6. High Throughput LC/MS/MS-based Quantification of 25-hydroxy Vitamin D2, 25-hydroxy Vitamin D3 and 3-epi-25 hydroxy Vitamin D3 in Dried Blood Spots: Aiming to Cover Entire Clinical Practice Human Population Range, Infants to Elderly

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Suma Ramagiri MDS Analytical Technologies

Suma Ramagiri1, Sylvie Beaudet1, Adam Latawiec1, Lisa Sapp2, Tania Sasaki2, Robert Ellis1 1MDS Analytical Technologies, 71 Four Valley Drive, Concord, ON, Canada, L4K 4V8, 2Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404 USA.

Vitamin D is known for its role in prevention of wide range of diseases. Its deficiency can lead to childhood rickets, cardiovascular diseases, various forms of cancers, type I and II diabetes and even schizophrenia. Universally accepted means to monitor vitamin D deficiency is by quantitative measurement of 25-hydroxy vitamin D2 (25-OH Vit D2) and 25-hydroxy vitamin D3 (25-OH Vit D3) levels in serum, plasma or whole blood. Recent studies have shown that C-3 Epimers of 25-OH Vit D2/D3 account for a significant proportion of total circulating 25-OH Vit D in infants, complicating accurate measurement and interpretation of Vitamin D Status. An LC/MS/MS based assay covering entire human population; neonatal to elderly patients is needed. Also specially focusing on pediatric and diabetic patients, an LC/MS/MS method is desirable in dried blood spots, which is a more convenient sampling technique. Here, we have developed a simple, accurate and high throughput LC/MS/MS based assay to monitor 25-OH Vit D2, 25-OH Vit D3 and 3-epi-OH Vit D3 in dried blood spots. The LC/MS/MS assay was developed on AB SCIEX QTRAP® 5500. Calibration curve ranging from 1 ng/mL-100 ng/mL was prepared in dried blood spots and evaluated for its linearity, accuracy and precision. 25-OH Vit D2-d6 was used as an internal standard. Two sample extraction methods were evaluated, one with simple protein precipitation using acetonitrile and the other with protein precipitation followed by liquid-liquid extraction using isopropyl alcohol. The current LC/MS/MS assay showed linearity (R> 0.99) with accuracy ranging from 80-120% and % CV below 15. Both extraction methods had good sample recovery (75-125%). The above LC/MS/MS based assay quantifying simultaneously 25-OH Vit D2, 25-OH Vit D3 and 3-epi-25-OH Vit D3 in dried blood spot looks promising and could open new opportunities in Vitamin D deficiency testing in neonatal screening and also in diabetic patients. References: 1. Zerwekh JE , Ann Clin Biochem 41:272–281, 2004. 2. Ravinder J. Singh, Robert L. Taylor, G. Satyanarayana Reddy, and Stefan K. G. Grebe; Clin Endocrinol Metab 91: 3055–3061, 2006 3. Rinaldo P, Tortorelli S, MaternD. Curr Opin Pediatr, 16: 427–33, 2004.

Email: suma.ramagiri@sciex.com

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