Rapid Analysis of 25-Hydroxyvitamin D₂and D₃by Liquid Chromatography–Tandem Mass Spectrometry and Association of Vitamin D and Parathyroid Hormone Concentrations in Healthy Adults

Abstract

Measurement of 25-hydroxyvitamin D (25OH-vitD) is used to assess vitamin D status. We developed a high-sensitivity measurement method for 25OH-vitD and assessed the relationship between 25OH-vitD and parathyroid hormone (PTH) in healthy adults. Aliquots (100 microL) of serum were spiked with internal standard, proteins were precipitated, and samples were analyzed by liquid chromatography-tandem mass spectrometry using 2-dimensional chromatographic separation. Total imprecision was less than 10%, and the limit of quantitation was 1.0 ng/mL. We determined the distribution of concentrations of 25OH-vitD(2) and 25OH-vitD(3) in healthy adults using samples collected during winter and summer and evaluated the association between 25OH-vitD and PTH. The difference between median concentrations of 25OH-vitD in samples collected during winter and summer was 11 ng/mL (27 nmol/L). Statistically significant differences in concentrations of PTH were observed between groups of samples with 25OH-vitD less than 11 (27 nmol/L) and 11 to 15 ng/mL (27-37 nmol/L) and between groups with 25 to 30 (62-75 nmol/L) and more than 40 ng/mL (100 nmol/L). Among the advantages of this method are its high sensitivity and specificity.

Measurement of Thyroglobulin by Liquid Chromatography–Tandem Mass Spectrometry in Serum and Plasma in the Presence of Antithyroglobulin Autoantibodies

“…Multidimensional separations are widely used in the analysis of small molecules (18–21). Using the same concept, a 2D separation was optimized for the targeted peptide using two chromatographic columns with complementary retention mechanisms under UPLC conditions.…”

“…The separation conditions were optimized to provide a good separation from other endogenous peaks, while maintaining a short analysis time. While the instrumentation and the method utilizing multi-dimensional chromatography are more complex, 2D separations have number of advantages over separation using single analytical column, (a) in a well-developed method orthogonal mechanisms of chromatographic separation on the 1 st and 2 nd columns could remove peaks that would remain unresolved using single retention mechanism; (b) peaks eluting outside of the window of the peak transfer from the 1 st to the 2 nd column are directed to waste; (c) reduced potential for ion suppression; (d) longer life of analytical column, and consequently more robust methods; and finally (e) reduced analysis time (reconditioning of the 1 st column takes place while separation takes place on the 2 nd column, and vice versa) (19–21). …”

Performance characteristics of six automated 25-hydroxyvitamin D assays: Mind your 3s and 2s

“…Additionally, clinically relevant comparisons were made to identify the effect of 25(OH)D 2 on these assays. Importantly, these comparisons encompassed a recalibration of an existing assay (Siemens Centaur) (5) as well as the first A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 6 description of two assays that have recently been FDA cleared for use in the United States (Beckman Coulter's Access2 and UniCel DxI 800). While other reports have described comparisons between 25(OH)D assays, most include only a small number of assays and do not evaluate the performance characteristics of each platform.…”

Development and validation of a quantitative assay based on tandem mass spectrometry

“…Two papers on tacrolimus and vitamin D determination are worth citing as a model validation covering issues addressed in the current paper. 66,67 To standardize the outcome of the process, laboratories may find it useful to summarize the information in a form designed for the purpose of assay verification (see Supplementary information). This document should be adjusted by deleting parameters not relevant to the instrumentation.…”