Presence of higenamine in beetroot containing 'foodstuffs' and the implication for WADA-relevant anti-doping testing.

Higenamine is an alkaloid found within plant species including some that are used in traditional Asian and Chinese herbal medicines. Identified as having mixed mode adrenergic receptor activity, higenamine is present within some nutritional supplements marketed for stimulant and/or weight loss. Its inclusion within nutritional supplements can be via its natural presence within botanical ingredients or as a synthetic additive, often added in mg amounts. The World Anti-doping Agency (WADA) prohibited list has contained higenamine since 2017 as banned at all times in the beta-2 agonist (S3) category, with a reporting level of 10 ng/ml for the free parent form in urine. In this study, an investigation into the content of beetroot or beetroot-containing foodstuffs and supplement products was conducted. Higenamine was confirmed as present within the majority of foodstuffs and supplements, with experimental evidence that higenamine can arise within beetroot extracts through heating. The results in this paper demonstrate the first reported evidence of a link between beetroot and this WADA prohibited substance. To investigate the link between intake and excretion, concentrated beetroot drinks were consumed by six individuals and higenamine quantified in their urine. Free higenamine was detected in the urine of all individuals, with maximum measured concentration in samples of less than 1% of the current WADA reporting limit. Although the risk of an inadvertent doping violation by consumption of the foodstuffs and products investigated in this study is low, beetroot as a source of higenamine should be considered by athletes.

Analysis of supplements available to UK consumers purporting to contain selective androgen receptor modulators.

Selective androgen receptor modulators (SARMs) are compounds with specific androgenic properties investigated for the treatment of conditions such as muscle wasting diseases. The reported androgenic properties have resulted in their use by athletes, and consequently they have been on the World Anti-Doping Agency prohibited list for more than a decade. SARMs have been investigated by pharmaceutical companies as potential drug candidates, but to date no SARM has demonstrated sufficient safety and efficacy to gain clinical approval by either the European Medicines Agency or the U.S. Food and Drug Administration. Despite their lack of safety approval, SARMs are often illegally marketed as dietary supplements, available for consumers to buy online. In this study, a range of supplement products marketed as SARMs were purchased and analyzed using high resolution accurate mass - mass spectrometry to evaluate the accuracy of product claims and content labeling. This study found discrepancies ranging from a supplement in which no active ingredients were found, to supplements containing undeclared prohibited analytes. Where SARMs were detected, discrepancies were observed between the concentrations measured and those detailed on the product packaging. The outcome of this experiment highlights the high risk of such supplement products to consumers. The inaccurate product claims give rise to uncertainty over both the dose taken and the identity of any of these unapproved drugs. Even for supplements for which the product labeling is correct, the lack of complete toxicity data, especially for combinations of SARMs taken as stacks, means that the safety of these supplements is unknown.

Biomarker-estimated flavan-3-ol intake is associated with lower blood pressure in cross-sectional analysis in EPIC Norfolk.

Flavan-3-ols are a group of bioactive compounds that have been shown to improve vascular function in intervention studies. They are therefore of great interest for the development of dietary recommendation for the prevention of cardio-vascular diseases. However, there are currently no reliable data from observational studies, as the high variability in the flavan-3-ol content of food makes it difficult to estimate actual intake without nutritional biomarkers. In this study, we investigated cross-sectional associations between biomarker-estimated flavan-3-ol intake and blood pressure and other CVD risk markers, as well as longitudinal associations with CVD risk in 25,618 participants of the European Prospective Investigation into Cancer (EPIC) Norfolk cohort. High flavan-3-ol intake, achievable as part of an habitual diet, was associated with a significantly lower systolic blood pressure (- 1.9 (- 2.7; - 1.1) mmHg in men and - 2.5 (- 3.3; - 1.8) mmHg in women; lowest vs highest decile of biomarker), comparable to adherence to a Mediterranean Diet or moderate salt reduction. Subgroup analyses showed that hypertensive participants had stronger inverse association between flavan-3-ol biomarker and systolic blood pressure when compared to normotensive participants. Flavanol intake could therefore have a role in the maintenance of cardiovascular health on a population scale.

Selection of a surrogate matrix for the quantification of an endogenous analyte in dried whole blood.

Aim: A surrogate matrix is needed to quantify 25-hydroxyvitamin D3 in dried whole blood (DWB). To date, there has been limited guidance on approaches for quantification of endogenous analytes in atypical matrices, such as DWB. Methods: Different surrogate matrices were investigated in a systematic process using an LC-MS/MS assay. Performance assessment was made using quality controls of DWB with different hematocrits. Results & conclusion: Suitability of both phosphate-buffered saline containing bovine serum albumin and washed red blood cells recombined with phosphate-buffered saline containing bovine serum albumin as a surrogate matrix was demonstrated across a range of concentrations and hematocrits representative of expected endogenous analyte samples.

Evaluation of (-)-epicatechin metabolites as recovery biomarker of dietary flavan-3-ol intake.

