Mycotoxins in Plant-Based Dietary Supplements: Hidden Health Risk

Jul 13, 2015 - *E-mail: [email protected]. Tel.: +420 220 443 142. Fax: +420 220 443 184. University of Chemistry and Technology, Prague, F...
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Mycotoxins in Plant-Based Dietary Supplements: Hidden Health Risk for Consumers Zdenka Veprikova,† Milena Zachariasova,*,† Zbynek Dzuman,† Alena Zachariasova,† Marie Fenclova,† Petra Slavikova,† Marta Vaclavikova,† Katerina Mastovska,‡ Daniel Hengst,‡ and Jana Hajslova† †

Department of Food Analysis and Nutrition, University of Chemistry and Technology, Technicka 3, 16628 Prague 6, Czech Republic ‡ Covance Laboratories, Nutritional Chemistry and Food Safety, 3301 Kinsman Blvd., Madison, Wisconsin 53704, United States S Supporting Information *

ABSTRACT: Mycotoxin contamination of dietary supplements represents a possible risk for human health, especially in the case of products intended for people suffering from certain health conditions. The aim of this study was to assess the extent of this problem based on analyses of a wide set of herbal-based dietary supplements intended for various purposes: (i) treatment of liver diseases (milk thistle); (ii) reduction of menopause effects (red clover, flax seed, and soy); and (iii) preparations for general health support (green barley, nettle, goji berries, yucca, etc.) The analytical method including 57 mycotoxins was based on a QuEChERS-like (quick, easy, cheap, effective, rugged, safe) approach and ultrahigh performance liquid chromatography coupled with tandem mass spectrometry. The main mycotoxins determined were Fusarium trichothecenes, zearalenone and enniatins, and Alternaria mycotoxins. Co-occurrence of enniatins, HT-2/T-2 toxins, and Alternaria toxins was observed in many cases. The highest mycotoxin concentrations were found in milk thistle-based supplements (up to 37 mg/kg in the sum). KEYWORDS: mycotoxins, dietary supplements, milk thistle, ultraperformance liquid chromatography, tandem mass spectrometry, standard addition method

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INTRODUCTION Botanical-based dietary supplements are used as a concentrated source of vitamins, minerals, and other biologically active substances. The most widespread plants with high content of antioxidants, used as raw materials for dietary supplements production, include, e.g., milk thistle (Silybum marianum L.), ginseng root (Panax ginseng C.A. Mey.), ginkgo biloba (Ginkgo biloba L.), green barley (Hordeum vulgare L.), accerola (Malpighia glabra L.), goji berries (Lycium chinense Mill.), boswellia (Boswellia serrata Roxb. Ex Colebr.), yucca (Yucca spp. L.), guarana (Paullinia cupana Kunth ex H.B.K.), and many others.1 Many beneficial health effects are attributed to their use. For example, milk thistle (S. marianum L.) is usually claimed to function as a liver protectant and regenerator of the whole organism because of high concentrations of silymarin, which is a complex of flavonoidic antioxidants.2 However, the majority of the positive health claims used by dietary supplement manufacturers are in the process of evaluation by expert panels of the European Food Safety Authority (EFSA).3 Botanical-based preparations on a market are mostly formulated as teas, oils, or tablets and capsules containing dried ethanolic extracts of active compounds. Unfortunately, common practices employed for their production do not avoid coisolation of various potential contaminants, which may be present in the raw material and possess similar physicochemical properties as targeted biologically active plant components. In this context, the ubiquitous microscopic filamentous fungi affecting plants and mycotoxins, their toxic secondary metabolites, are very relevant. A number of publications describing the presence of Fusarium, Alternaria, or Asperigillus © 2015 American Chemical Society

