Capillary gas chromatography of amino acids, including asparagine and glutamine: sensitive gas chromatogaphic—mass spectrometric and selected ion monitoring gas chromatographic—mass spectrometric detection of the N,O(S)- tert.-butyldimethylsilyl derivatives

Abstract

Amino acids and the amino amides glutamine and asparagine can be simulataneously derivatized to the corresponding N,O(S)- tert.-butyldimethylsilyl derivatives in a one-step reaction with N-methyl-N-( tert.-butyldimethylsilyl)trifluoroacetamide in acetonitrile. The solution is used directly for gas chromatography (GC). Losses due to evaporation steps are avoided. Except for the more basic amino acids, derivatization occurs at room temperature. Lysine, arginine and histidine require additional heating at 150δC for 2.5 h in order to complete derivatization. The derivatization has high reproducibility. The response factors relative to norvaline or cycloleucine lie between 0.40 and 1.30. Arginine is the most difficult amino acid to derivatize. The sizwe of the tert.-butyldimethylsilyl (TBDMS) groups prevents multiple silylation of the nitrogen atoms. Only a single peak is observed for each compound Twenty-seven amino acid (and glutamine and asparagine) derivatives were simultaneously chromatographed and well separated in a single run on a 25 m × 0.20 mm I.D. glass capillary column coated with OV-1. The TBDMS derivatives possess very characteristics. EI mass spectra at 70 eV, with intense diagnostic ions. This makes them very appropriate for GC—mass spectrometric (MS) work and selected ion monitoring GC—MS at the picomole level. The detection limit for arginine as the TBDMS derivative is less than 0.3 ng. The usefulness of the method is illustrated by the detection of amino acids in a peptide hydrolysate obtained from 1 μg of bovin insulin B-chain.