A specific LC/ESI-MS/MS method for determination of 25-hydroxyvitamin D3 in neonatal dried blood spots containing a potential interfering metabolite, 3-epi-25-hydroxyvitamin D3 |
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Authors: | Higashi Tatsuya Suzuki Masahiro Hanai Junji Inagaki Shinsuke Min Jun Zhe Shimada Kazutake Toyo'oka Toshimasa |
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Affiliation: | School of Pharmaceutical Sciences and Global COE Program, University of Shizuoka, Shizuoka, Japan. higashi@u-shizuoka-ken.ac.jp |
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Abstract: | Vitamin D deficiency in an infant is associated with a wide range of adverse health outcomes in later life. A method for the quantification of 25‐hydroxyvitamin D3 [25(OH)D3, the best‐established indicator of vitamin D status] in neonatal dried blood spots (DBSs) using LC/ESI‐MS/MS has been developed and validated. The method employed two steps of derivatization, a Diels–Alder reaction with 4‐phenyl‐1,2,4‐triazoline‐3,5‐dione followed by acetylation, to enhance the detectability of 25(OH)D3 in ESI‐MS/MS and to separate 25(OH)D3 from 3‐epi‐25‐hydroxyvitamin D3 [3‐epi‐25(OH)D3], a potent interfering metabolite. 25(OH)D3 was extracted from two DBS punches (3 mm in diameter, equivalent to 5.3 μL of whole blood), purified using an Oasis HLB® cartridge, and subjected to derivatization prior to analysis with LC/ESI‐MS/MS. 25‐Hydroxyvitamin D4 was used as the internal standard. This method was reproducible (intra‐ and inter‐assay RSDs, <6.9%) and accurate (analytical recovery, 95.2–102.7%), and the LOQ was 3.0 ng/mL. The developed method enabled specific quantification of 25(OH)D3 in neonatal DBSs and detection of vitamin D deficiency without interference from 3‐epi‐25(OH)D3. |
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Keywords: | Derivatization Dried blood spot 3‐Epi‐25‐hydroxyvitamin D3 25‐Hydroxyvitamin D3 LC/ESI‐MS/MS |
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