Pierre-Luc Mallet (Presenter)
University of Montreal, CIUSSS-CHUS
Bio: Education: June 2014 – June 2016 (expected completion date of the program) Postdoctoral diploma in clinical biochemistry Montreal University, Sherbrooke University Hospital Centre (Quebec) 2010-2014 Philosophiae doctor’s degree in biochemistry Sherbrooke University, Sherbrooke (Quebec) 2007-2009 Master’s degree in Biology, including a specialty in microbiology Sherbrooke University, Sherbrooke (Quebec) 2007-2009 Micro-program diploma in scientific interactions Sherbrooke University, Sherbrooke (Quebec) 2004-2007 Bachelor’s degree in biotechnology, including a specialty in molecular biology Sherbrooke University, Sherbrooke (Quebec) 2003-2004 Health sciences program Moncton University, Campus of Edmundston (New-Brunswick)
Authorship: Pierre-Luc Mallet(1,2) and Guy Fink(1,3)
(1) Dept. of clinical biochemistry, CIUSSS-CHUS, Sherbrooke, Can; (2) Dept. of biochemistry and molecular medicine, U. of Montreal, Can; (3) Dept. of biochemistry, U. of Sherbrooke, Can
| Short Abstract Development and validation of a LC-ESI-MS/MS quantification method of 25-hydroxyvitamin D2&D3 and of their C3-epimer (25-OH-D) has been performed to overcome the issue with the overestimation of vitamin D levels cause by the occurrence of C3-epimers. Briefly, samples were deproteinized using ZnSO4 and MeOH, extracted using heptane, dried down, and finally resolubilized in the mobile phase (MeOH 68 %/ formic acid 0.1 %). Liquid chromatography was performed using isocratic gradient with a pentafluorophenyl column. CV and Bias were respectively inferior to 20 % and to 15 % for 3 nM of 25-OH-D. Method comparison was performed with another lab using an LC-ESI-MS/MS technique and we have obtained a slope and intercept of respectively 0.998 and 0.05 nM for total 25-OH-D. This LC-ESI-MS/MS method allows quantification of 25-OH-D2&D3 and of both C3-25-OH-D2&D3 which a few labs offer. |
Long Abstract
Introduction
Vitamin D is important for bone health and recently has been linked to a much wider role in the pathology of diabetes, cancer and autoimmune, neurodegenerative, mental and cardiovascular diseases. Vitamin D is extensively metabolized in human, more than 50 metabolites are described in the literature. Vitamin D undergo 25-hydroxylation in the liver and 25-hydroxyvitamin-vitaminD2&D3 (25-OH-D2&D3) are the most abundant circulating metabolites. Therefore, they are used as a measurable criterion to assess vitamin D level status. 25-OH-D2&D3 are further hydroxylated in position 1 at the kidney to form the active metabolites, 1,25-(OH)2-D2&D3. These active metabolites have a lower binding affinity with vitamin D binding protein, resulting in low half-life and therefore low levels in human blood. Furthermore, 1,25-(OH)2-D2&D3 metabolites has been shown to not systematically correlated with the vitamin D level status of patients (1).
LC-MS/MS is referred as the gold standard method for measurement of 25-OH-D2&D3. Nevertheless, a serious problem is the occurrence of C3-epimers for all vitamin D compounds. C3-epimers result from the reversal of the stereochemical configuration of the C3-bound hydroxyl group from α to β. These C3-epimers has lower biological activity (1). Generally, levels of theses C3-epimers are higher in neonates and infants samples. However, these C3-epimers are detected in 99 % of samples ranging from neonates to participants aged over 80 years. Furthermore, the proportion of these C3-epimers can account for up to 25 % of vitamin D levels (2). Therefore, the National Health and Nutrition Examination Survey (NHANES) suggested that all quantitative LC-MS/MS assays for vitamin D metabolites have the capability to separate those C3-epimers to avoid overestimation of vitamin D levels (1). Moreover, the Roche Elecsys vitamin D test used on our automaton underestimated the 25-OH-D2 concentration, which is an issue with vitamin D2 supplemented patients who has undergone bariatric surgery.
Here in this project, to handle those issues, we have performed the development and validation of an LC-ESI-MS/MS method to quantitate 25-OH-D2&D3 and of both C3-25-OH-D2&D3 which a few labs offer.
