Quantification of 1,25-Dihydroxyvitamin D2 and D3 in Serum Using Liquid Chromatography-Tandem Mass Spectrometry

Abstract

1,25-Dihydroxyvitamin D is the active form of vitamin D and plays a critical role in the maintenance of calcium and phosphorous metabolism of the human body. Measurement of 1,25-dihydroxyvitamin D in serum can aid in clinical diagnosis and/or management of renal disease, sarcoidosis, and rare inherited diseases. We present here an effective and accurate method for measuring 1,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D2 by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) after immunoaffinity extraction. The MS/MS is operated in multiple reaction mode with positive electrospray. Quantification is based on peak area ratios of the analytes to respective deuterated internal standards. This method offered a linear range from 4.0 to 160.0 pg/mL with analytical recovery of 89.9-115.5 % for both 1,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D2.

Application of Cookson‐type reagents for biomedical HPLC and LC/MS analyses: a brief overview

“…The derivatization using Cookson‐type reagents can enhance the assay sensitivity, leading to a reduction of the sample volume. For the 1,25(OH) 2 D 3 assay, both the skillful pretreatment techniques, such as the immunoaffinity extraction, and the derivatization are usually necessary (Mahlow, Bunch, & Wang, ), but an LC/MS/MS method not requiring any derivatization was recently developed for the simultaneous quantification of 1,25(OH) 2 D 3 and 1,25(OH) 2 D 2 in serum (Fang et al, ). The developed method was based on coordination ion spray–MS using Li + as an additive, which showed the lower limit of quantification (LLOQ) of 10 pg/mL using a 0.5 mL sample aliquot.…”

Simultaneous determination of vitamin D metabolites 25(OH)D3 and 1α,25(OH)2D3 in human plasma using liquid chromatography tandem mass spectrometry

“…Partly because 1α,25(OH) 2 D 3 circulates at low picomolar concentration ranges with highly lipophilic and plasma protein binding properties [23] , [24] , measurement in the human body is challenging. Many assays for vitamin D 3 metabolites have been published, including enzyme-linked immunoassay, radioimmunoassay [25] , [26] , high-performance liquid chromatography [27] , [28] , and liquid chromatography coupled with mass spectrometry (LC-MS/MS), the latter of which is considered the “gold standard” for the determination of vitamin D 3 metabolite levels [29] , [30] , [31] , [32] , [33] . Currently, the majority of methods reported in the literature usually use derivatization to improve the ionization efficiency of 25(OH)D 3 and 1α,25(OH) 2 D 3 , which is financially costly and time consuming [31] , [33] .…”