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

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.