FEMTOMOLE DETECTION OF AMINO-ACIDS AND DIPEPTIDES BY GAS-CHROMATOGRAPHY NEGATIVE-ION CHEMICAL-IONIZATION MASS-SPECTROMETRY FOLLOWING ALKYLATION WITH PENTAFLUOROBENZYL BROMIDE

Amino acids and di- and tripeptides were derivatized by extractive alkylation using pentafluorobenzyl bromide (PFBBr) followed by reaction with heptafluorobutyric anhydride (HFBA) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). Good chromatographic separation and the formation of intense diagnostic ions were observed for the derivative when examined using gas chromatography-negative-ion chemical ionization mass spectrometry (GC-NICI-MS). Of the 20 amino acids investigated, only Arg and Glu could not be detected by this method. Also, dipeptides which included neutral amino acid residues were derivatized with more success than those containing either acidic or basic residues. Each of the amino acids or dipeptides formed one major derivative with the exception of Asn which formed two derivatives with either one or two HFB groups. This derivatization method was optimized with respect to the reaction temperature, reaction time, and choice of derivatizing reagents. Recoveries of derivatized [H-3]-labeled Phe, Lys, and Thr were 76, 55, and 34%, respectively. Linearity was observed from 10 to 2000 mu g of Ala per vial; selected-ion monitoring provided a detection limit of less than 150 fg with a signal-to-noise (SIN) ratio of 80 to 1. This method has proven to work well with urine samples and shows great promise for the detection of small peptides at low levels.