Determination of plasma pipecolic acid by an easy and rapid liquid chromatography-tandem mass spectrometry method

Pipecolic acid (PA) is an important biochemical marker for the diagnosis of peroxisomal disorders. PA is also a factor responsible for hepatic encephalopathy and a possible biomarker for pyridoxine-dependent seizures. We developed an easy and rapid PA quantification method, by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), requiring no derivatization and applicable to small sample volumes. Plasma (100 μl) is extracted with 500 μl acetonitrile (ACN) containing 2 μmol/l [(2)H5]-phenylalanine as internal standard, vortexed and centrifuged. The supernatant is analyzed by HPLC-MS/MS in positive-ion mode using multiple reaction monitoring scan type. HPLC column is a Luna HILIC (150×3.0mm; 3 μ 200A): Buffer A: ammonium formate 5 mmol/l; Buffer B: ACN/H20 90:10 containing ammonium formate 5 mmol/l. PA retention time is 4.86 min. Recovery was 93.8%, linearity was assessed between 0.05 and 50 μmol/l (R(2)=0.998), lower limit of detection was 0.010 μmol/l and lower limit of quantification was 0.050 μmol/l. Coefficient of variation was 3.2% intra-assay and 3.4% inter-assay, respectively. Clinical validation was obtained by comparing PA plasma values from 5 patients affected by peroxisomal disorders (mean, 23.38 μmol/l; range, 11.20-37.1 μmol/l) to 24 ages related healthy subjects (mean, 1.711 μmol/l; range, 0.517-3.580 μmol/l).