Definition of system suitability and proficiency testing acceptance criteria for the standardization of quality control methodologies of botanical bioactives: A case study for the development of AOAC Official Method of Analysis 2020.05 flavanols and procyanidins.

Flavanols and procyanidins are bioactives found in many foods including cocoa. The characterization of cocoa flavanols and procyanidins (CF) has historically been challenged by the lack of commercially available standards and weak chromatographic separation performances. The recent release of a reference material and the final authorization of an AOAC Official Method of Analysis (AOAC 2020.05) technically enables the standardization of CF testing. However, the practical implementation of CF testing for routine testing remains challenging for new laboratories, highlighting the need to define guidelines and acceptance criteria to verify normal method performance and user proficiency. This study leveraged the data generated from the recent multi-laboratory validation to define normal method performances. While the challenges associated with HILIC separation can be alleviated through a thorough system equilibration, monitoring system performances remains critical to routine operation and a laboratory's ability to generate reliable data. Guidelines for routine analysis were developed for system precision and bracketing standard recovery, as well as acceptance criteria for the analysis of the reference material. These guidelines not only complete a body of work that provide accessible, reliable, and robust CF analysis solution to research and quality laboratories, but also provide an example to facilitate the establishment and implementation of future international testing standards for botanical bioactives.

Impact of polyphenol oxidase on the bioavailability of flavan-3-ols in fruit smoothies: a controlled, single blinded, cross-over study.

Flavan-3-ols are bioactive compounds found in a variety of fruits and vegetables (F&V) that have been linked to positive health benefits. Increasing habitual flavan-3-ol intake is challenged by the generally low consumption of F&V. While smoothies are a commonly endorsed, consumer-accepted means to increase the daily intake of these important foods, fruits used for smoothie preparation can have a high polyphenol oxidase (PPO) activity and thus potentially affect the content and bioavailability of flavan-3-ols. To assess whether or not consuming freshly prepared smoothies made with different PPO-containing fruit impacts the bioavailability of the flavan-3-ols, a controlled, single blinded and cross-over study was conducted in healthy men (n = 8) who consumed a flavan-3-ol-containing banana-based smoothie (high-PPO drink), a flavan-3-ol-containing mixed berry smoothie (low-PPO drink) and flavan-3-ols in a capsule format (control). The peak plasma concentration (Cmax) of flavan-3-ol metabolites after capsule intake was 680 ± 78 nmol L-1, which was similar to the levels detected after the intake of the low PPO drink. In contrast, the intake of the high PPO drink resulted in a Cmax of 96 ± 47 nmol L-1, 84% lower than that obtained after capsule intake. In a subsequent study (n = 11), flavan-3-ols were co-ingested with a high-PPO banana drink but contact prior to intake was prevented. In this context, plasma flavan-3-ol levels were still reduced, suggesting an effect possibly related to post-ingestion PPO activity degrading flavan-3-ols in the stomach. There was a substantial range in the PPO activity detected in 18 different fruits, vegetables and plant-derived dietary products. In conclusion, bioavailability of flavan-3-ols, and most likely other dietary polyphenol bioactives, can be reduced substantially by the co-ingestion of high PPO-containing products, the implications of which are of importance for dietary advice and food preparation both at home and in industrial settings.

Cocoa Flavanol Supplementation and the Effect on Insulin Resistance in Females Who Are Overweight or Obese: A Randomized, Placebo-Controlled Trial.

