AbstractThe assessment of urinary α‐aminoadipic semialdehyde (α‐AASA) has become the diagnostic laboratory test for pyridoxine dependent seizures (PDS). α‐AASA is in spontaneous equilibrium with its cyclic form Δ1‐piperideine‐6‐carboxylate (P6C); a molecule with a heterocyclic ring structure. Ongoing diagnostic screening and monitoring revealed that in some individuals with milder ALDH7A1 variants, and patients co‐treated with a lysine restricted diet, α‐AASA was only modestly increased. This prompted us to investigate the diagnostic power and added value of the assessment of urinary P6C compared to α‐AASA. Urine samples were diluted to a creatinine content of 0.1 mmol/L, followed by the addition of 0.01 nmol [2H9]pipecolic acid as internal standard (IS) and 5 μL was injected onto a Waters C18 T3 HPLC column. Chromatography was performed using water/methanol 97/3 (v/v) including 0.03 % formic acid by volume with a flow rate of 150 μL/min and detection was accomplished in the multiple reaction monitoring mode: P6C m/z 128.1 > 82.1; [2H9]pipecolic acid m/z 139.1 > 93.1. Due to the dualistic nature of α‐AASA/P6C, and the lack of a proper internal standard, the method is semi quantitative. The intra‐assay CVs (n = 10) for two urine samples of proven PDS patients with only modest P6C increases were 4.7% and 8.1%, whereas their inter‐assay CVs (n = 10) were 16 and 18% respectively. In all 40 urine samples from 35 individuals with proven PDS, we detected increased levels of P6C. Therefore, we conclude that the diagnostic power of the assessments of urinary P6C and α‐AASA is comparable.