1. VITAMIN D BLOODSPOT ANALYSES BY LC-MS/MS:
Method validation and a field study revealing 42% deficiency in Dutch pharmacists
Jac J. van der Heijden1, Jos P.M. Wielders2, Wim A. Buurman1
(1) DBSL, Brightlands Chemelot Campus, Geleen, the Netherlands, (2) Meander Medical Center, Amersfoort, the Netherlands
Introduction
Dried blood spotting (DBS) is a minimally invasive sampling technique based on finger puncture where blood samples are spotted onto a filter paper and dried. The method is particular suitable for field studies and for sampling small children. We describe the validation of a method for quantitative measurement of 25-hydroxycholecalciferol (25OHD3) in dried blood spots by HPLC-MS/MS. Our method is also suitable for quantification of 3-epi-25hydroxy vitamin D3. In addition, we present data generated in a field study using DBS analyses of 25OHD3 levels in a population of Dutch pharmacists.
Materials and methods
The LC-MS/MS method used is a further development of Eyles’ method (Clinica Chimica Acta 2009;403: ) employing UHPLC and an Agilent 6460C triple-quadrupole MS/MS with an ESI source and tri-deutero 25OHD3 as internal standard. Bloodspot punches are extracted with an alkaline surfactant solution, allowing release of 25OHD3 from D-binding protein, followed by hexane extraction and derivatization with PTAD. The method was calibrated with serum calibrators from LabQuality (Helsinki, Finland) mixed with washed erythrocytes to yield a hematocrit of 46%. For adult bloodspot samples, results are converted to plasma data applying a standard hematocrit of 46%. We tested imprecision, LOQ and method correlation with a LabQuality calibrated LC-MS/MS method (dr van den Ouweland CWZ, Nijmegen, the Netherlands) using 27 native serum samples. Free testing for 25OHD3 was offered at Dutch meetings of pharmacists in March and October Similar to American and German guidelines we consider 25OHD3 < 50 nmol/L as deficient.
Results
Initial experiments demonstrated equivalent results for standard 8 mm punches of DBS from 20 – 50 μl blood drops.
from baseline less than the inter-assay CV for DBS samples stored up to three months at room temperature, shielded from light.
The ability of our method to measure 25OHD3 as well as 3-epi-25OHD3 is shown in fig 2. No 25OHD2 is detected in this sample, which is actually a pediatric sample from the so-called kiDs study. The biological function of 3-epi-25OHD3 is yet unknown.
In a field study March 2014, we found 41.6% of 120 pharmacists, volunteering at a pharmaceutical booth, having 25OHD3 levels < 50 nmol/L. On a similar occasion early October 2014 and after a sunny summer 19.1% of 246 pharmacists were still deficient at 50 nmol/L. Over the year 23.5% of indigenous Dutch are < 50 nmol/L (vdMeer Thesis 2010) and 39 % of Dutch man < 50 nmol/L (Spiro Nutr Bull DOI: /nbu.12108). Pharmacist have no better vitamin D status than the average Dutchmen.
Extraction recovery was found to be between 86 and 96 %.
Inter-assay imprecision was calculated from 19 independent runs at three levels: CV’s were 9.8% at 48.3 nmol/L, 8.7% at 76.4 and 7.6% at nmol/L. Mean bias and correlation to the reference LC-MS/MS are shown in figures 1A and 1B.
We found a mean bias of 3.01 nmol/L and by Deming regression a correlation equation DBSL = 1.00 Ref method (R=0.936)
This implies that according to present standards (Clinica Chimica Acta 2009;408:8-13) our method fulfils the requirements of bias ≤ 5% and imprecision ≤ 10% for routine 25OHD measurement.
The LOQ is below10 nmol/L, inter-run CV is 13.9% at this level.
The stability of 25OHD3 in DBS is excellent. We found deviations
Fig 2 LC-MS/MS chromatogram showing the presence of 15 nmol/L
3-epi-25OHD3 and 91 nmol/L 25OHD3 in a dried bloodspot obtained from an infant A B
Fig 1A Correlation plot Fig 1B Bland-Altman plot
Conclusion It is demonstrated that this elegant bloodspot LC-MS/MS method is suitable for measuring 25-hydroxycholecalciferol (25OHD3) according to present standards, making it most suitable for field studies and pediatric sampling. As an example of a field study, we showed that even pharmacists have no better 25OHD3 status than the average Dutch population.
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