There is interest in the impact that dietary interventions can have on preventing the transition from insulin resistance to type 2 diabetes, including a suggestion that the bioactive components of cocoa may enhance fasting insulin sensitivity. However, a role for cocoa flavanols (CF) in reducing insulin resistance in the insulin-stimulated state, an important risk factor for cardiovascular disease, is unresolved. This study investigated whether CF consumption improved whole-body insulin-mediated glucose uptake ('M') in females with overweight/obesity, using a randomized, double-blinded, placebo-controlled, parallel-group design. Thirty-two premenopausal females (19-49 years; 27-35 kg·m-2) with elevated HOMA-IR (HOMA-IR >1.5) supplemented their habitual diet with two servings/day of a high-flavanol cocoa drink (HFC; 609 mg CF/serving; n = 16) or low-flavanol cocoa drink (LFC; 13 mg CF/serving; n = 16) for 4 weeks. Assessment of HOMA-IR and 'M' during a 3-h, 60 mIU insulin·m-2·min-1 euglycemic clamp was performed before and after the intervention. Data are the mean (SD). Changes to HOMA-IR (HFC -0.003 (0.57); LFC -0.0402 (0.86)) and 'M' (HFC 0.99 (7.62); LFC -1.32 (4.88) µmol·kg-1·min-1) after the intervention were not different between groups. Four weeks' consumption of ~1.2 g CF/day did not improve indices of fasting insulin sensitivity or insulin-mediated glucose uptake. A recommendation for dietary supplementation with cocoa flavanols to improve glycemic control is therefore not established.

Determination of Cocoa Flavanols and Procyanidins (by Degree of Polymerization DP1-7) in Cocoa-Based Products by Hydrophilic Interaction Chromatography Coupled With Fluorescence Detection: Collaborative Study.

BACKGROUND: Cocoa flavanols and procyanidins (CF) are flavonoids whose consumption is associated with health benefits, resulting in increasing attention from consumers, industry, researchers, and regulators. Methods that can provide appropriate characterization and quantification of the distinct mixture found in cocoa-based products thus offer important scientific and commercial value. OBJECTIVE: This study validated the precision of AOAC Official Method of AnalysisSM2020.05, which measures CF with a degree of polymerization DP1-7. METHOD: Method precision (repeatability and reproducibility) was evaluated for seven cocoa matrixes in blind duplicates with total CF content from 1.0 to 500 mg/g. Ten of the 12 laboratories from multiple sectors invited to implement the method returned data for statistical analysis. Precision was evaluated per AOAC INTERNATIONAL guidelines for collaborative studies using RSDr and RSDR as indicators of method repeatability and reproducibility. RESULTS: RSDr ranged from 1.6 to 4.8%, and RSDR ranged from 5.8 to 22.4%, demonstrating excellent within-laboratory repeatability and good method precision across different laboratories. RSDR values were below 10% with the exception of chocolate, potentially due to very low CF content and sampling inhomogeneity. CONCLUSIONS: These data demonstrate that acceptable method repeatability and reproducibility is achieved when measuring cocoa flavanols and procyanidins using AOAC Method 2020.05 and support the advancement of the AOAC Official Method of Analysis status to Final Action for evaluated matrixes. HIGHLIGHTS: This collaborative study evaluated the repeatability and reproducibility of AOAC Official Method of Analysis 2020.05.

Development and validation of HPLC-MS2 methodology for the accurate determination of C4-C8 B-type flavanols and procyanidins.

Cocoa flavanols and procyanidins (CFs), natural dietary bioactives, have been studied extensively over the past two decades for their potential health benefits. Research on their safety and efficacy is critically dependent upon on the ability to reliably characeterize the research materials that are utilized, and with growing consumer availability of CF-based products, reliable methods for the detection of potential adulteration are of increasing importance. This research focused on the development of a high performance liquid chromatography-tandem mass spectrometry method (HPLC-MS2) using primary standards and 13C-labelled procyanidins as internal standards. The ability of MS2 detection to discriminate A- and B-type procyanidins was demonstrated. Method performances were validated for degrees of polymerization up to four in seven model food matrices. Accuracy ranged from 90.9 to 125.4% and precision was < 10% at lower concentrations. Finally, the method was applied to cocoa-based samples and compared to the AOAC 2020.05 analytical protocol, supporting the use of NIST 8403 as reference material for HPLC-MS2 analysis.

Evolution of cocoa flavanol analytics: impact on reporting and cross-study comparison.

