A sensitive LC/MS/MS assay of 25OH vitamin D3 and 25OH vitamin D2 in dried blood spots.
BACKGROUND
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).


METHODS
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.


RESULTS
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).


CONCLUSIONS
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.
Eyles
Darryl
D
Queensland Centre for Mental Health Research, The Park Centre for Mental Health, Wacol, Q4076, Australia. eyles@uq.edu.au
Anderson
Cameron
C
Ko
Pauline
P
Jones
Alun
A
Thomas
Andrew
A
Burne
Thomas
T
Mortensen
Preben Bo
PB
Nørgaard-Pedersen
Bent
B
Hougaard
David Michael
DM
McGrath
John
J
D015652:25-Hydroxyvitamin D 2 / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D001774:Blood Chemical Analysis / Q000379:methods*; D002112:Calcifediol / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D002138:Calibration; D002853:Chromatography, Liquid; D006801:Humans; D007231:Infant, Newborn; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry; D013997:Time Factors; D014230:Triazoles / Q000737:chemistry
D014230:Triazoles; D015652:25-Hydroxyvitamin D 2; C053540:4-phenyl-1,2,4-triazoline-3,5-dione; D002112:Calcifediol