10. A Sensitive and Robust LC-MSMS Assay for Vitamin D2 and D3 Using Human Serum Calibrators
Tue 4:54 PM - PosterSplash Track 1
Alex Cherkasskiy
*Alex Cherkasskiy, *Matt Willetts, *Rebecca Pitts, *Joe Trometer, *Hari Nair, #Alla Ostrinskaya.

*PerkinElmer Inc, Waltham, MA
#Ostrinskaya Blas Cerda, Ostrinskaya
The increase in demand for Vitamin D testing has been widely reported in the technical and mainstream media. Vitamin D deficiency is now considered to be common throughout most of the world. This has been attributed to lack of exposure to UV light from the sun due to the increased use of sunscreens and sedentary indoor lifestyles common across the developed world.

Here we present a sensitive, robust assay for the determination of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in serum using LC-MSMS. The assay includes a simple derivatization step which allows lower performance instruments to be utilized. The performance of the assay was evaluated on a Waters Quattro Micro mass spectrometer using calibrators made from human charcoal stripped serum spiked with varying concentrations D2 and D3 analytes. The limit of quantitation was 1.3 ng/mL for both analytes. The assay was linear from 1.3 – 200 ng/mL (R2 >0.99). Recoveries were determined to be >75% for 25-hydroxyvitamin D3 and >90% for 25-hydroxyvitamin D2. The assay was precise with within run and between run %CVs less than 15% across 7 runs, two instruments and 3 operators.

We are an active participant in the DEQAS vitamin D proficiency testing scheme ( and results from the assay using the in-house produced calibrators were within 10% of LC-MS results submitted by other laboratories participating in the scheme. Performance of the assay was also assessed with the new NIST reference material (SRM 972). Results show excellent agreement for both 25-hydroxyvitamin D3 (5%) and 25-hydroxyvitamin D2 (12%) with the certified concentration values included on the certificate of analysis for the materials.