Cocoa flavanols (CF) are a group of dietary bioactives that have been studied for their potential health benefits for over two decades. In this time, multiple methods for CF testing have evolved, introducing the potential for differences in reported CF content. The reliable characterization of CF content in food and test materials used in clinical studies is critical to comparisons of research studies over time, as well as critical to enabling the systematic reviews and meta-analyses required to support dietary recommendations of bioactives. In this work, we compared two analytical methods that have been widely applied to characterize materials used in clinical research and a method newly recognized by AOAC as the official method for CF analysis. Differences in accuracy of -36% to +20% were observed when comparing CF contents determined with these methods, supporting the notion that CF values determined across methods are not directly comparable. To address differences, a linear regression model was developed to predict CF values. This approach was cross-validated and directly applied to the conversion of CF values published in key scientific papers on the benefits of CF. This work provides a valid tool to compare CF values reported across these different methods and enables comparisons and interpretation of studies investigating the bioactivity of CF.

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.

Safety and efficacy of cocoa flavanol intake in healthy adults: a randomized, controlled, double-masked trial.

BACKGROUND: Evidence from dietary intervention studies shows that the intake of flavanols and procyanidins can be beneficial for cardiovascular health. Nevertheless, there is a clear need for advancing our understanding with regard to safe amounts of intake for these bioactives. OBJECTIVE: The aim was to investigate in healthy adults the effects of cocoa flavanol (CF) intake amount and intake duration on blood pressure, platelet function, metabolic variables, and potential adverse events (AEs). DESIGN: This investigation consisted of 2 parts. Part 1 was an open-label, intake-amount escalation study, in which 34 healthy adults (aged 35-55 y) consumed escalating amounts of CFs, ranging from 1000 to 2000 mg/d over 6 wk. Primary outcomes were blood pressure and platelet function, select metabolic variables, and the occurrence and severity of AEs. Secondary outcomes included plasma concentrations of CF-derived metabolites and methylxanthines. On the basis of the outcomes of study part 1, and assessing the same outcome measures, part 2 of this investigation was a controlled, randomized, double-masked, 2-parallel-arm dietary intervention study in which healthy participants (aged 35-55 y) were asked to consume for 12 consecutive weeks up to 2000 mg CFs/d (n = 46) or a CF-free control (n = 28). RESULTS: Daily intake of up to 2000 mg CFs/d for 12 wk was not associated with significant changes in blood pressure or platelet function compared with CF-free controls in normotensive, healthy individuals who exhibited a very low risk of cardiovascular disease. There were no clinically relevant changes in the metabolic variables assessed in either of the groups. AEs reported were classified as mild in severity and did not significantly differ between study arms. CONCLUSION: The consumption of CFs in amounts up to 2000 mg/d for 12 wk was well tolerated in healthy men and women. This trial was registered at clinicaltrials.gov as NCT02447770 (part 1) and NCT02447783 (part 2).

Recommendations on reporting requirements for flavonoids in research.

Numerous observational and intervention-based human studies support the notion of a beneficial role for dietary flavonoids in human health. Despite these studies, it is not yet possible to make dietary recommendations with regard to the types and amounts of flavonoids to be consumed. The inherent diversity of flavonoid structure, chemistry, and natural distribution in foods lends itself to errors in reporting the types and/or amounts of flavonoids consumed, as well as incomplete recognition of requirements for intervention studies that aim to assess their benefits in a clinical setting. A need exists for guidelines that facilitate the design and reporting of flavonoid research. With a focus on clinical studies, this article 1) outlines limitations commonly encountered in the field of flavonoid research, including the inconsistent use of nomenclature, inappropriate analytic methods, inconsistent use of existing flavonoid databases, and the lack of full consideration in the design of test materials for intervention trials, and 2) provides guidance for future studies with a focus on clinical intervention trials. Adoption of this guidance will facilitate more accurate and interpretable research that will support the development of dietary recommendations regarding the intake of flavonoids.

