What is a food and what is a medicinal product in the European Union? Use of the benchmark dose (BMD) methodology to define a threshold for "pharmacological action".

The decision criterion for the demarcation between foods and medicinal products in the EU is the significant "pharmacological action". Based on six examples of substances with ambivalent status, the benchmark dose (BMD) method is evaluated to provide a threshold for pharmacological action. Using significant dose-response models from literature clinical trial data or epidemiology, the BMD values were 63mg/day for caffeine, 5g/day for alcohol, 6mg/day for lovastatin, 769mg/day for glucosamine sulfate, 151mg/day for Ginkgo biloba extract, and 0.4mg/day for melatonin. The examples for caffeine and alcohol validate the approach because intake above BMD clearly exhibits pharmacological action. Nevertheless, due to uncertainties in dose-response modelling as well as the need for additional uncertainty factors to consider differences in sensitivity within the human population, a "borderline range" on the dose-response curve remains. "Pharmacological action" has proven to be not very well suited as binary decision criterion between foods and medicinal product. The European legislator should rethink the definition of medicinal products, as the current situation based on complicated case-by-case decisions on pharmacological action leads to an unregulated market flooded with potentially illegal food supplements.

Application of 19F NMR Spectroscopy for Content Determination of Fluorinated Pharmaceuticals.

A simple, rapid, and selective quantitative nuclear magnetic resonance spectroscopic method was evaluated for the determination of the content of fluorinated pharmaceuticals. 19F NMR spectra were either obtained in dimethylsulfoxide-d6 or aqueous buffer, using trifluoroacetic acid as internal standard. Quantification of 13 fluorine-containing pharmaceuticals spanning various pharmacological classes was accomplished using the proposed method. The method was found to be fit for purpose (interday precision 1.2% relative standard deviation) and may thus be applied for routine analysis and quality control of fluorine-containing pharmaceuticals due to its simplicity, nondestructive sample measurement, reliability, and high specificity. Therefore, 19F NMR may serve as a suitable analytical tool for the identification and selective determination of fluorinated pharmaceuticals used as reference materials and bulk samples.

Evaluation of Calligonum azel Maire, a North African desert plant, for its nutritional potential as a sustainable food and feed.

This study is the first to evaluate the potential of Calligonum azel Maire as a food ingredient. The plant materials flowers, leaves, stems and roots were analyzed by wet-chemical and instrumental methods for major and minor composition. The highest protein and sugar contents were recorded in the flowers (17.8% and 1.6% respectively), while the fat content was most abundant in the leaves (2.9%). Hexane extracts screened by gas chromatography (GC) for fatty acid composition showed 14 major compounds such as oleic acid, linoleic acid and palmitic acid, but also ω3 and ω6 unsaturated fatty acids. The essential oils extracted by hydrodistillation were screened by GC/MS and 31 compounds were identified including estragole, naphthalene, anethole, phytol and curcumene. The HPLC-DAD screening revealed that the methanolic extracts do not contain any pharmacologically active compound with adverse properties, but quercetin, hordenine and vanillin were abundant in the flowers, leaves and roots. Proton nuclear magnetic resonance (1H NMR) spectroscopy showed that glucose, sucrose and fructose were abundant in acetone and methanol extracts, while urea and glycerol were abundant in the aqueous extracts. A sensory evaluation of the aqueous infusions showed properties of the flowers reminiscent of other herbal teas. In conclusion, our results corroborate the suitability of the traditionally described use of Calligonum azel Maire as food and feed. The further study of this plant as a sustainable nutritional ingredient appears to be worthwhile.

Determination of the purity of pharmaceutical reference materials by 1H NMR using the standardless PULCON methodology.

A fast and reliable nuclear magnetic resonance spectroscopic method for quantitative determination (qNMR) of targeted molecules in reference materials has been established using the ERETIC2 methodology (electronic reference to access in vivo concentrations) based on the PULCON principle (pulse length based concentration determination). The developed approach was validated for the analysis of pharmaceutical samples in the context of official medicines control, including ibandronic acid, amantadine, ambroxol and lercanidipine. The PULCON recoveries were above 94.3% and coefficients of variation (CVs) obtained by quantification of different targeted resonances ranged between 0.7% and 2.8%, demonstrating that the qNMR method is a precise tool for rapid quantification (approximately 15min) of reference materials and medicinal products. Generally, the values were within specification (certified values) provided by the manufactures. The results were in agreement with NMR quantification using an internal standard and validated reference HPLC analysis. The PULCON method was found to be a practical alternative with competitive precision and accuracy to the classical internal reference method and it proved to be applicable to different solvent conditions. The method can be recommended for routine use in medicines control laboratories, especially when the availability and costs of reference compounds are problematic.

Rapid assessment of the illegal presence of 1,3-dimethylamylamine (DMAA) in sports nutrition and dietary supplements using 1H NMR spectroscopy.

1,3-Dimethylamylamine (DMAA) is a stimulant that can be found in pre-workout sports nutrition and dietary supplements. This practice is illegal because DMAA is not a safe food ingredient but rather an unapproved medicinal compound due to its pharmacological action. In order to determine the DMAA content in such products, a nuclear magnetic resonance (NMR) spectroscopic method was developed and validated (DMAA was quantified as DMAA-HCl). For quantification, the collective integral from two of the methyl groups of the molecule in the range δ 0.92-0.84 ppm was used. The method was linear over the examined range of 1-21 g/kg (R(2) = 0.9937). The recoveries from spiked concentrations (0.1-6 g/kg) ranged between 85% and 105% (96% on average), with a relative standard deviation (RSD) of 1% for an authentic sample. The detection limit was 0.03 g/kg and the quantification limit was 0.08 g/kg (calculated for 75 mg sample weight). The actual DMAA-HCl content in the sample was quantified using calibration curves (external standardization) or 3,5-dinitrobenzoic acid as single-point internal standard. The developed NMR methodology was applied for the analysis of 16 products, from which 9 samples were found positive (the DMAA-HCl concentration varied between 3.1 g/kg and 415 g/kg). The method can be recommended for routine use in food testing, customs or doping control laboratories.

