POST-CHROMATOGRAPHIC DETECTION OF OLIGOPEPTIDES USING TERBIUM-SENSITIZED LUMINESCENCE

Abstract

This report investigated the potential use of oligopeptide complexation with terbium III as a high-performance liquid chromatography method with time-resolved spectrofluorimetric detection for analysis of milk-derived products. The study focused on glycyl-leucyl-phenylalanine, a compound with immunostimulatory properties isolated from milk casein, and also considered tyrosine and tryptophan residues in peptides. As the water content of the mobile phase was highly restrictive for detection, acetate ions were introduced as a potentiation agent. Only post-chromatographic complexation proved feasible, because passage through a column destroyed oligopeptide-terbium chelation. Validation of glycyl-leucylphenylalanine, glycyl-phenylalanine, and leucyl-phenylalanine assays showed linearity in the 8 × 10⁻⁵ to 5 × 10⁻⁴mol l⁻¹ range (correlation coefficients of 0.96, 0.99 and 0.99 respectively). Results for the assay at 10⁻⁵ mol l⁻¹ showed a 7% intra-day (repeatability) coefficient of variation (CV) and a 15% inter-day (reproducibility) CV. No interferences were observed in the presence of various water-soluble vitamins present in whey. This method, when tested in the bromatology field on yoghurts with different milk origins, showed that goat wheys had phenylalanine, tyrosine, and tryptophan residues twice those of cow wheys and three times those of sheep or soja bean wheys.

Keywords:

• High-performance liquid chromatography
• Luminescence
• Oligopeptide
• Terbium
• Complexes

ACKNOWLEDGMENTS

The authors are grateful to Mrs. Pierre-Eugène for technical assistance.