A sensitive LC/MS/MS assay of 250H vitamin D-3 and 250H vitamin D-2 in dried blood spots
ABSTRACT Low levels of 25 hydroxyvitamin D (25OHD) during early development is associated with a range of adverse health outcomes. While a number of methods exist to measure 25OHD in sera, none have been specifically developed to examine dried blood spots (DBS).
We describe an assay where 25 hydroxyvitamin D(3) (25OHD3) and 25 hydroxyvitamin D(2) (25OHD2) are extracted from 3.2 mm DBS punches, derivatised with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) prior to analysis with LC/MS/MS. We assessed assay precision, relative accuracy and examined the impact of storage conditions in samples stored for up to 22 years.
The new assay had good accuracy and precision, and was highly sensitive, being capable of detecting <1 nmol/l 25OHD3 and 2 nmol/l 25OHD2. CDER sensitivity criteria were slightly higher at 7.7 nmol/l for 25OHD3 and 10.7 nmol/l for 25OHD2. The mean 25OHD3 concentration in 118 archived DBS was 20.8+/-11.4, (4.8 to 67.8 nmol/l). 25OHD2 was detected in only two of these samples. 25OHD3 concentrations were significantly higher in DBS collected in summer compared to winter (p<0.0001).
Both 25OHD3 and 25OHD2 can be reliably quantified in archived 3.2 mm dried blood spots. We can not be certain that the levels we measure in archived samples are exactly the same as when they were collected. However, the fact that the DBS levels reflect the well-known seasonal variation in this vitamin and when corrected for sera, values fall within the normal range for 25OHD3, means that DBS are a useful tissue repository for testing a range of hypotheses linking developmental hypovitaminosis D and adverse health outcomes.
- SourceAvailable from: Bernhard T Baune
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- "The assay method is highly sensitive, and can measure 25(OH)D3 in DBS stored for between 8 and 22 years (Eyles et al., 2009). With this method, assay calibrants are extracted and derived in the same way as clinical samples, using multi-point calibration curves (Eyles et al., 2009). Co-twins were plated in adjacent wells on the same plate. "
ABSTRACT: Cytokines and vitamin D both have a role in modulating the immune system, and are also potentially useful biomarkers in mental illnesses such as major depressive disorder (MDD) and schizophrenia. Studying the variability of cytokines and vitamin D in a healthy population sample may add to understanding the association between these biomarkers and mental illness. To assess genetic and environmental contributions to variation in circulating levels of cytokines and vitamin D (25-hydroxy vitamin D: 25(OH)D3), we analyzed data from a healthy adolescent twin cohort (mean age 16.2 years; standard deviation 0.25). Plasma cytokine measures were available for 400 individuals (85 MZ, 115 DZ pairs), dried blood spot sample vitamin D measures were available for 378 individuals (70 MZ, 118 DZ pairs). Heritability estimates were moderate but significant for the cytokines transforming growth factor-β1 (TGF-β1), 0.57 (95% CI 0.26-0.80) and tumor necrosis factor-receptor type 1 (TNFR1), 0.50 (95% CI 0.11-0.63) respectively. Measures of 25(OH)D3 were within normal range and heritability was estimated to be high (0.86, 95% CI 0.61-0.94). Assays of other cytokines did not generate meaningful results. These potential biomarkers may be useful in mental illness, with further research warranted in larger sample sizes. They may be particularly important in adolescents with mental illness where diagnostic uncertainty poses a significant clinical challenge.Twin Research and Human Genetics 12/2014; 18(01):1-8. DOI:10.1017/thg.2014.70 · 1.92 Impact Factor
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- "Accessing blood samples in this population however may be particularly difficult. The measurement of 25OHD 3 in archived samples such as dried blood spots (Eyles et al., 2009, 2010) with prospective diagnoses in this sample type may be one way of addressing this question in a sufficiently large cohort (Eyles, 2010). "
