E. Struys, C. Jakobs
Jun 1, 1999
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0
Influential Citations
9
Citations
Journal
Journal of Inherited Metabolic Disease
Abstract
Pipecolic acid is an important marker for peroxisomal disorders next to bile acids, very long-chain fatty acids, phytanic and pristanic acid. The two chiral forms of pipecolic acid have di†erent origins : D-pipecolic acid derives both from intestinal bacteria and diet, whereas L-pipecolic acid is an endogenous intermediate of the L-lysine pathway. We here present an enantiomeric analysis of Dand L-pipecolic acid using a chiral capillary gas chromatography column and mass fragmentography, wherein derivatization and sample preparation are based on earlier published technology (Kok et al 1987 ; Zee et al 1992). To 100 kl of plasma, 1 nmol D/L acid was added as internal stan[2H11]pipecolic dard followed by addition of 500 kl phosphate bu†er (1 mol/L, pH 11) and 50 kl methylchloroformate to form N-methylcarbamate derivatives. After extraction with ethyl acetate, the extract was dried and pentaÑuorobenzyl (PFB) derivatives were formed. From the Ðnal hexane extract, 1 kl was used for gas chromatography with electron capture detection (GC-ECD) and a second 1kl was used for gas chromatographyÈmass spectrometry (GC-MS) analysis. Enantiomeric separation was performed on a CP Chiralsil-Dex CB (25 m] 0.25 mm, Ðlm thickness 0.25km) analytical column (Chrompack, Middelburg, The Netherlands). The oven temperature was programmed from 100¡C (1 min), with a ramp of 1¡C, to 160¡C, followed by a fast ramp to 240¡C. Mass fragmentography was performed in the negative chemical ionization (NCI) mode with ammonia as reagent gas. The masses measured were m/z 186 (endogenous pipecolic acid) and m/z 195 ([2H9]pipecolic acid). Individual enantiomerically pure standards were used to determine the elution order of Dand L-pipecolic acid. The separation was optimized by testing di†erent temperature ramps and carrier gas Ñows. The best analytical performance was achieved using a temperature ramp of 1.0¡C/min with a carrier gas Ñow of 3 ml/min. In control and Zellweger plasma samples only the L-isomer of pipecolic acid was observed, which is in agreement with results published by others (Armstrong et al 1993). The method was also applied to a plasma sample of a patient with previously detected elevations of total pipecolic acid and phytanic acid but with normal bile acids and very long-chain fatty acids (Baumgartner et al 1998). Using our procedure we could unambiguously conclude that only the L-isomer of pipecolic acid was present in this plasma sample. The stability of the N-methylcarbamate-PFB deriv-