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Improved method for direct high-performance liquid chromatography assay of angiotensin-converting enzyme-catalyzed
Jianping Wu1, Rotimi E Aluko, Alister D Muir
1Agriculture and Agri-Food Canada, Saskatoon Research Station, SK, Canada.
Abstract:
A rapid and sensitive assay was developed for determination of the activity of angiotensin-converting enzyme (ACE) in the presence of inhibitory peptides present in soybean protein hydrolysates. The method utilizes reversed-phase high-performance liquid chromatography (HPLC) to separate and quantify hippuryl-histidyl-leucine (HHL) and hippuric acid (HA). HHL and HA were separated on a Symmetry C18 column by gradient elution that used mixtures of trifluoroacetic acid TFA)-acetonitrile and TFA-water as solvents. Analytical time and baseline separation of HA from HHL were improved over previous HPLC methods. In comparison to the standard spectrophotometric method, the new HPLC method obviates the need for ethyl acetate extraction of HA but requires direct injection of the ACE reaction mixture onto the HPLC column.