction.
We developed an HPLC method using a phenyl-hexyl column to investigate interfering isomers of 1α,25(OH)2D.
Using this method, 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) derivatization product of 1α,25(OH)2D was found to be present as 2 epimers, which were separated chromatographically with an area ratio of 2:1. PTAD derivatized metabolite of 25-hydroxyvitamin D3 [i.e., 4β,25-dihydroxyvitamin D3 (4β,25(OH)2D3)] eluted out between 6R and 6S epimers of derivatized 1α,25(OH)2D3. If not chromatographically resolved, 4β,25(OH)2D can affect 1α,25(OH)2D quantitation. In a method comparison study, it was found that the presence of 4β,25(OH)2D produced positive bias up to 127% on 1α,25(OH)2D3 quantitation.
The LC-MS/MS method we developed without an immunoextraction procedure was able to resolve the major interference peak from 1α,25(OH)2D and achieved reliable quantitation of 1α,25(OH)2D.
1α,25-二羟基维生素D[1α,25(OH)2D]的循环浓度非常低，如果在分析前不进行分离，多种异构体的存在可能导致定量不准确。基于抗体的免疫提取程序有时用于在LC-MS/MS分析前去除1α,25(OH)2D的结构相关异构体。然而，免疫提取增加了样品制备时间和成本。此外，一些二羟基维生素D代谢物不能通过免疫提取完全去除。
我们开发了一种使用苯基己基柱的HPLC方法来研究1α,25(OH)2D的干扰异构体。
使用该方法，发现1α,25(OH)2D的4-苯基-1,2,4-三唑啉-3,5-二酮(PTAD)衍生化产物以2种差向异构体形式存在，通过色谱分离，面积比为2:1。25-羟基维生素D3的PTAD衍生化代谢物[即4β,25-二羟基维生素D3(4β,25(OH)2D3)]在衍生化的1α,25(OH)2D3的6R和6S差向异构体之间洗脱出来。如果不进行色谱分离，4β,25(OH)2D会影响1α,25(OH)2D的定量。在一项方法比较研究中，发现4β,25(OH)2D的存在会使1α,25(OH)2D3的定量产生高达127%的正偏差。
我们开发的无需免疫提取程序的LC-MS/MS方法能够分离1α,25(OH)2D的主要干扰峰，并实现1α,25(OH)2D的可靠定量。
