Determination of new biomarkers for diagnosis of pyridoxine dependent epilepsy in human plasma and urine by liquid chromatography-mass spectrometry.

Background: Pyridoxine-dependent epilepsy (PDE) is a rare inborn error of lysine metabolism. To date, diagnosis of PDE relies on the quantification of α-AminoAdipic SemiAldehyde (α- AASA), Piperideine-6-Carboxylate (P6C) and Pipecolic acid (PA) in urine or plasma from patients with overt symptoms. However, these biomarkers are not specific, and their biochemical analysis is challenged by their instability and technical limitations. We set-up and validated a method for the quantification of two new biomarkers of PDE (2S,6S- and 2S,6R-oxopropylpiperidine-2-carboxylic acid, 2- OPP, and 6-oxopiperidine2-carboxylic acid, 6-oxoPIP) on human urine and plasma by LC-MS/MS, to overcome the diagnostic and technical challenges of old biomarkers.

Methods: We analysed urine and plasma samples by LC-MS/MS, and validated the method in both biological matrices.

Results: We performed the biomarkers extraction from a 10 µL aliquot of urine or plasma in around 15 min using water 100 % for urine, and a solution of water/methanol 50 % for plasma, respectively. The analytical method was validated and gave good linearity (r > 0.999) in the range 0-15 µmol/L for 2-OPP and 0-25 µmol/L for 6-oxoPIP. In both matrices, the biomarkers were stable at different storage temperatures tested.

Conclusions: We set-up and validated a reliable method and confirmed its clinical applicability on real samples from PDE patients. This method could be used as routine test for the diagnosis and monitoring of PDE.