Methods
Calibrators, internal standards and quality controls
The calibrators 25-OH-D3 and 25-OH-D2 (Cerilliant, Round Rock, TX), C3-epimer-25-OH-D3 and C3-epimer-25-OH-D2 (Isosciences, King of Prussia, PA) and the internal standards 25-OH-D3-2H3, 25-OH-D2-2H3 and C3-epimer-25-OH-D3-2H3 (Isosciences, King of Prussia, PA) were all prepared in BSA 1 %/NaCl 0.9 %. The quality controls Precicontrol varia (Roche, Laval, QC) were used. External quality controls from the vitamin D External Quality Assessment Scheme (DEQUAS) were used to validate our LC-MS/MS method with peers.
Sample preparation
Briefly, the internal standard working solution was mixed to the samples. Then, ZnSO4 and MeOH were added to the samples for protein precipitation. Thereafter, heptane was added to samples for liquid-liquid extraction. Following centrifugation, the supernatants were transferred to new microtubes and dried down. Resolubilization was performed with the mobile phase (68 % MeOH/ 0.1 % formic acid). Finally, solutions were transferred into a vial, then put into the 1290 infinity LC system autosampler (Agilent, Santa Clara, CA). All solvents were at least HPLC grade.
LC-MS/MS conditions
HPLC was performed using isocratic condition with 68 % MeOH/ 0.1 % formic acid to a flow of 0.8 ml/min. A Pursuit 3 PFP 100 X 3.0 mm column (Agilent, Santa Clara, CA) was used for chromatography at 45ºC. A 6460 triple quad MS/MS (Agilent, Santa Clara, CA) equipped with a jet stream ESI (Agilent, Santa Clara, CA) were used to monitor MRM analysis.
Results
After optimization of MRM transitions and MS/MS source parameters for higher sensitivity, liquid chromatography was optimized for the separation of 25-OH-D2&D3 from their C3-epimer. Resolution factors (Rs) of 1.27 for 25-OH-D2 and of 1.59 for 25-OH-D3 were obtained against their respective C3-epimer. Carryover analysis has revealed that the lower concentration of 25-OH-D that can be measured and would not be affected by carryover was found to be at 1 nM. Matrix effect was verified using the post-extraction addition method and has revealed no interference. Linearity was verified up to 500 nM for 25-OH-D2&D3 and their C3-epimer and all have shown a value of more than 0.99 for the R2. Intra- and inter- day coefficient of variation (CV) and bias analysis was performed with five replicates for each concentration over 5 days. Criteria of acceptability were fixed at less than 20 % for the CV and less than 15 % for the bias, which has leaded to a 3 nM concentration value has our lower limit of quantification. Method comparison was performed with another lab using an LC-ESI-MS/MS technique to quantify PTAD-derivated 25-OH-D. Deming regression analysis has shown a slope and an intercept of respectively 0.998 and 0.05 nM for total 25-OH-D. Bland-Altman plot has showed no point over 20 % difference and a mean at 0.53 % with a 95 % confidence interval with an upper limit of 15.83 % and a lower limit of -14.77 %. Evaluation of the standard deviation index (SDI) for DEQUAS samples #471-485 in comparison with the LC-MS/MS method group has shown none over 2.
Conclusions
Validation steps were based in part on the Clinical and Laboratory Standards Institute C62-A document (3). To further the validation process interference of hemolysis, icterus and lipemia, freeze-thaw cycles and storage stability will be verified. This LC-ESI-MS/MS method will suppress vitamin D level overestimation due to interference by the C3-epimers. In addition, it will also give better quantification of the vitamin D level status of patients who have undergone bariatric surgery and are supplemented in vitamin D2. Furthermore, the quantification of C3-epimers will enable clinical research collaboration to deepen our knowledge on theirs physiological functions. Finally, the implantation of an automated sample preparation workstation will be developed and validated. It will enable to reduce the sample preparation time, the coefficient of variation and the gross error.
References & Acknowledgements:
1 - Volmer DA, Mendes LR, Stokes CS. Mass Spectrom Rev. 2015 Jan-Feb;34(1):2-23.
2 - Lensmeyer G, Poquette M, Wiebe D, Binkley N. J Clin Endocrinol Metab. 2012 Jan;97(1):163-8.
3 - Clarke W and al. CLSI. 2014. Liquid chromatography-mass spectrometry methods; Approved guideline. C62-A.
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