Determination of l-Pipecolic Acid in Plasma Using Chiral Liquid Chromatography-Electrospray Tandem Mass Spectrometry

Abstract

Background: l-Pipecolic acid (L-PA), an important biochemical marker for the diagnosis of peroxisomal disorders, is usually determined as the racemate. We developed a chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of L-PA in plasma. Methods: We used a narrow bore chiral macrocyclic glycopeptide teicoplanin column for the enantioseparation of d-pipecolic acid (D-PA) and L-PA and interfaced the column directly to the electrospray source of a tandem mass spectrometer. We used phenylalanine-d5 as internal standard added to 50 μL of plasma followed by deproteinization, evaporation, and injection. The analysis was performed in the selected-reaction monitoring mode using two transitions: m/z 130→m/z 84 for PA, and m/z 171→m/z 125 for phenylalanine-d5. L-PA eluted at 7 min, and D-PA eluted at 11.7 min, whereas phenylalanine-d5 eluted at 6 min. The turnaround time for the assay was 20 min. Results: Linear calibration curves were obtained in the range of 0.5–80 μmol/L. At a plasma concentration of 1.0 μmol/L, the signal-to-noise ratio was 50:1. The intra- and interassay variations were 3.1–7.9% and 5.7–13%, respectively, at concentrations of 1–50 μmol/L. Mean recoveries of L-PA added to plasma were 95% (5 μmol/L) and 102% (50 μmol/L). The method clearly distinguished between healthy individuals and peroxisomal disease patients. Conclusions: The novel LC-MS/MS method is simple, rapid, and stereoselective, and uses only 50 μL of plasma, no derivatizing reagents, and a commercially available internal standard. Sample preparation is not complex and is faster than for all other methods.

Evaluation of C26:0‐lysophosphatidylcholine and C26:0‐carnitine as diagnostic markers for Zellweger spectrum disorders

“…For some patients, additional data on peroxisomal parameters in blood was available. Plasma VLCFA, phytanic acid, pristanic acid (Vreken et al 1998), plasma bile acids (Bootsma et al 1999), pipecolic acid (Rashed et al 2001), and plasmalogens in erythrocytes (Dacremont and Vincent 1995) were measured in the Laboratory Genetic Metabolic Diseases in the AMC as part of standard diagnostic procedures or standard patient care.…”

Laboratory diagnosis of disorders of peroxisomal biogenesis and function: a technical standard of the American College of Medical Genetics and Genomics (ACMG)

“…Amino acid analysis performed with traditional methods, like ion exchange chromatography, can detect L-pipecolic acid when present in large amounts; 106 but several more sensitive methods have been developed using mass spectrometry combined with gas or liquid chromatography. 74,76,77,[107][108][109] Pipecolic acid can be measured by GC-MS in negative 74,77 or positive 107 mode following derivatization of the amine group with methyl chloroformate, acidic ethyl acetate extraction, and further derivatization with pentafluorobenzyl bromide. Some LC-MS/MS methods do not require sample derivatization, thus requiring smaller sample volumes and less processing time.…”

“…Some LC-MS/MS methods do not require sample derivatization, thus requiring smaller sample volumes and less processing time. 108,109 A deuterated stable isotope, like 2 PAd 11 or 2 PA-d 9 , is utilized as internal standard. Quantitation can be performed by stable isotope dilution or using a calibration curve.…”

Significance of the Natural Occurrence of L‐ Versus D‐Pipecolic Acid: A Review

“…was performed using capillary zone electrophoresis, thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), chiral liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS), and gas chromatography-mass spectrometry (GC-MS). 17,23,[33][34][35][36] Also, mono-and bienzyme-based amperometric biosensors using physical immobilization of L-amino acid oxidase and/or horseradish peroxidase or D-amino acid oxidase and horseradish peroxidase for assay of L-pipecolic and D-pipecolic acid, respectively, 37 or enantioselective potentiometric membrane electrode based on macrocyclic glycopeptide antibiotic vancomycin 38 were used. All of the above-mentioned methods are simple, rapid, and stereoselective and require only a small sample volume.…”

“…L-pipecolic acid was isolated from a hot-water extract of S. aspratus with a 0.02% yield, 109 which is an order of magnitude higher than the maximum concentration of L-pipecolic acid reported in plasma of patients with peroxisomal abnormalities. 36 Metabolism of the L-versus D-enantiomers of pipecolic acid differs between organs and animal species. L-pipecolic acid oxidation is the highest in kidney cortex and lower in liver, heart, and brain of rabbit and monkey.…”