Misleading measures in Vitamin D analysis: A novel LC-MS/MS assay to account for epimers and isobars

Abstract

BackgroundRecently, the accuracies of many commercially available immunoassays for Vitamin D have been questioned. Liquid chromatography tandem mass spectrometry (LC- MS/MS) has been shown to facilitate accurate separation and quantification of the major circulating metabolite 25-hydroxyvitamin-D3 (25OHD3) and 25-hydroxyvitamin-D2 (25OHD2) collectively termed as 25OHD. However, among other interferents, this method may be compromised by overlapping peaks and identical masses of epimers and isobars, resulting in inaccuracies in circulating 25OHD measurements. The aim of this study was to develop a novel LC-MS/MS method that can accurately identify and quantitate 25OHD3 and 25OHD2 through chromatographic separation of 25OHD from its epimers and isobars.MethodsA positive ion electrospray ionisation (ESI) LC-MS/MS method was used in the Multiple Reaction Monitoring (MRM) mode for quantification. It involved i) liquid-liquid extraction, ii) tandem columns (a high resolution ZORBAX C18 coupled to an ULTRON chiral, with guard column and inlet filter), iii) Stanozolol-D3 as internal standard, and iv) identification via ESI and monitoring of three fragmentation transitions. To demonstrate the practical usefulness of our method, blood samples were collected from 5 healthy male Caucasian volunteers; age range 22 to 37 years and 25OHD2, 25OHD3 along with co-eluting epimers and analogues were quantified.ResultsThe new method allowed chromatographic separation and quantification of 25OHD2, 25OHD3, along with 25OHD3 epimer 3-epi-25OHD3 and isobars 1-α-hydroxyvitamin-D3 (1αOHD3), and 7-α-hydroxy-4-cholesten-3-one (7αC4). The new assay was capable of detecting 0.25 ng/mL of all analytes in serum.ConclusionsTo our knowledge, this is the first specific, reliable, reproducible and robust LC-MS/MS method developed for the accurate detection of 25OHD (Vitamin D). The method is capable of detecting low levels of 25OHD3 and 25OHD2 together with chromatographic separation from the co-eluting epimers and isobars and circumvents other instrumental/analytical interferences. This analytical method does not require time-consuming derivatisation and complex extraction techniques and could prove very useful in clinical studies.

Mass Spectrometric Profiling of Vitamin D Metabolites beyond 25-Hydroxyvitamin D

“…Similarly, exogenous compounds such 1␣-hydroxyvitamin D 3 can inflate measured 25(OH)D concentrations when given to patients during management of secondary hyperparathyroidism in chronic kidney disease (13 ). Finally, the C-3 epimer, 3-epi-25(OH)D, will also give overestimated 25(OH)D concentrations if not properly resolved by chromatography (vide infra) (7,15,16 (17 ).…”

“…These primarily relate to protein binding, ionization properties, and stabilities and interferences from isomers and isobars, as recently reviewed (7 ). Briefly, the endogenous properties of vitamin D, such as its lipophilic nature and tight binding to DBP and albumin, have the potential to cause substantial matrix effects during sample extraction, which is usually performed using protein precipitation (PP), liquid-liquid extraction (LLE), solid-phase extraction (SPE), or combination of these techniques (13,14 ). Stable isotope standards of vitamin D compounds are essential to correct for analytical issues caused by ion suppression effects (7 ).…”

Triple Quadrupole Versus High Resolution Quadrupole-Time-of-Flight Mass Spectrometry for Quantitative LC-MS/MS Analysis of 25-Hydroxyvitamin D in Human Serum

“…7α-Hydroxy-4-cholestene-3-one is another constitutional isomer of 25(OH)D 3 and would therefore likely present a serious tandem mass spectrometric challenge if it co-eluted with 25(OH)D 3 . It was shown to be easily resolved from 25(OH)D 3 by reversed-phase chromatography, however, and eluted much earlier than 25(OH)D 3 [22]. Finally, the 3-epimer species of 25(OH)D 3 , which has been shown to be present in serum samples of adult subjects at considerable levels [8,22,24], was also well resolved from its 25(OH)D 3 variant [19] and therefore did not constitute an interference in our LRMS and HRMS assays.…”

“…It was shown to be easily resolved from 25(OH)D 3 by reversed-phase chromatography, however, and eluted much earlier than 25(OH)D 3 [22]. Finally, the 3-epimer species of 25(OH)D 3 , which has been shown to be present in serum samples of adult subjects at considerable levels [8,22,24], was also well resolved from its 25(OH)D 3 variant [19] and therefore did not constitute an interference in our LRMS and HRMS assays. Figure S1 (Supplementary Material) illustrates the separation of 25(OH)D 3 and 3-epi-25(OH)D 3 for a CLD patient sample, showing~5% contribution of the epimer relative to the 25(OH)D 3 signal.…”

“…This compound was previously described as a potential isobar/ isomer in the analysis of 25(OH)D 3 [6]; it was detected in human serum before and shown to elute after 25(OH)D 3 [6]. Other isobaric interferences of 25(OH)D 3 described in the literature are 7α-hydroxy-4-cholestene-3-one (C 27 H 44 O 2 ) [9,22,23] and 1,2-didecanoyl-sn-glycerol (C 23 H 44 O 5 ) [6]. 1,2-Didecanoyl-sn-glycerol exhibited much stronger retention on the reversed-phase column than 25(OH)D 3 [6].…”