GC-MS analysis of nonprotein amino acids in Gymnocladus dioicus as N(O,S)-isobutyloxycarbonyl silyl derivatives.

Two different derivatives of novel nonprotein amino acids in Gymnocladus dioicus seeds are prepared for gas chromatographic (GC) and mass spectrometric (MS) analysis. The interfering components in a water extract of the seeds are removed by picric acid treatment and multistep washing with organic solvents followed by N(O,S)-isobutyloxycarbonylation combined with solid-phase extraction. The resulting N(O,S)-isobutyloxycarbonyl (isoBOC) amino acids are then converted to either tert-butyldimethylsilyl (TBDMS) or trimethylsilyl (TMS) derivatives, which are analyzed by dual capillary column GC and GC-MS. The characteristic ions in the mass spectra of N(O,S)-isoBOC TBDMS and N(O,S)-isoBOC TMS derivatives enable confirmation of molecular weight and existence of functional groups containing active protons. Temperature programmed retention index sets measured on DB-5 and DB-17 capillary columns (J&W Scientific; Folsom, CA) are useful for distinguishing among structural isomers. gamma-Methylglutamic acid, diastereoisomers of 3-hydroxy-4-methylglutamic acid, cis and trans isomers of 5-hydroxypipecolic acid, and some free protein amino acids are identified in Gymnocladus dioicus seeds.