Single-Laboratory Validation for the Determination of Cocoa Flavanols and Procyanidins (by Degree of Polymerization DP1-7) in Cocoa-Based Products by Hydrophilic Interaction Chromatography Coupled with Fluorescence Detection: First Action 2020.05.

BACKGROUND: Flavanols and procyanidins are complex bioactives found in many foods such as cocoa. As their consumption is associated with health benefits, cocoa flavanols and procyanidins are receiving increasing attention from consumers, industry, researchers, and regulators. OBJECTIVE: The objective of this study is to validate a method using hydrophilic interaction chromatography (HILIC) with fluorescence detection (FLD) and a commercially available reference material for the determination of flavanols and procyanidins (CF) in cocoa-based products. METHODS: Method performances were evaluated for cocoa matrices with CF content that ranged from 0.8 to 500 mg/g, which included low CF matrices (milk and dark chocolate, cocoa powder, and liquor) and high CF matrices (cocoa extract and dietary supplement products). The method was validated in a single-laboratory by determining sensitivity, selectivity, linearity, stability, robustness, accuracy, and precision for each of the matrices. RESULTS: The method was validated for cocoa matrices with CF content that ranged from 0.8 to 500 mg/g. Accuracy ranged from 86 to 99% and repeatability (RSDr) from 1.5 to 8.6% for CF. CONCLUSIONS: Analytical performances acquired through this single-laboratory validation study for a wide range of cocoa-based matrices demonstrate that this method is fit-for-purpose for the determination of flavanols and procyanidins in cocoa-based products. HIGHLIGHTS: Hydrophilic interaction chromatography (HILIC) with fluorescence detection was successfully used to determine total CF content in multiple product types. Single-laboratory method validation results demonstrate that the method is fit for purpose for cocoa-based matrices containing <0.8 to 500 mg/g of CF.

Reliable, accessible and transferable method for the quantification of flavanols and procyanidins in foodstuffs and dietary supplements.

Flavanols and procyanidins are plant-derived bioactives that are receiving increasing attention because of their potential health benefits. Analytical tools that can accurately identify and reproducibly quantify these bioactives are critical to researchers for test material characterization, as well as to the food industry and regulators, notably for product labeling. However, the chemical complexity of procyanidins, and the absence of analytical standards have prevented the development of methods that could serve the needs of these different sectors. This report describes the development and validation of a reliable, accessible and transferable method for the quantification of flavanol monomers and procyanidins in cocoa-derived dietary supplements and foodstuffs. To accomplish this, flavanols and procyandins from cocoa, one of the most studied dietary sources of these compounds, were used as a model system. To overcome limitations related to the absence of analytical standards, a cocoa extract was thoroughly characterized for use as a calibrant. It was then used in the development and validation of a method based on reliable and accessible instrumentation, namely HPLC coupled with fluorescence detection. The resulting method permitted the quantification of flavanols and procyanidins in amounts ranging from 2 to 500 mg g-1, with high precision (%RSD 0.2 to 1.9%) and accuracy (100.7 to 102.9%). The method was successfully applied to assess the flavanol and procyanidin content of different cocoa-based commercial products. Furthermore, the high precision of the methods showed the feasibility of using principal component analysis of flavanol and procyanidin profiles to discriminate cocoa-derived products by origin and manufacturing processes. A feature that offers advantages in monitoring product authenticity/adulteration. Overall, these findings support the application of this method for the routine analysis of cocoa flavanols and procyandins.

A Fluorescence Quenching Analysis of the Binding of Fluoroquinolones to Humic Acid.

Fluorescence quenching was used to investigate the interaction of six fluoroquinolones with humic acid. Static quenching was observed for the binding of ciprofloxacin, enoxacin, fleroxacin, levofloxacin, norfloxacin, and ofloxacin to humic acid. The equilibrium binding constants were found from Stern-Volmer plots of the data. The quenching experiments were repeated over a temperature range of 25-45 ℃ and van't Hoff plots were generated. From these linear plots, thermodynamic values were calculated for Δ H, Δ G, and Δ S for each of the fluoroquinolones. The equilibrium binding constants were found to be <1 for all the antibiotics studied. The calculated ΔH values were all negative and ranged from -9.5 to -27.6 kJ/mol. The high water solubility of the antibiotics and low ΔH of binding suggests that the antibiotics will be transported easily through the environment. Finally, whether the fluoroquinolones are in a protonated, deprotonated, or partially protonated state is found to correlate to the strength of binding to humic acid.

Silanediol inhibitors of angiotensin-converting enzyme. Synthesis and evaluation of four diastereomers of Phe[Si]Ala dipeptide analogues.

Four stereoisomers of a Phe-Ala silanediol dipeptide mimic have been evaluated as inhibitors of angiotensin-converting enzyme (ACE) and compared to ketone-based inhibitors reported by Almquist et al. One stereogenic center of the isomers was derived from the individual enantiomers of methyl 3-hydroxy-2-methylpropionate, with separation of diastereomers after introduction of the second stereogenic center. The diastereomeric identities were established by X-ray crystallography of an intermediate. Inhibition of ACE by three of the silanediol diastereomers (IC(50) = 3.8-207 nM) closely paralleled that of the corresponding diastereomeric ketones (IC(50) = 1.0-46 nM). The fourth diastereomer, corresponding to the least inhibitory ketone (IC(50) = 3200 nM), exhibited an unexpected level of inhibition in the silanediol (IC(50) = 72 nM), suggesting an alternative mode of binding to the enzyme.