Simultaneous determination of Angiotensin I-converting enzyme inhibitory peptides in tryptic casein hydrolysate by high-performance liquid chromatography combined with a replicate heart-cut column-switching technique.

A replicate heart-cut column-switching HPLC method combined with two switching valves was newly developed for the simultaneous determination of three antihypertensive peptides (Ala-Phe, Tyr-Pro, and Trp-Tyr) in tryptic casein hydrolysate in one run-in assay. After a first separation on an octadecyl silane (ODS) column, heart-cuts of each peptide were individually separated on a subsequent analytical ODS column: 26% acetonitrile for Ala-Phe and Tyr-Pro (32% for Trp-Tyr) in 0.1% trifluoroacetic acid containing 10 mM sodium 1-octanesulfonate at 0.8 mL/min. Ala-Phe, Tyr-Pro, and Trp-Tyr in casein hydrolysate were determined within 70 min to be 0.377 +/- 0.037 mg/g, 2.50 +/- 0.26 mg/g, and 0.096 +/- 0.008 mg/g, respectively.