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Semeraro M.,Bambino Ges Childrens Research Hospital | Muraca M.,Bambino Ges Childrens Research Hospital | Catesini G.,Bambino Ges Childrens Research Hospital | Inglese R.,Bambino Ges Childrens Research Hospital | And 4 more authors.
Clinica Chimica Acta

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 (100μl) is extracted with 500μl acetonitrile (ACN) containing 2μmol/l [2H5]-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; 3μ 200A): Buffer A: ammonium formate 5mmol/l; Buffer B: ACN/H20 90:10 containing ammonium formate 5mmol/l. PA retention time is 4.86min.Recovery was 93.8%, linearity was assessed between 0.05 and 50μmol/l (R2=0.998), lower limit of detection was 0.010μmol/l and lower limit of quantification was 0.050μmol/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). © 2014 Elsevier B.V. Source

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