Sensitive determination of N-terminal prolyl peptides by high-performance liquid chromatography with laser-induced fluorescence detection

Short-chain Peptides with an N-terminal proline (Pro-Gly, Pro-Ile, Pro-Gly-Gly, Pro-Leu-Gly-NH2, and Pro-Thr-Pro-Ser-NH2, etc.) were determined by HPLC with laser-induced fluorescence (LIF) detection. The Peptides were quantitatively labelled with 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) at 50 degrees C after 1 h in a 0.1 M borax (pH 9.3)-acetonitrile mixture. The rate of reaction decreases inversely with the molecular weight of the Peptides. The mean value of fluorescent emission of the resulting DBD-peptides and DBD-peptide amides was 573 nm (excitation, 453 nm). The proline Peptides, including bioactive Peptides such as Pro-Leu-Gly-NH2 (release inhibitor of melanocyte-stimulating hormone), Pro-Thr-Pro-Ser-NH2 (IgA1 proteinase inhibitor) and Pro-Asp-Val-Asp-His-Val- Phe-Leu-Arg-Phe-NH2 [FMRF amide-like (Phe-Met-Arg-Phe-NH2) neuropeptide], were well separated by reversed-phase HPLC with water-acetonitrile containing 0.1% trifluoroacetic acid (TFA). The acetonitrile concentration in the mobile phase had a profound effect upon the retention times, and the capacity factors (k') were dependent on the hydrophobicity of the Peptides. The structure of DBD-Pro-Leu-Gly-NH2 was identified by LC-atmospheric pressure chemical ionization MS. The chromatographic detection limits (S/N = 2) of the Peptides with a 15-mW argon-ion laser at 488 nm were in the 6-28 fmol range. The detection limits were improved to 2-5 fmol with a microbore column. The detectability was two orders of magnitude higher than with a conventional fluorescence detector using xenon arc lamp.