Determination of total homocysteine in dried blood spots using high performance liquid chromatography for homocystinuria newborn screening

Background: The most widely used method for newborn screening for homocystinuria (HCU) is a semi-quantitative bacterial inhibition assay for measuring methionine concentration in dried blood spots (DBS). Because this method has resulted in a number of missed cases due to many factors, we developed a high performance liquid chromatography (HPLC) method with fluorescence detection to measure total homocysteine (tHcy) in DBS which might be useful for newborn screening for HCU. Methods: One disk of DBS 3 mm in diameter was sonicated in 10 min. The extract was reduced with dithioerythritol and was derivatized with 4-aminosulfonyl-7fluoro-2,1,3-benzoxadiazole before injection into HPLC. Results: This method showed good linearity (r=0.996), precision (coefficient of variation range 2.7-5%), and excellent correlation coefficient between DBS and serum tHcy, both in control (r=0.932) and patient samples (r=0.952). By this method, the mean tHcy concentration in DBS of preterm newborns, full-term newborns, and adults was 1.4+/-1.0, 2.5+/-1.6, and 4.9+/-1.5 mumol/L, respectively. The mean tHcy DBS concentration in two cases of cystathionine-beta-synthase deficiency and one case of 5,10-methylentetrahydrofolate reductase deficiency was 22.7+/-2.88, 29.3+/-1.90, and 41.3 mumol/L, respectively. Conclusions: The present method, which is rapid, user friendly and reliable, seems applicable to newborn screening of HCU in place of methionine measurement.