Diastereoisomeric determination of R‐alanine in bacteria using capillary gas chromatography and positive/negative ion mass spectrometry

The possibilities of using combined capillary gas chromatography mass spectrometry for highly sensitive and selective determinations of enantiomeric alanines of bacterial origin have been evaluated. The alanines were separated as their diastereoisomeric N‐heptafluorobutyryl‐2‐butyl ester derivatives on a 25 m fused silica capillary column coated with SE‐54 as stationary phase. The mass spectra of the derivative obtained by chemical ionization using methane or ammonia (positive ions) and methane or Isobutane (negative ions) as reagent gases have been recorded. The highest sensitivity of detection was achieved by selected ion monitoring in the negative ion mode (detection limit about 0.8 pg). The method was tested by measuring the R‐alanine content in (a) Escherichia coli cultures and (b) natural bacterial communities associated with plant roots. In (a) the recorded detection limit implied possibilities to detect R‐alanine corresponding to 10³−10⁴ E. coli cells. The precision for quantifying R‐alanine in the cultures was 14% (coefficient of variation). (b) demonstrated an application where the R/(R + S) ratio of alanine is low. It was shown that accurate determinations of this ratio in the picogram range could be effected down to 0.4%.