Rapid detection and identification of angiotensin-converting enzyme inhibitors by on-line liquid chromatography–biochemical detection, coupled to electrospray mass spectrometry

An analytical method based on on-line liquid chromatography–biochemical detection (LC–BCD) coupled to electrospray mass spectrometry was developed for the detection and identification of angiotensin-converting enzyme (ACE) inhibitors in complex mixtures, such as hydrolyzed whey proteins. ACE inhibitory activity was detected by coupling a homogeneous, substrate conversion based bioassay on-line to high-performance liquid chromatography (HPLC). Chemical information was obtained by directing part of the HPLC effluent towards a mass spectrometer. After correlating the biochemical and chemical data, the accurate molecular masses of the bioactive peptides were used as search queries in protein databases. Combined with the recorded mass spectrometry (MS)–MS fingerprints, bioactive peptides were selected from the database search results. The results of LC–BCD–MS analyses were verified by establishing a bioactivity balance. Reference samples, containing several peptides at concentration levels similar to those observed in the hydrolyzed milk samples, were analyzed by LC–BCD–MS. High recoveries of biological activity were obtained, indicating that the correct ACE inhibitors were identified and that no co-elution of significantly bioactive molecules had occurred. Approximately, 30 ACE inhibitors were detected and identified. IC50 values of ACE inhibitors, reported in literature, ranged between 43 and 580 μM.