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

Abstract 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 H 5 ]-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 × 3.0 mm; 3 μ 200A): Buffer A: ammonium formate 5 mmol/l; Buffer B: ACN/H 2 0 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).