Data from dietary intervention studies suggest that intake of (-)-epicatechin mediates beneficial vascular effects in humans. However, population-based investigations are required to evaluate associations between habitual intake and health and these studies rely on accurate estimates of intake, which nutritional biomarkers can provide. Here, we evaluate a series of structurally related (-)-epicatechin metabolites (SREM), particularly (-)-epicatechin-3'-glucuronide, (-)-epicatechin-3'-sulfate and 3'-O-methyl-(-)-epicatechin-5-sulfate (SREMB), as flavan-3-ol and (-)-epicatechin intake. SREMB in urine proved to be a specific indicator of (-)-epicatechin intake, showing also a strong correlation with the amount of (-)-epicatechin ingested (R2: 0.86 (95% CI 0.8l; 0.92). The median recovery of (-)-epicatechin as SREMB in 24 h urine was 10% (IQR 7-13%) and we found SREMB in the majority of participants of EPIC Norfolk (83% of 24,341) with a mean concentration of 2.4 ± 3.2 µmol/L. Our results show that SREMB are suitable as biomarker of (-)-epicatechin intake. According to evaluation criteria from IARC and the Institute of Medicine, the results obtained support use of SREMB as a recovery biomarker to estimate actual intake of (-)-epicatechin.

Evaluation at scale of microbiome-derived metabolites as biomarker of flavan-3-ol intake in epidemiological studies.

The accurate assessment of dietary intake is crucial to investigate the effect of diet on health. Currently used methods, relying on self-reporting and food composition data, are known to have limitations and might not be suitable to estimate the intake of many bioactive food components. An alternative are nutritional biomarkers, which can allow an unbiased assessment of intake. They require a careful evaluation of their suitability, including: (a) the availability of a precise, accurate and robust analytical method, (b) their specificity (c) a consistent relationship with actual intake. We have evaluated human metabolites of a microbiome-derived flavan-3-ol catabolite, 5-(3',4'-dihydroxyphenyl)-[gamma]-valerolactone (gVL), as biomarker of flavan-3-ol intake in large epidemiological studies. Flavan-3-ols are widely consumed plant bioactives, which have received considerable interest due to their potential ability to reduce CVD risk. The availability of authentic standards allowed the development of a validated high-throughput method suitable for large-scale studies. In dietary intervention studies, we could show that gVL metabolites are specific for flavan-3-ols present in tea, fruits, wine and cocoa-derived products, with a strong correlation between intake and biomarker (Spearman's r = 0.90). This biomarker will allow for the first time to estimate flavan-3-ol intake and further investigation of associations between intake and disease risk.

Development and validation of a high-throughput assay for the quantification of multiple green tea-derived catechins in human plasma.

A rapid, accurate and robust method for the determination of catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (Cg), epicatechin gallate (ECg), gallocatechin gallate (GCg) and epigallocatechin gallate (EGCg) concentrations in human plasma has been developed. The method utilizes protein precipitation following enzyme hydrolysis, with chromatographic separation and detection using reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS). Traditional issues such as lengthy chromatographic runtimes, sample and extract stability, and lack of suitable internal standards have been addressed. The method has been evaluated using a comprehensive validation procedure, confirming linearity over appropriate concentration ranges, and inter/intra-batch precision and accuracies within suitable thresholds (precisions within 13.8% and accuracies within 12.4%). Recoveries of analytes were found to be consistent between different matrix samples, compensated for using suitable internal markers and within the performance of the instrumentation used. Similarly, chromatographic interferences have been corrected using the internal markers selected. Stability of all analytes in matrix is demonstrated over 32 days and throughout extraction conditions. This method is suitable for high-throughput sample analysis studies.

Development, validation and evaluation of an analytical method for the determination of monomeric and oligomeric procyanidins in apple extracts.

There is a lack of data for individual oligomeric procyanidins in apples and apple extracts. Our aim was to develop, validate and evaluate an analytical method for the separation, identification and quantification of monomeric and oligomeric flavanols in apple extracts. To achieve this, we prepared two types of flavanol extracts from freeze-dried apples; one was an epicatechin-rich extract containing ∼30% (w/w) monomeric (-)-epicatechin which also contained oligomeric procyanidins (Extract A), the second was an oligomeric procyanidin-rich extract depleted of epicatechin (Extract B). The parameters considered for method optimisation were HPLC columns and conditions, sample heating, mass of extract and dilution volumes. The performance characteristics considered for method validation included standard linearity, method sensitivity, precision and trueness. Eight laboratories participated in the method evaluation. Chromatographic separation of the analytes was best achieved utilizing a Hilic column with a binary mobile phase consisting of acidic acetonitrile and acidic aqueous methanol. The final method showed linearity for epicatechin in the range 5-100µg/mL with a correlation co-efficient >0.999. Intra-day and inter-day precision of the analytes ranged from 2 to 6% and 2 to 13% respectively. Up to dp3, trueness of the method was >95% but decreased with increasing dp. Within laboratory precision showed RSD values <5 and 10% for monomers and oligomers, respectively. Between laboratory precision was 4 and 15% (Extract A) and 7 and 30% (Extract B) for monomers and oligomers, respectively. An analytical method for the separation, identification and quantification of procyanidins in an apple extract was developed, validated and assessed. The results of the inter-laboratory evaluation indicate that the method is reliable and reproducible.