Development of a fast liquid chromatography/mass spectrometry screening method for angiotensin-converting enzyme (ACE) inhibitors in complex natural mixtures like snake venom.

A new robust high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS)-based screening method for angiotensin-converting enzyme (ACE)-inhibiting substances in crude samples is described. The ACE assay is carried out in a typical offline setup by incubation of the samples with ACE and angiotensin I (AI), followed by stopping the reaction with acetonitrile containing val(5)-AI serving as internal standard (I.S.). AI and the product angiotensin II (AII) are extracted from the incubation mixture by turbulent-flow chromatography (TFC) applied in backflush mode as online solid-phase extraction and are directly quantified by ESI(+)-MS. The presence of ACE inhibitors (ACEi) is detected by an increase in AI signal intensity and a corresponding decrease of AII signal, as compared to the blank assay. The overall time of analysis of the TFC/ESI-MS method was 5 min, thus making the described setup suitable for a rapid screening method. The assay was validated using a known ACE inhibitor and the IC(50) values found were in good accordance with a common HPLC/UV method and literature data. The method was successfully applied for the screening of size-exclusion chromatography fractions of the venom of the pitviper Bothrops moojeni. Three of 18 analyzed fractions inhibited ACE, due to peptides present as components of this snake venom. These compounds were extracted from the two most-active fractions by means of TFC and isolated by means of HPLC. Three peptides with ACE inhibitory activity were characterized and their structures were elucidated with ESI-MS/MS-based de novo sequencing to be ZKWPPGKVPP, ZKWPRPGPEIPP and ZNWPRPGPEIPP, respectively (Z = pyroglutamic acid).