title: Improved enantiomer resolution and quantification of free D-amino acids in serum and urine by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry.
abstract: The potential of comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) in the quantitative analysis of amino acid enantiomers (AAEs) as their methyl chloroformate (MCF) derivatives in physiological fluids was investigated. Of the two column sets tested, the combination of an Rt-γDEXsa chiral column with a polar ZB-AAA column provided superior selectivity. Twenty AAEs were baseline resolved including L-Leu and D-Ile, which had failed separation by one-dimensional chiral GC-quadrupole-MS (GC-qMS). Lower limits of quantification (LLOQ) were in the range of 0.03-2 μM. Reproducibility of the analysis of a serum specimen in octaplicate ranged from 1.3 to 16.6%. The GC×GC-TOFMS method was validated by analyzing AAEs in 48 urine and 43 serum specimens, respectively, and by comparing the results with data obtained by a previously validated GC-qMS method. Mean recoveries ranged from 78.4% for D-Leu to 116.4% for D-Pro in urine and 72.2% for L-Thr to 129.4% for L-Ile in serum. The method was applied to the comparison of AAE serum levels in patients suffering from liver cirrhosis to a control group. Significantly increased D-AA concentrations were found for the patient group, whereas L-AA levels were slightly decreased.
authors.affiliation: Institute of Functional Genomics, University of Regensburg, Regensburg, Germany. magdalena.waldhier@klinik.uni-regensburg.de
authors.forename: Magdalena C
authors.initials: MC
authors.lastname: Waldhier
authors.forename: Martin F
authors.initials: MF
authors.lastname: Almstetter
authors.forename: Nadine
authors.initials: N
authors.lastname: Nürnberger
authors.forename: Michael A
authors.initials: MA
authors.lastname: Gruber
authors.forename: Katja
authors.initials: K
authors.lastname: Dettmer
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Oefner
mesh_terms: D000596:Amino Acids / Q000097:blood* / Q000737:chemistry / Q000652:urine*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D016014:Linear Models; D008103:Liver Cirrhosis / Q000378:metabolism; D055442:Metabolome; D055432:Metabolomics / Q000379:methods*; D015203:Reproducibility of Results; D013237:Stereoisomerism
chemical_list: D000596:Amino Acids