Identification and Quantification of Free Amino Acids Using Gas Chromatography Coupled with Vacuum Ultraviolet Absorption Spectroscopy

Abstract

Amino acids serve a variety of important biological functions so their identification and quantification is integral to a number of different fields including the diagnosis of inborn errors of metabolism, food science, metabolomics and biomedical research. There are several existing methodologies for their identification and quantification; however, there remains a need for improvement in areas such as speed of analysis, robustness, and reproducibility. GC‐VUV, a novel technology, provides a new 2D‐detection technique in a field that has been dominated by mass spectrometry for decades. The GC‐VUV detector is unique in its ability to probe molecular electronic transitions. Virtually all molecules absorb in the 125–240 nm energy range and thus provide unique absorbance spectra that can be used for identification. The distinctiveness of the spectra enable closely related structural isomers and isobaric compounds to be readily distinguished. Subsequent quantitative analysis follows well established Beer‐Lambert Law principles allowing for simple, predictable and accurate quantitation via spectral deconvolution without the addition of internal standards. The objective of this work was to develop a method for the detection and quantification of free amino acids as their chloroformate derivatives using gas chromatography (GC) coupled with vacuum ultraviolet (VUV) absorption spectroscopy. In a procedure requiring less than 5 minutes, stock concentrations of individual amino acids were derivatized using isobutanol and isobutylchloroformate, with pyridine as a catalyst. The derivatized amino acids were then extracted into chloroform and injected into the GC. Initially, the derivatized amino acids were run individually so their resultant spectra could be ‘banked’ in the VUV spectral library. Subsequently, the individual amino acids were pooled and the resulting amino acid mixture was derivatized and injected into the GC. All 19 of the amino acids in the mixture were identified and quantified, including the isomers leucine and isoleucine and the nearly isobaric molecules lysine and glutamine. The simplicity, speed, robustness and reproducibility of this approach has the potential to overcome current methodological limitations and, thus, to provide a novel analytic method for use in amino acid applications.