An LC-MS/MS-Based Method for the Quantification of Pyridox(am)ine 5'-Phosphate Oxidase Activity in Dried Blood Spots from Patients with Epilepsy.

We report the development of a rapid, simple, and robust LC–MS/MS-based enzyme assayusing dried blood spots(DBS) for the diagnosis of pyridox(am)ine 5′-phosphate oxidase ( PNPO) deficiency (OMIM 610090). PNPOdeficiency leads to potentially fatal early infantile epileptic encephalopathy, severe developmental delay, and other features of neurological dysfunction. However, upon prompt treatment with high doses of vitamin B6, affected patients can have a normal developmental outcome. Prognosis of these patients is therefore reliant upon a rapid diagnosis. PNPOactivity was quantified by measuring pyridoxal5′-phosphate (PLP) concentrations in a DBS before and after a 30 min incubation with pyridoxine5′-phosphate (PNP). Samples from 18 PNPOdeficient patients (1 day–25 years), 13 children with other seizure disorders receiving B6 supplementation (1 month–16 years), and 37 child hospital controls (5 days–15 years) were analyzed. DBS from the PNPO-deficient samples showed enzyme activity levels lower than all...