Cost-Effective HPLC-UV Method for Quantification of Vitamin D<sub>2</sub> and D<sub>3</sub> in Dried Blood Spot: A Potential Adjunct to Newborn Screening for Prophylaxis of Intractable Paediatric Seizures

Abstract

25-Hydroxyvitamin D (25-(OH)D) deficiency is recently been described as one of the multiple factors responsible for pediatric seizures. 25-Hydroxyvitamin D3 and 25-Hydroxyvitamin D2 are the well-known markers to determine Vitamin D status. In this work we report the development of a sensitive and cost effective HPLC technique for the quantification of the vitamin D metabolites from dried blood spot samples (DBS). The metabolites were extracted using acetonitrile–methanol–0.1% formic acid (60 : 20 : 20 (v/v)) and analyzed on an Acclaim C18 column (150 × 4.6 mm i.d., 3 µm) at a flow rate of 1 mL/min. The method was linear in the range of 10–80 ng/mL. Limit of detection and limit of quantification (LOQ) of the method were 5 and 10 ng/mL respectively. Extensive stability studies demonstrated the analytes to be stable in stock and matrix with a percent change within the acceptable range of ±15%. Comparison of the newly developed HPLC-DBS method with the reported LC-MS-DBS and electrochemiluminescence immunoassay (ECLIA) methods followed by Bland–Altman analysis demonstrated a bias of 0.08 and −0.14, respectively proving the methods are comparable. Application of the developed method to a pediatric seizure cohort depicted 46.6% of cases as deficient and 26.6% as insufficient for 25-(OH)D. Among deficient cases 8 samples were below 10 ng/mL and exact amount was not calculated since these were below the LOQ levels. The mean ± standard deviation (S.D.) in the remaining 6 deficient cases was 13.22 ± 2.80 ng/mL. The levels in healthy infants were 33.9 ± 6.11 ng/mL. The method can be used routinely for assessing 25-(OH)D deficiency in newborn.