ABSTRACT: Increasingly vitamin D deficiency is being associated with a number of psychiatric conditions. In particular for disorders with a developmental basis, such as autistic spectrum disorder and schizophrenia the neurobiological plausibility of this association is strengthened by the preclinical data indicating vitamin D deficiency in early life affects neuronal differentiation, axonal connectivity, dopamine ontogeny and brain structure and function. More recently epidemiological associations have been made between low vitamin D and psychiatric disorders not typically associated with abnormalities in brain development such as depression and Alzheimer's disease. Once again the preclinical findings revealing that vitamin D can regulate catecholamine levels and protect against specific Alzheimer-like pathology increase the plausibility of this link. In this review we have attempted to integrate this clinical epidemiology with potential vitamin D-mediated basic mechanisms. Throughout the review we have highlighted areas where we think future research should focus.Frontiers in Neuroendocrinology 07/2012; 34(1). DOI:10.1016/j.yfrne.2012.07.001 · 7.58 Impact Factor
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- "PPE (MeCN) → RPSPE →derivatization (PTAD) a) YMC-Pack Pro C18 RS (5 μm, 150 × 2.0 mm, YMC) b) MeOH-10 mM ammonium formate (5:1 v/v) + 5 mM methylamine c) 0.2 mL/min a) API 2000 b) positive ESI c) 607 [M +CH 3 NH 3 ] + →298 m/z 5–125 pmol/L ∼8 min Newman et al. (2009) 1) 25(OH)D 2 2) 25(OH)D 3 [ 2 H 6 -25(OH)D 3 ] a) 6-mm disks from dried blood spot → sonication→PPE (MeOH) →LLE (hexane) b) Human serum (0.01 mL) →PPE (MeOH) →LLE (hexane) a) Varian Pursuit 3u PFP (3 μm, 50 × 2.0 mm, Varian) b) Gradient: 70-95% MeOH with 0.1% formic acid-2 mM ammonium acetate c) 0.2 mL/min a) Varian 320-MS TQ b) positive ESI c) 1) 413 [M + H] + →377 and 395 m/z 2) 401 [M + H] + →365 and 383 m/z 1) ND 2) 8.75–187.5 nmol/L 10 min Eyles et al. (2009) 1) 25(OH)D 2 2) 25(OH)D 3 [ 2 H 3 -25(OH)D 3 ] 3.2-mm disks from dried blood spot →PPE (MeCN) → derivatization (PTAD) a) Zorbax SB-C18 (5 μm, 50 × 2.1 mm, Agilent) b) MeCN-0.1% formic acid (63:37 v/v) c) 0.35 ml/min a) AB API 4000 QTRAP b) positive ESI c) 1) 570 [M+H-H 2 O] + →298 m/z 2) 558 [M+H-H 2 O] + →298 m/z 1) 10.7-121.3 nmol/L 2) 7.7-125 nmol/L ∼3 min Holmoy et al. (2010) 1) 25(OH)D 2 2) 25(OH)D 3 [ 2 H 6 -25(OH)D 3 ] a) Cerebrospinal fluid (0.4 mL) → MeOH b) Human serum (0.15 mL) → IPA + butylated hydroxytoluene a) Zorbax SB-C18 (1.8 μm, 50 × 4.6 mm, Agilent) b) MeOH-H 2 O (75:25 v/v) + 0.1% formic acid c) 1.5 mL/min a) Agilent b) positive APCI c) 1) [M + H-H 2 O] + = 395 m/z 2) [M + H-H 2 O] + = 383 m/z a) 0.04–10 nmol/L b) 5–400 nmol/L 7 min Kissmeyer et al. (2001) "
ABSTRACT: Clinical testing for vitamin D nutritional status has experienced tremendous growth in the past several years, driven by research results linking various diseases with low serum 25-hydroxyvitamin D [25(OH)D] levels. Meanwhile, interest in the pathophysiological mechanism elucidation and pharmaceutical applications requires measurement of vitamin D metabolites and analogues. Liquid chromatography-mass spectrometry (LC-MS) has been increasingly utilized in these applications. In this work, our objective was to critically review the progress of LC-MS application in measuring vitamin D metabolites and analogues in biological fluids. The LC-MS methods included were selected from those searchable in PubMed up to January 2010. LC-MS has unique advantages in measuring various vitamin D metabolites and analogues due to its flexibility, sensitivity, and specificity. Despite some controversies over serum 25(OH)D tests, LC-MS will be used for standardizing serum 25(OH)D assays using reference materials available from the National Institute of Standards and Technology.Clinical biochemistry 01/2011; 44(1):66-76. DOI:10.1016/j.clinbiochem.2010.05.007 · 2.28 Impact Factor