Determination of L-pipecolic acid in plasma using chiral liquid chromatography-electrospray tandem mass spectrometry.
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 microL 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 micromol/L. At a plasma concentration of 1.0 micromol/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 micromol/L. Mean recoveries of L-PA added to plasma were 95% (5 micromol/L) and 102% (50 micromol/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 microL of plasma, no derivatizing reagents, and a commercially available internal standard. Sample preparation is not complex and is faster than for all other methods.
Rashed
M S
MS
Metabolic Screening Laboratory, King Faisal Specialist Hospital and Research Centre, MBC-03, PO Box 3354, Riyadh 11211, Saudi Arabia.
Al-Ahaidib
L Y
LY
Aboul-Enein
H Y
HY
Al-Amoudi
M
M
Jacob
M
M
D015415:Biomarkers / Q000097:blood; D002853:Chromatography, Liquid; D006801:Humans; D013058:Mass Spectrometry; D010875:Pipecolic Acids / Q000097:blood*; D015203:Reproducibility of Results; D013237:Stereoisomerism
D015415:Biomarkers; D010875:Pipecolic Acids; C031345:pipecolic acid