Cocoa flavanol consumption improves cognitive function, blood pressure control, and metabolic profile in elderly subjects: the Cocoa, Cognition, and Aging (CoCoA) Study--a randomized controlled trial.

BACKGROUND: Recent evidence has indicated that flavanol consumption may have many health benefits in humans, including improved cognitive activities. OBJECTIVE: The aim was to evaluate the effect of flavanol consumption on cognitive performance in cognitively intact elderly subjects. DESIGN: This was a double-blind, controlled, parallel-arm study conducted in 90 elderly individuals without clinical evidence of cognitive dysfunction who were randomly assigned to consume daily for 8 wk a drink containing 993 mg [high flavanol (HF)], 520 mg [intermediate flavanol (IF)], or 48 mg [low flavanol (LF)] cocoa flavanols (CFs). Cognitive function was assessed at baseline and after 8 wk by using the Mini-Mental State Examination (MMSE), the Trail Making Test (TMT) A and B, and the Verbal Fluency Test (VFT). RESULTS: The changes in MMSE score in response to the 3 different treatments were not different. In contrast, there was a positive impact of the intervention on specific aspects of cognitive function. Mean changes (±SEs) in the time required to complete the TMT A and B after consumption of the HF (-8.6 ± 0.4 and -16.5 ± 0.8 s, respectively) and IF (-6.7 ± 0.5 and -14.2 ± 0.5 s, respectively) drinks significantly (P < 0.0001) differed from that after consumption of the LF drinks (-0.8 ± 1.6 and -1.1 ± 0.7 s, respectively). Similarly, VFT scores significantly improved among all treatment groups, but the magnitude of improvement in the VFT score was significantly (P < 0.0001) greater in the HF group (7.7 ± 1.1 words/60 s) than in the IF (3.6 ± 1.2 words/60 s) and LF (1.3 ± 0.5 words/60 s) groups. Significantly different improvements in insulin resistance (P < 0.0001), blood pressure (P < 0.0001), and lipid peroxidation (P = 0.001) were also observed for the HF and IF groups in comparison with the LF group. Changes in insulin resistance explained ∼17% of changes in composite z score (partial r² = 0.1703, P < 0.0001). CONCLUSIONS: This dietary intervention study provides evidence that regular CF consumption can reduce some measures of age-related cognitive dysfunction, possibly through an improvement in insulin sensitivity. These data suggest that the habitual intake of flavanols can support healthy cognitive function with age.

Considerations on an approach for establishing a framework for bioactive food components.

Bioactive food components have shown potential health benefits for more than a decade. Currently there are no recommended levels of intake [i.e., Dietary Reference Intakes (DRIs)] as there are for nutrients and fiber. DRIs for essential nutrients were based on requirements for each specific nutrient to maintain normal physiologic or biochemical function and to prevent signs of deficiency and adverse clinical effects. They were later expanded to include criteria for reducing the risk of chronic degenerative diseases for some nutrients. There are many challenges for establishing recommendations for intakes of nonessential food components. Although some nonessential food components have shown health benefits and are safe, validated biomarkers of disease risk reduction are lacking for many. Biomarkers of intake (exposure) are limited in number, especially because the bioactive compounds responsible for beneficial effects have not yet been identified or are unknown. Furthermore, given this lack of characterization of composition in a variety of foods, it is difficult to ascertain intakes of nonessential food components, especially with the use of food-frequency questionnaires designed for estimating intakes of nutrients. Various intermediary markers that may predict disease outcome have been used as functional criteria in the DRI process. However, few validated surrogate endpoints of chronic disease risk exist. Nonvalidated intermediary biomarkers of risk may possibly predict clinical outcomes, but more research is needed to confirm the associations between cause and effect. One criterion for establishing acceptable intermediary outcome indicators may be the maintenance of normal physiologic function throughout adulthood, which presumably would lead to reduced chronic disease risk. Multiple biomarkers of outcomes that demonstrate the same health benefit may also be helpful. It would be beneficial to continue to refine the process of setting DRIs by convening a workshop on establishing a framework for nonessential food components that would take into consideration intermediary biomarkers indicative of optimal health.

