Enhanced 3-epi-25-hydroxyvitamin D₃ signal leads to overestimation of its concentration and amplifies interference in 25-hydroxyvitamin D LC-MS/MS assays

Abstract

Background: 3-epi-25-hydroxyvitamin D 3 (3-epi-25OHD 3 ) interferes in most liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for 25-hydroxyvitamin D (25OHD). The clinical significance of this is unclear, with concentrations from undetectable to 230 nmol/L reported. Many studies have quantified 3-epi-25OHD 3 based on 25OHD 3 calibrators or other indirect methods, and we speculated that this contributes to the observed variability in reported 3-epi-25OHD 3 concentrations. Methods: We compared continuous MS/MS infusions of 3-epi-25OHD 3 and 25OHD 3 solutions, spiked both analytes into the same serum matrix and analysed patient samples to assess the effect of three different quantitation methods on 3-epi-25OHD 3 concentration. Experiments were performed on an LC-MS/MS system using a phenyl column which does not resolve 3-epi-25OHD 3 , and a modified method utilizing a Zorbax SB-CN column that chromatographically resolves 3-epi-25OHD 3 from 25OHD 3 . Results: A greater 3-epi-25OHD 3 signal, compared with 25OHD 3 , was observed during equimolar post-column continuous infusion of analyte solutions, and following analysis of a serum pool spiked with both analytes. 3-epi-25OHD 3 signal enhancement was dependent on mobile phase composition. Compared with 3-epi-25OHD 3 calibrators, indirect quantitation methods resulted in up to 10 times as many samples having 3-epi-25OHD 3 concentrations ! 10 nmol/L, and an approximately fourfold increase in the maximum observed 3-epi-25OHD 3 concentration to 95 nmol/L. Conclusions: Enhanced 3-epi-25OHD 3 signal leads to overestimation of its concentrations in the indirect quantitation methods used in many previous studies. The enhanced signal may contribute to greater interference in some 25OHD LC-MS/MS assays than others. We highlight that equimolar responses cannot be assumed in LC-MS/MS systems, even if two molecules are structurally similar.

Measurement of serum 3-epi-25-hydroxyvitamin D3, 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in infant, paediatric and adolescent populations of Korea using ultra-performance liquid chromatography-tandem mass spectrometry

“…20 Because both 3-epi-25(OH)D 3 and the native form of 25(OH)D 3 have the same molecular weight and fragmentation pattern, the presence of 3-epi-25(OH)D 3 would lead to an overestimation of 25(OH)D 3 in various methods currently used for vitamin D measurements that do not separate this isomeric form. 21 Furthermore, existing LC-MS/MS methods that were developed for C-3 epimer separation require long chromatography separation times, extensive sample preparation or high-resolution mass spectrometer. 15,[21][22][23] In this study, we separated the 3-epi-25(OH)D 3 form successfully using an UPLC-MS/MS method with a PerkinElmer Kit and a Kinetex XB-C18 column for 11 min of run time.…”

“…21 Furthermore, existing LC-MS/MS methods that were developed for C-3 epimer separation require long chromatography separation times, extensive sample preparation or high-resolution mass spectrometer. 15,[21][22][23] In this study, we separated the 3-epi-25(OH)D 3 form successfully using an UPLC-MS/MS method with a PerkinElmer Kit and a Kinetex XB-C18 column for 11 min of run time. This method offers an improvement in sample preparation step and in the throughput compared with previous methods with longer run times.…”

“…10,25 On the other hand, the LC-MS/MS method, which cannot separate 3-epi-25(OH)D 3 , would be expected to overestimate the concentration of 25(OH)D in the infant group, owing to the considerable amounts of 3-epi-25(OH)D 3 in their circulation.There is a limitation in our study. Flynn et al21 demonstrated enhanced 3-epi-25(OH)D 3 signal relative to equimolar 25(OH)D 3 during infusions and in spiked human serum. They insisted enhanced signal caused overestimation of 3-epi-25(OH)D 3 concentrations when quantified using 25(OH)D 3 calibrators as an indirect quantitation method, and the 3-epi-25(OH)D 3 signal enhancement was dependent on mobile phase composition.…”

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

“…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. Note that the peak areas of the two epimers are not directly comparable because of significant response factor differences between the two epimer species, as recently described by van den Ouweland et al [25] and Flynn et al [26], requiring a dedicated stable isotope standard for 3-epi-25(OH)D 3 [12].…”

Mass spectrometry for steroids

“…This issue of the Annals includes several articles reporting analysis of steroids using mass spectrometry (MS). [1][2][3] Many papers on the use of MS for steroid measurement and assay interference have been published recently in the Annals, reflecting a growing appreciation of the power of this physicochemical technology. [4][5][6][7][8][9] Formerly, MS was primarily used for steroids in clinical research and reference centres.…”