Regulatory landscape of dietary supplements and herbal medicines from a global perspective.

The number of Individuals that use dietary supplements and herbal medicine products are continuous to increase in many countries. The context of usage of a dietary supplement varies widely from country-to-country; in some countries supplement use is just limited to general health and well-being while others permit use for medicinal purposes. To date, there is little consensus from country to country on the scope, requirements, definition, or even the terminology in which dietary supplement and herbal medicines categories could be classified. Transparent science-based quality standards for the ingredients across these regulatory frameworks/definitions becomes even more important given the international supply chain. Meanwhile, there has been a rapid advancement in emerging technologies and data science applied to the field. This review was conceived at the Global Summit on Regulatory Sciences that took place in Beijing on September 2018 (GSRS2018) which is organized by Global Coalition for Regulatory Science Research (GCRSR) that consists of the global regulatory agencies from over ten countries including the European Union. This review summarizes a significant portion of discussions relating to a longitudinal comparison of the status for dietary supplements and herbal medicines among the different national jurisdictions and to the extent of how new tools and methodologies can improve the regulatory application.

Detection of Total Ergot Alkaloids in Cereal Flour and in Bread by a Generic Enzyme Immunoassay Method.

Four sets of polyclonal antibodies against ergot alkaloids ergometrine, ergotamine, α-ergocryptine, and ergocornine were produced and characterized in a competitive direct or indirect enzyme immunoassay (EIA). Standard curve LODs were 0.03 ng/mL (ergometrine EIA) to 2.0 ng/mL (ergocornine EIA). Three EIAs were highly specific, whereas the ergometrine EIA had a broad specificity pattern and reacted, albeit weakly, with all seven major ergot alkaloids and their epimeric forms. Using the ergometrine EIA, a generic test system was established in which total ergot alkaloids are quantified by a standard curve for a toxin mixture composed of three alkaloids that matched the ergot alkaloid composition in naturally contaminated rye and wheat products. Sample extraction with acetonitrile-phosphate-buffered saline at pH 6.0 without further cleanup was sufficient for EIA analysis. The LODs for total ergot alkaloids were 20 ng/g in rye and wheat flour and 14 ng/g in bread. Recoveries were 85-110% (RSDs of 0.1-11.7%) at a concentration range of 50-1000 ng/g. The total ergot alkaloid EIA was validated by comparison with HPLC-fluorescence detection. Although some under- and overestimation by the total ergot alkaloid EIA was observed, it was suitable for the reliable identification of positive samples at 10-20 ng/g and for the determination of total ergot alkaloids in a concentration range between 100 and 1000 ng/g.

Enzyme immunoassay for tenuazonic acid in apple and tomato products.

The Alternaria mycotoxin tenuazonic acid was derivatized with succinic anhydride and conjugated to keyhole limpet hemocyanin (KLH) and to horseradish peroxidase (HRP), respectively. The KLH conjugate was used to produce polyclonal antibodies in rabbits. A competitive direct enzyme immunoassay (EIA) for tenuazonic acid was established, which was moderately sensitive for tenuazonic acid [50% inhibition concentration (IC(50)): 320 ± 130 ng/mL] but strongly reacted with tenuazonic acid acetate (IC(50): 23.3 ± 7.5 ng/mL). Therefore, an optimized EIA protocol was established, which employed acetylation of standard and sample extract solutions. The mean standard curve detection limit (IC(30)) for tenuazonic acid acetate was 5.4 ± 2.0 ng/mL, enabling detection limits for tenuazonic acid in apple and tomato products of 25-50 ng/g (150 ng/g in tomato paste). Recoveries in a concentration range of 50-2000 ng/g were 60-130% in apple juice and tomato juice and 40-150% in other tomato products. Tenuazonic acid was detected in apple juice and tomato products from German retail shops at levels of 50-200 ng/g. In conclusion, this novel EIA for tenuazonic acid could be useful within a screening program for Alternaria mycotoxins in food.

Widespread occurrence of low levels of alternariol in apple and tomato products, as determined by comparative immunochemical assessment using monoclonal and polyclonal antibodies.