Method for the determination of catechin and epicatechin enantiomers in cocoa-based ingredients and products by high-performance liquid chromatography: First Action 2013.04.

Single-laboratory validation data previously published in the Journal of AOAC INTERNATIONAL 95(2), 500-507 (2012) was reviewed by the Stakeholder Panel on Strategic Food Analytical Methods Expert Review Panel (ERP) at the AOAC INTERNATIONAL Mid-Year Meeting held on March 12-14, 2013 in Rockville, MD. The ERP determined the data presented met the established standard method performance requirement and approved the method as AOAC Official First Action on March 14, 2013. Using high-performance liquid chromatography (HPLC), flavanol enantiomers, (+)- and (-)-epicatechin and (+)- and (-)-catechin, are eluted isocratically using ammonium acetate and methanol mobile phase. The mobile phase is applied to a modified beta-cyclodextrin chiral stationary phase and the flavanols detected by fluorescence. Using several cocoa-based matrices, recoveries for the four enantiomers ranged from 82.2-102.1% at a 50% spike level, and 80.4-101.1% at a 100% spike level. Precision was determined to be 1.46-3.22% for (-)-epicatechin, 3.66-6.90% for (+)-catechin, 1.69-6.89% for (-)-catechin. (+)-Epicatechin was not detected in any of the samples used for this work, so precision could not be determined for this molecule.

Analysis of cocoa flavanols and procyanidins (DP 1-10) in cocoa-containing ingredients and products by rapid resolution liquid chromatography: single-laboratory validation.

Recently, a multilaboratory validation (MLV) of AOAC Official Method 2012.24 for the determination of cocoa flavanols and procyanidins (CF-CP) in cocoa-based ingredients and products determined that the method was robust, reliable, and transferrable. Due to the complexity of the CF-CP molecules, this method required a run time exceeding 1 h to achieve acceptable separations. To address this issue, a rapid resolution normal phase LC method was developed, and a single-laboratory validation (SLV) study conducted. Flavanols and procyanidins with a degree of polymerization (DP) up to 10 were eluted in 15 min using a binary gradient applied to a diol stationary phase, detected using fluorescence detection, and reported as a total sum of DP 1-10. Quantification was achieved using (-)-epicatechin-based relative response factors for DP 2-10. Spike recovery samples and seven different types of cocoa-based samples were analyzed to evaluate the accuracy, precision, LOD, LOQ, and linearity of the method. The within-day precision of the reported content for the samples was 1.15-5.08%, and overall precision was 3.97-13.61%. Spike-recovery experiments demonstrated recoveries of over 98%. The results of this SLV were compared to those previously obtained in the MLV and found to be consistent. The translation to rapid resolution LC allowed for an 80% reduction in analysis time and solvent usage, while retaining the accuracy and reliability of the original method. The savings in both cost and time of this rapid method make it well-suited for routine laboratory use.

Determination of Flavanols and Procyanidins (DP 1-10) in Cocoa-Based Ingredients and Products by UHPLC: First Action 2013.03.

Single-laboratory validation data were reviewed by the Expert Review Panel (ERP) of the Stakeholder Panel on Strategic Food Analytical Methods at the AOAC Mid-Year Meeting, March 12-14, 2013, in Rockville, MD. The ERP determined that the data presented met established standard method performance requirements and adopted a method for determination of flavanols and procyanidins (DP 1-10) in cocoa-based ingredients and products by ultra-HPLC as AOAC Official First Action Method 2013.03 on March 14, 2013. The flavanols and procyanidins (DP 1-10) are eluted using a binary gradient (solvents A and B) consisting of 98 + 2 (v/v) acetonitrile-glacial acetic acid (A) and 95 + 3 + 2 (v/v/v) methanol-water-glacial acetic acid (B). The mobile phase is applied to a diol stationary phase. Detection occurs using fluorescence detection. Recovery of flavanols and procyanidins (DP 1-10) from both high- and low-fat matrixes was 98.4-99.8%. Precision was determined for seven different sample types (cocoa extract, cocoa nib, natural cocoa powder, cocoa liquor, alkalized cocoa powder, dark chocolate, and milk chocolate).

