HPLC analysis of gonadotropin-releasing hormone and its analogues using benzoin as a fluorogenic reagent

Benzoin, which reacts specifically with arginine-containing peptides, was u sed as a pre-column fluorogenic reagent in the HPLC analysis of gonadotropi n-releasing hormone (GnRH) and its synthetic analogues in an attempt to imp rove their detection sensitivity. The reaction kinetics of benzoin with GnR H and its structurally-related analogues, which all contain an arginine res idue in the peptide chain, were investigated in this study. A fluorescence detector (ex 325 nm/em 435 nm) was used to detect benzoin-arginine derivati ves. Another set of excitation and emission wavelengths (289 nm/344 nm), wh ere fluorescent activity results from the aromatic amino acid residues, suc h as Tyrosine and Tryptophan, was used for comparison. The derivatization r eaction and the fluorescent derivative product were highly affected by pept ide concentration and heating time. The reactivity of arginine at position 8 was found to depend on the primary structure of GnRH and its analogues. H owever, it was observed that GnRH and its analogues were extremely unstable , as indicated by the labile nature of GnRH and its analogues under the der ivatization conditions used.