High-performance liquid chromatography of N-terminal tryptophan-containing peptides with precolumn fluorescence derivatization with glyoxal.
A precolumn fluorescence derivatization method combined with high-performance liquid chromatography is described for the sensitive and selective determination of N-terminal tryptophan-containing peptides. The peptides and tryptophan were converted into fluorescent derivatives with glyoxal in a moderately acidic medium (pH 4.5). The derivatives were separated on a reversed-phase column with isocratic elution with an aqueous mobile phase composed of acetonitrile, methanol and phosphate buffer (pH 6.0), and subsequently detected by fluorimetry. The derivatization technique provided the respective N-terminal tryptophan-containing oligopeptides with single fluorescent peaks in chromatography. The detection limits for the peptides were 55-382 fmol per 100-microliters injection volume at a signal-to-noise ratio of 3. The method also allowed the facile detection of an N-terminal tryptophyl fragment in the enzyme reaction mixture of dynorphin A with trypsin.
Kai
M
M
Faculty of Pharmaceutical Sciences, Kyushu University 62, Fukuoka, Japan.
Kojima
E
E
Ohkura
Y
Y
Iwasaki
M
M
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005453:Fluorescence; D006037:Glyoxal; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D014364:Tryptophan / Q000032:analysis*
D010455:Peptides; D006037:Glyoxal; D014364:Tryptophan