Electronic cigarettes: overview of chemical composition and exposure estimation.

BACKGROUND: Electronic cigarettes (e-cigarettes) are advertised to tobacco users as a tool to decrease cigarette consumption and to reduce toxic exposure associated with conventional tobacco smoking. Little is known about the compounds contained in such products, their exposure and long-term health effects. METHODS: NMR spectroscopy was used to ascertain the content of several constituents of e-cigarette liquids including nicotine, solvents and some bioactive flavour compounds. Risk assessment was based on probabilistic exposure estimation and comparison with toxicological thresholds using the margin of exposure (MOE) approach. RESULTS: In 54 samples of e-cigarette liquids, the average nicotine content was 11 mg/ml. Only 18 from 23 samples were confirmed as nicotine-free samples and in one e-cigarette liquid nicotine was not detected while being declared on the labelling. Major compounds of e-cigarette liquids include glycerol (average 37 g/100 g), propylene glycol (average 57 g/100 g) and ethylene glycol (average 10 g/100 g). Furthermore, 1,3-propanediol, thujone and ethyl vanillin were detected in some samples. The average exposure for daily users was estimated as 0.38 mg/kg bw/day for nicotine, 8.9 mg/kg bw/day for glycerol, 14.5 mg/kg bw/day for 1,2-propanediol, 2.1 mg/kg bw/day for ethylene glycol, and below 0.2 mg/kg bw/day for the other compounds. The MOE was below 0.1 for nicotine, but all other compounds did not reach MOE values below 100 except ethylene glycol and 1,2-propanediol. CONCLUSIONS: NMR spectroscopy is a useful and rapid method to simultaneously detect several ingredients in e-cigarette liquids. From all compounds tested, only nicotine may reach exposures that fall into a high risk category with MOE <1. Therefore, e-cigarette liquid products should be subjected to regulatory control to ensure consistent nicotine delivery. Solvents with more favourable toxicological profiles should be used instead of ethylene glycol and 1,2-propanediol, which may fall into a risk category with MOE < 100.

Standardless 1H NMR determination of pharmacologically active substances in dietary supplements and medicines that have been illegally traded over the internet.

Dietary supplements and medicines are widely marketed over the Internet. Such products may be counterfeited and lack some or all of the labelled ingredients, or, in the case of lifestyle supplements, illegally contain pharmacologically active substances, such as anorectic or androgenic compounds. The market control - especially in the case of customs seizures - is complex, as reference substances necessary for identification and calibration in traditional high performance liquid chromatography (HPLC) or gas chromatography-mass spectrometry (GC-MS) analysis are often unavailable, or extremely expensive. In this study, we introduce a 400 MHz (1) H NMR methodology, which allows identification and quantitative estimation even without such pure compound standards. The identification can be based on literature spectra, or if these data are unavailable, by applying computational NMR spectra prediction. For standardless NMR determination, simple peak-area comparison of the target compound with the TSP reference was used. The applicability was demonstrated for a wide range of compounds, such as mesterolone, oxymetholone, sibutramine, monacolin K, vinpocetine, evodiamine, caffeine, kavain, and dehydroepiandrosterone. The average relative standard deviations were 5.0% for peak area comparison, and 3.3% for external calibration with standard substance. The method uncertainty is therefore higher in standardless determination, but acceptable for the purpose of proving the presence or absence of pharmacologically active substances. The limit of detection of 0.5-2 mg/kg is sufficient for the purpose. NMR is ideally suited to controlling dietary supplements or illegal medicines as it provides qualitative and at least semi-quantitative information more rapidly (measurement time 20 min) than with any other currently available spectroscopic or chromatographic method.

NMR evaluation of total statin content and HMG-CoA reductase inhibition in red yeast rice (Monascus spp.) food supplements.

BACKGROUND: Red yeast rice (i.e., rice fermented with Monascus spp.), as a food supplement, is claimed to be blood cholesterol-lowering. The red yeast rice constituent monacolin K, also known as lovastatin, is an inhibitor of the hydroxymethylglutaryl-CoA (HMG-CoA) reductase. This article aims to develop a sensitive nuclear magnetic resonance (NMR) method to determine the total statin content of red yeast rice products. METHODS: The total statin content was determined by a 400 MHz 1H NMR spectroscopic method, based on the integration of the multiplet at δ 5.37-5.32 ppm of a hydrogen at the hexahydronaphthalene moiety in comparison to an external calibration with lovastatin. The activity of HMG-CoA reductase was measured by a commercial spectrophotometric assay kit. RESULTS: The NMR detection limit for total statins was 6 mg/L (equivalent to 0.3 mg/capsule, if two capsules are dissolved in 50 mL ethanol). The relative standard deviations were consistently lower than 11%. The total statin concentrations of five red yeast rice supplements were between 1.5 and 25.2 mg per specified daily dose. A dose-dependent inhibition of the HMG-CoA reductase enzyme activity by the red yeast rice products was demonstrated. CONCLUSION: A simple and direct NMR assay was developed to determine the total statin content in red yeast rice. The assay can be applied for the determination of statin content for the regulatory control of red yeast rice products.