Flavanol and procyanidin content (by degree of polymerization 1-10) of chocolate, cocoa liquors, cocoa powders, and cocoa extracts: first action 2012.24.

An international collaborative study was conducted on an HPLC method with fluorescent detection for the determination of flavanols and procyanidins in chocolate and cocoa-containing materials. The sum of the oligomeric fractions with degree of polymerization 1-10 was the determined content value. Sample materials included dark and milk chocolates, cocoa powder, cocoa liquors, and cocoa extracts. The content ranged from approximately 2 to 500 mg/g (defatted basis). Thirteen laboratories--representing commercial, industrial, and academic institutions in six countries--participated in this interlaboratory study. Fourteen samples were sent as blind duplicates to the collaborators. Results for 12 laboratories yielded repeatability RSD (RSDr) values below 10% for all materials analyzed, ranging from 4.17 to 9.61%. Reproducibility RSD (RSDR) values ranged from 5.03 to 12.9% for samples containing 8.07 to 484.7 mg/g material analyzed. In one sample containing a low content of flavanols and procyanidins (approximately 2 mg/g), the RSDR was 17.68%.

Determination of flavanol and procyanidin (by degree of polymerization 1-10) content of chocolate, cocoa liquors, powder(s), and cocoa flavanol extracts by normal phase high-performance liquid chromatography: collaborative study.

An international collaborative study was conducted on an HPLC method with fluorescent detection (FLD) for the determination of flavanols and procyanidins in materials containing chocolate and cocoa. The sum of the oligomeric fractions with degree of polymerization 1-10 was the determined content value. Sample materials included dark and milk chocolates, cocoa powder, cocoa liquors, and cocoa extracts. The content ranged from approximately 2 to 500 mg/g (defatted basis). Thirteen laboratories representing commercial, industrial, and academic institutions in six countries participated in the study. Fourteen samples were sent as blind duplicates to the collaborators. Results from 12 laboratories yielded repeatability relative standard deviation (RSDr) values that were below 10% for all materials analyzed, ranging from 4.17 to 9.61%. The reproducibility relative standard deviation (RSD(R)) values ranged from 5.03 to 12.9% for samples containing 8.07 to 484.7 mg/g. In one sample containing a low content of flavanols and procyanidins (approximately 2 mg/g), the RSD(R) was 17.68%. Based on these results, the method is recommended for Official First Action for the determination of flavanols and procyanidins in chocolate, cocoa liquors, powder(s), and cocoa extracts.

Benefits in cognitive function, blood pressure, and insulin resistance through cocoa flavanol consumption in elderly subjects with mild cognitive impairment: the Cocoa, Cognition, and Aging (CoCoA) study.