This study investigated the production of polyclonal (pAB) antibodies and the first time production of monoclonal (mAB) antibodies against the mycotoxin alternariol, and their implementation in enzyme immunoassay (EIA) for the rapid determination of alternariol in foods. Both EIAs were highly sensitive, with detection limits (IC20) of 35 ± 6.9 pg/mL (mAb EIA) and 59 ± 16 pg/mL (pAb EIA). Food products (n = 109; apple and tomato products, white wine) from German retail shops were analyzed. At a detection limit of 1-2 µg/kg, alternariol at 1-13 µg/kg was found with high frequency in apple (67%) and tomato (93%) products. Tomatoes with visible signs of Alternaria infection, stored at room temperature for up to 4 weeks, contained alternariol at levels up to 50 mg/kg, as determined by EIA and HPLC-FLD. It is concluded that the alternariol immunoassays present a versatile screening tool which could facilitate food control for Alternaria toxins.

Mycotoxins in horse feed.

A total of 62 samples of commercial horse feed preparations (complementary feeds) containing cereal mixtures ("muesli" or mash, n = 39; pelleted feeds, n = 12), and plain horse feed grains (maize, n = 5; oats, n = 4; barley, n = 2) were purchased from 21 different producers/distributors from the German market. All samples were analysed by competitive enzyme immunoassays (EIA) for six different mycotoxins (mycotoxin groups). Analytes (detection limit, mean recovery) were: deoxynivalenol (DON, 10 µg/kg, 84%), zearalenone (ZEA, 5 µg/kg, 93%), fumonisin B1 (FB1, 2 µg/kg, 113%), T-2 toxin (T-2, 0.1 µg/kg, 71%), sum of T-2 + HT-2 toxin (T-2/HT2, 0.2 µg/kg, 97%), ochratoxin A (OTA, 0.2 µg/kg, 67%), and total ergot alkaloids (Generic Ergot Alkaloids "GEA", 30 µg/kg, 132%). All samples contained DON (16-4,900 µg/kg, median 220 µg/kg), T-2/HT-2 (0.8-230 µg/kg, median 24 µg/kg), and T-2 (0.3-91 µg/kg, median 7 µg/kg). ZEA was detected in 98% of the samples (7-310 µg/kg, median 61 µg/kg). Most samples (94%) were positive for FB1 (2-2,200 µg/kg, median 27 µg/kg). Ergot alkaloids were detected in 61% of samples (28-1,200 µg/kg, median 97 µg/kg), OTA was found in 42% of samples (0.2-4 µg/kg, median 0.35 µg/kg). The results demonstrate that a co-contamination with several mycotoxins is very common in commercial horse feed from the German market. The toxin concentrations were in most cases well below the levels which are usually considered as critical or even toxic. The highest mycotoxin concentrations were mostly found in single-grain cereal feed: the maximum values for DON and FB1 were found in maize, the highest T-2/HT-2 toxin concentrations were found in oats, and the highest concentration of ergot alkaloids was found in barley. In composed feeds, no correlation between cereal composition and mycotoxin levels could be found.

Production and characterization of antibodies against fumigaclavine A.

Polyclonal antibodies against fumigaclavine A (FuA) were obtained from rabbits after immunization with a FuA-keyhole limpet hemocyanine conjugate prepared by formaldehyde condensation. Using these antibodies and a FuA-bovine serum albumine conjugate, a competitive indirect enzyme immunoassay (EIA) was established. The antiserum obtained from one rabbit enabled highly sensitive detection of FuA, with an IC50 level and detection limit of the standard curve of 3.3 ng/ml and approx. 1 ng/ml, respectively. The EIA was very specific for FuA, with 1.3% cross-reactivity with FuB. Several other lysergic acid derivatives (ergonovine, ergotamine, alpha-ergocryptine) were tested but did not cross-react in the FuA EIA. This is the first description of antibodies against FuA and the first development of an EIA for FuA.

Rapid methods for deoxynivalenol and other trichothecenes.

Method development for deoxynivalenol (DON) and other trichothecenes in recent years was driven by the analytical necessities arising from its widespread (and increasing) occurrence in foods and feeds. This has resulted in the establishment of guideline levels for animal feed, tolerable daily intake (TDI) levels for humans, and most importantly, in the prospect of low-tolerance levels for these toxins in foods in the near future. In order to ensure reliable determination of the toxin content at the tolerance levels, routine analytical methods must have detection limits of less than the tolerance level. This paper intends to give an overview of current analytical developments of rapid testing for deoxynivalenol and other trichothecene mycotoxins, with a special focus on antibody-based techniques. This includes high-throughput instrumental analysis for the laboratory environment, as well as rapid visual tests for on-site testing. The applicability of rapid tests within an integrated detection system for mycotoxins in foods is discussed.