title: 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)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).
authors.affiliation: Department of Laboratory Medicine, Bambino Gesù Children's Research Hospital, IRCCS, Rome, Italy. Electronic address: semeraro.michela@gmail.com.
authors.forename: Michela
authors.initials: M
authors.lastname: Semeraro
authors.affiliation: Department of Laboratory Medicine, Bambino Gesù Children's Research Hospital, IRCCS, Rome, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Muraca
authors.affiliation: Department of Laboratory Medicine, Bambino Gesù Children's Research Hospital, IRCCS, Rome, Italy.
authors.forename: Giulio
authors.initials: G
authors.lastname: Catesini
authors.affiliation: Department of Laboratory Medicine, Bambino Gesù Children's Research Hospital, IRCCS, Rome, Italy.
authors.forename: Rita
authors.initials: R
authors.lastname: Inglese
authors.affiliation: Department of Laboratory Medicine, Bambino Gesù Children's Research Hospital, IRCCS, Rome, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Iacovone
authors.affiliation: Research Unit of Multifactorial Diseases, Obesity and Diabetes, Bambino Gesù Children's Research Hospital, IRCCS, Rome, Italy.
authors.forename: Gloria Maria
authors.initials: GM
authors.lastname: Barraco
authors.affiliation: Research Unit of Multifactorial Diseases, Obesity and Diabetes, Bambino Gesù Children's Research Hospital, IRCCS, Rome, Italy.
authors.forename: Melania
authors.initials: M
authors.lastname: Manco
authors.affiliation: Division of Metabolism and Research Unit of Metabolic Biochemistry, Bambino Gesù Children's Research Hospital, IRCCS, Rome, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Boenzi
authors.affiliation: Division of Metabolism and Research Unit of Metabolic Biochemistry, Bambino Gesù Children's Research Hospital, IRCCS, Rome, Italy.
authors.forename: Carlo
authors.initials: C
authors.lastname: Dionisi-Vici
authors.affiliation: Department of Laboratory Medicine, Bambino Gesù Children's Research Hospital, IRCCS, Rome, Italy.
authors.forename: Cristiano
authors.initials: C
authors.lastname: Rizzo
mesh_terms: D000293:Adolescent; D000328:Adult; D015415:Biomarkers / Q000097:blood; D002138:Calibration; D002648:Child; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004827:Epilepsy / Q000097:blood / Q000175:diagnosis; D005260:Female; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D007540:Isonipecotic Acids / Q000302:isolation & purification; D057230:Limit of Detection; D008297:Male; D009557:Nipecotic Acids / Q000302:isolation & purification; D018901:Peroxisomal Disorders / Q000097:blood / Q000175:diagnosis; D010875:Pipecolic Acids / Q000097:blood* / Q000302:isolation & purification; D012016:Reference Values; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*; D055815:Young Adult
chemical_list: D015415:Biomarkers; D007540:Isonipecotic Acids; D009557:Nipecotic Acids; D010875:Pipecolic Acids; C030278:nipecotic acid; C031345:pipecolic acid
keywords: Hepatic encephalopathy; Liquid chromatography–tandem mass spectrometry; Peroxisomal disorders; Pipecolic acid; Pyridoxine-dependent seizures