Flavanol consumption is favorably associated with cognitive function. We tested the hypothesis that dietary flavanols might improve cognitive function in subjects with mild cognitive impairment. We conducted a double-blind, parallel arm study in 90 elderly individuals with mild cognitive impairment randomized to consume once daily for 8 weeks a drink containing ≈990 mg (high flavanols), ≈520 mg (intermediate flavanols), or ≈45 mg (low flavanols) of cocoa flavanols per day. Cognitive function was assessed by Mini Mental State Examination, Trail Making Test A and B, and verbal fluency test. At the end of the follow-up period, Mini Mental State Examination was similar in the 3 treatment groups (P=0.13). The time required to complete Trail Making Test A and Trail Making Test B was significantly (P<0.05) lower in subjects assigned to high flavanols (38.10±10.94 and 104.10±28.73 seconds, respectively) and intermediate flavanols (40.20±11.35 and 115.97±28.35 seconds, respectively) in comparison with those assigned to low flavanols (52.60±17.97 and 139.23±43.02 seconds, respectively). Similarly, verbal fluency test score was significantly (P<0.05) better in subjects assigned to high flavanols in comparison with those assigned to low flavanols (27.50±6.75 versus 22.30±8.09 words per 60 seconds). Insulin resistance, blood pressure, and lipid peroxidation also decreased among subjects in the high-flavanol and intermediate-flavanol groups. Changes of insulin resistance explained ≈40% of composite z score variability through the study period (partial r(2)=0.4013; P<0.0001). To the best of our knowledge, this is the first dietary intervention study demonstrating that the regular consumption of cocoa flavanols might be effective in improving cognitive function in elderly subjects with mild cognitive impairment. This effect appears mediated in part by an improvement in insulin sensitivity.

Method for the determination of catechin and epicatechin enantiomers in cocoa-based ingredients and products by high-performance liquid chromatography: single-laboratory validation.

A single-laboratory validation study was performed for an HPLC method to identify and quantify the flavanol enantiomers (+)- and (-)-epicatechin and (+)- and (-)-catechin in cocoa-based ingredients and products. These compounds were eluted isocratically with an ammonium acetate-methanol mobile phase applied to a modified beta-cyclodextrin chiral stationary phase and detected using fluorescence. Spike recovery experiments using appropriate matrix blanks, along with cocoa extract, cocoa powder, and dark chocolate, were used to evaluate accuracy, repeatability, specificity, LOD, LOQ, and linearity of the method as performed by a single analyst on multiple days. In all samples analyzed, (-)-epicatechin was the predominant flavanol and represented 68-91% of the total monomeric flavanols detected. For the cocoa-based products, within-day (intraday) precision for (-)-epicatechin was between 1.46-3.22%, for (+)-catechin between 3.66-6.90%, and for (-)-catechin between 1.69-6.89%; (+)-epicatechin was not detected in these samples. Recoveries for the three sample types investigated ranged from 82.2 to 102.1% at the 50% spiking level, 83.7 to 102.0% at the 100% spiking level, and 80.4 to 101.1% at the 200% spiking level. Based on performance results, this method may be suitable for routine laboratory use in analysis of cocoa-based ingredients and products.

Influence of sugar type on the bioavailability of cocoa flavanols.

The beneficial effects of cocoa on vascular function are mediated by the absorption of monomeric flavanols into the circulation from the small intestine. As such, an understanding of the impact of the food matrix on the delivery of flavanols to the circulation is critical in assessing the potential vascular impact of a food. In the present study, we investigated the impact of carbohydrate type on flavanol absorption and metabolism from chocolate. A randomised, double-blind, three-arm cross-over study was conducted, where fifteen volunteers were randomly assigned to either a high-flavanol (266 mg) chocolate containing maltitol, a high-flavanol (251 mg) chocolate with sucrose or a low-flavanol (48 mg) chocolate with sucrose. Test chocolates were matched for micro- and macronutrients, including the alkaloids theobromine and caffeine, and were similar in taste and appearance. Total flavanol absorption was lower after consumption of the maltitol-containing test chocolate compared with following consumption of its sucrose-containing equivalent (P = 0·002). Although the O-methylation pattern observed for absorbed flavanols was unaffected by sugar type, individual levels of unmethylated ( - )-epicatechin metabolites, 3'-O-methyl-epicatechin and 4'-O-methyl-epicatechin metabolites were lower for the maltitol-containing test chocolate compared with the sucrose-containing equivalent. Despite a reduction in the total plasma pool of flavanols, the maximum time (T max) was unaffected. The present data indicate that full assessment of intervention treatments is vital in future intervention trials with flavanols and that carbohydrate content is an important determinant for the optimal delivery of flavanols to the circulation.