Carl Jenkinson (Presenter)
University of Birmingham
Bio: Postdoctoral Research Fellow based on the Institute of Metabolism and Systems Research at the University of Birmingham.
Authorship: Carl Jenkinson, I MA, Jennifer Tamblyn, Martin Hewison
Institute of Metabolism and Systems Research, University of Birmingham, Birmingham, B15 2TT, UK
| Short Abstract Clinical analysis of vitamin D continues to be focussed primarily on the highly abundant circulating 25-hydoxyvitamin D (25OHD) despite being an inactive form. However quantitation of the active hormonal form 1,25(OH)2D3 may provide further information on the physiology of vitamin D in health and disease. Circulating concentrations of 1,25(OH)2D3 range between 15-75 pg/mL and often cannot be measured by LC-MS/MS owing to LLOQ levels above these ranges. The aim of this project was to determine the optimum derivatization approach for measuring dihydroxy metabolites of vitamin D, including 1,25(OH)2D3 to ensure accurate routine measurements at endogenous concentrations. |
Long Abstract
Introduction
A number of endocrine metabolites circulate at low endogenous concentrations making accurate quantitation challenging. Often ionization by tandem mass spectrometry does not achieve the lower limits of quantitation (LLOQ) required to routinely quantify analytes with reference ranges below 100-200 pg/mL. The metabolic pathway of vitamin D undergoes complex metabolism producing many biologically important metabolites notably 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), and catabolites such as 24,25-dihydroxvitamin D3 (24,25(OH)2D3). Clinical analysis of vitamin D continues to be focussed primarily on the highly abundant circulating 25-hydoxyvitamin D (25OHD) despite being an inactive form. However quantitation of the active hormonal form 1,25(OH)2D3 may provide further information on the physiology of vitamin D in health and disease. Circulating concentrations of 1,25(OH)2D3 range between 15-75 pg/mL and often cannot be measured by LC-MS/MS owing to LLOQ levels above these ranges. An approach to enhance ionization and lowering LLOQ concentrations is to use a derivatization agent to enhance signal strength. The aim of this project was to determine the optimum derivatization approach for measuring dihydroxy metabolites of vitamin D, including 1,25(OH)2D3 to ensure accurate routine measurements at endogenous concentrations.
Methods
Analysis was performed by comparing three derivatization agents described previously to improve 1,25(OH)2D3 detection, PTAD, DMEQ-TAD and Amplifex Diene Reagent. The optimal reaction conditions were identified by comparing derivatization concentrations, reaction time and temperature. LC-MS/MS methods were developed for each derivatized compound on a Waters AQCUITY UPLC coupled to a Waters Xevo TQ-S mass spectrometer. Routine serum analysis was performed with each derivitization agent to ensure standardization between measurements with each method. A biphenyl column separated compounds of equal mass.
Results
Each of the derivitization agents enhanced the LLOQ of the dihydroxy metabolites of vitamin D analysed. The LLOQ concentrations obtained for 1,25(OH)2D3 were; underivatized [40 pg/mL], PTAD [12.5 pg/mL], DMEQ-TAD [8 pg/mL] and amplifex [6 pg/mL]. Analysis of routine serum samples revealed no bias between methods for quantitation of 1,25(OH)2D3 between each derivatization method. In each case these methods were able to detect 1,25(OH)2D3, however when using the underivatized method a number of lower level circulating concentrations below 40 pg/mL could not be measured.
Conclusions & Discussion
The methods described provide a comparison for accurate analysis of routine dihydroxyvitamin D metabolites, which will provide further clinical assessment to the role of vitamin D in health and disease.
References & Acknowledgements:
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