Cost-effective HPLC-UV method for quantification of Vitamin D2 and D3 in dried blood spot: A potential adjunct to newborn screening for prophylaxis of intractable paediatric seizures

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Chemical and Pharmaceutical Bulletin


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 C column (150×4.6mm i.d., 3µm) at a flow rate of 1mL/min. The method was linear in the range of 10–80ng/mL. Limit of detection and limit of quantification (LOQ) of the method were 5 and 10ng/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 10ng/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±2.80ng/mL. The levels in healthy infants were 33.9±6.11ng/mL. The method can be used routinely for assessing 25-(OH)D deficiency in newborn. 18

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