A sensitive LC/MS/MS assay of 250H vitamin D-3 and 250H vitamin D-2 in dried blood spots

Queensland Centre for Mental Health Research, The Park Centre for Mental Health, Wacol, Q4076, Australia.
Clinica chimica acta; international journal of clinical chemistry (Impact Factor: 2.76). 03/2009; 403(1-2):145-51. DOI: 10.1016/j.cca.2009.02.005
Source: PubMed

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.

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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. "
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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). "
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