No abstract

An HPLC-MRM-MS method was developed for the quantification of 17 small ACE inhibiting (ACEI) peptides in plasma samples collected from human volunteers after the consumption of a peptide-enriched drink. The assay shows the high selectivity and sensitivity necessary to monitor small changes in the levels of the ACEI peptides after consumption of drinks developed to effect lowering of the blood pressure. Four different sample preparation methods were tested and evaluated. The final sample preparation method selected is simple and effective and consists mainly of the removal of proteins by acidification and heating, followed by a large volume injection. Additional sample preparation steps such as solid phase extraction and liquid/liquid partitioning were studied. Although they resulted in cleaner extracts, losses of specific peptides such as SAP were frequently seen. The isotope labeled form of one of the peptides to be quantified, [U(13)C]IPP, was used as an internal standard. The limit of detection of the assay is below 0.01 ng ml(-1). The limit of quantification is between 0.05 and 0.2 ng ml(-1), which is approximately 10% of the expected peptide concentration in plasma based on a normal diet. The intra- and inter-day relative standard deviations for all peptides have shown to be below 25% and the method has an accuracy of better than 75%. The long-term stability is good. At least 200 samples could be analysed before the system had to be cleaned. The assay has been successfully applied to blood samples collected from volunteers during a human trial.

A two-dimensional chromatographic method with mass spectrometric detection has been developed for identification of small, hydrophilic angiotensin I-inhibiting peptides in enzymatically hydrolysed milk proteins. The method involves the further separation of the poorly retained hydrophilic fraction from a standard C18 reversed-phase column on a hydrophilic interaction liquid chromatography (HILIC) column. The latter column is specifically designed for the separation of hydrophilic compounds. Narrow fractions collected from the HILIC column were analysed for their angiotensin I-converting enzyme (ACE) inhibiting potential in an at-line assay. Fractions showing significant inhibition of ACE were analysed by LC–MS for structure elucidation. With this method the main peptides responsible for ACE-inhibition in the hydrophilic part of a milk hydrolysate could be determined. The ACE-inhibiting peptides RP, AP, VK, EK, and EW explained more than 85% of ACE-inhibition by the hydrophilic fraction.

A fast at-line method was developed for the identification of ACE inhibiting (ACEI) peptides in protein hydrolysates. The method consists of activity measurements of fractions collected from a two-dimensional HPLC fractionation of the peptide mixture followed by MS identification of the peptides in the inhibiting fractions. The inhibition assay is based on the inhibiting effect of ACEI peptides on the hydrolytic scission of the substrate Hippuric acid-His-Leu (HHL) during the ACE-catalysed hydrolysis reaction. A fast LC method was developed for the quantification of Hippuric acid (H) and Hippuric acid-Histidine-Leucine (HHL), allowing a large number of fractions to be analysed within a reasonable time period. The method is sensitive and uses only standard laboratory equipment. The limit of detection is 0.34 M for the known ACEI peptide IPP. This is sufficiently sensitive for the identification of only moderately active peptides and/or ACEI peptides present at low concentrations. The relative standard deviation of the inhibition assay was 12% measured over a time period of 2 months. The IC50 value of IPP measured with the assay was 5.6 M, which is comparable to the values of 5 M and 5.15 M reported in literature for the standard Matsui method. The assay was successfully applied in the identification of ACEI peptides in enzymatically hydrolysed caseinate samples. Two new, not earlier published ACEI peptides were identified; MAP (␤-casein f102-104) and ITP (␣-s2-casein f119-121) with IC50 values of 3.8 M and 50 M, respectively.