fungi in herbs have been published. The critical points of the production technologies include formation and spreading of fungi in plants during growing, inappropriate harvesting, cleaning, transportation, and storage.4−6 In a study by Tournas et al., Asperigillus, Eurotium, Penicillium, Fusarium, and Alternaria fungi species were found in dietary supplement preparations made of milk thistle retailed in the US market.7 The fungi were determined in unprocessed herbs as well as whole seeds, seed powder, cut herb, herb powder, and not in alcohol- or oil-based extracts and capsules. In another publication, the same author group studied the content of mycotoxins aflatoxins (AFs) in milk thistle supplements.8 Fifteen out of the 83 investigated samples containedon aflatoxins at levels ranging from 0.04 to 2 μg/kg. A set of 7 samples of milk thistle-based dietary supplements was also analyzed by Arroyo-Manzanares et al.9 From the 15 investigated mycotoxins, only HT-2 toxin and T-2 toxins (HT-2, T-2) were detected in two samples. The levels of T-2 were 363.0 and 453.9 μg/kg and of HT-2, 826.9, and 943.7 μg/kg.9 Another group studied the mycotoxins content in milk thistle, chamomile, valerian, senna, rhubarb, and ginkgo biloba.10 They analyzed 84 samples and found 99% of them to be contaminated with T-2, 98% with zearalenone (ZEN), 96% with AFs, 63% with ochratoxin A (OTA), 62% with deoxynivalenol (DON), 61% with citrinin (CIT), and 13% with fumonisins (FBs). All samples in that study were contaminated Received: Revised: Accepted: Published: 6633

April 27, 2015 July 13, 2015 July 13, 2015 July 13, 2015 DOI: 10.1021/acs.jafc.5b02105 J. Agric. Food Chem. 2015, 63, 6633−6643

Article

Journal of Agricultural and Food Chemistry

(E-metrine); ergosine (E-sine); ergosinine (E-sinine); ergotamine (Eamine); ergotaminine (E-aminine); aflatoxins B1, B2, G1, G2 (AFB1, AFB2, AFG1, AFG2); altenuene (ATE); gliotoxin (GLIO); sterigmatocystin (STER); cyclopiazonic acid (CPA); CIT; meleagrin (MEL); MPA; OTA; patulin (PAT); paxilline (PAX); penicillic acid (PEN-Ac); penitrem A (PEN-A); roquefortine C (ROQ-C); verruculogen (Verruc); and stachybotrylactam (STACH) were purchased from Romer Labs (Austria). Analytical standards of mycotoxins alternariol (AOH), alternariol-methyl ether (AME), tenuazonic acid (TeA), and verrucarol (VER) were purchased from Sigma-Aldrich (United States). All the used analytical standards were certified by the manufacturers with defined purity (95.0−99.8%). HPLC grade acetonitrile, ammonium acetate (≥99.0%), ammonium formate (≥99.0%), sodium chloride (>99.5%), and magnesium sulfate (>99.5%) were obtained from Sigma-Aldrich (United States). HPLC grade methanol and hexane were purchased from Merck (Germany), formic acid from Penta Chrudim (Czech Republic), and Bondesil-C18 sorbent from Varian (United States). Water was purified by a Milli-Q water system (Millipore, Germany). Samples. Samples of dietary supplements based on various herbs or plants were collected from Czech and US retail markets. The detailed description of the preparations (matrix characterization, application form, recommended dosage, and the country of origin) is provided in the Supporting Information. Generally, the samples can be divided into three main groups: (i) 32 samples based on milk thistle, containing silymarin, intended to treat and maintain the health of liver; (ii) 9 samples based on red clover, soy, flax, and black cohosh, containing phytoestrogens and intended for reduction of menopausal effects; (iii) 28 samples based on other herbs and plants, e.g., green barley/wheat, nettle, goji, yucca, acerola, magnolia, guarana, orange peel, turmeric, black cohosh, bacopa monnieri, Chinese jujube, green tea leaf, boswellia serrate, containing various antioxidants and intended for general health improvement (immunity, vitality, cardiovascular system, brain functions, etc.). Sample Preparation. Prior to the extraction procedure, the tested samples were properly homogenized. Seeds, dried leaves, and uncoated tablets were homogenized directly using a mill Grindomix GM200 (Retsch, Germany). In the case of wrapped capsules, only the inside part containing the herbal extract was used for the analysis (the empty capsule was removed and weighed for the purposes of follow-up results recalculation). Samples were processed by the QuEChERS-based (quick, easy, cheap, effective, rugged, safe) method published by Dzuman et al.17 Representative sample (1.00 ± 0.01 g) was weighed into a 50 mL polytetrafluorethylene (PTFE) centrifuge tube (Merci, Czech Republic) and mixed with 10.0 mL of 1% aqueous formic acid. Matrix was allowed to soak for 30 min, followed by a 30 min extraction by shaking with 10.0 mL of acetonitrile using an automatic shaker HS 250 basic (IKA Labortechnik, Germany). After that, 1.00 g of sodium chloride and 4.00 g of magnesium sulfate were added and the tube was shaken again for 1 min by hand. After centrifugation (5 min at 15 800g using ROTINA 35R, Hettich, Germany), a 2.00 mL aliquot of the upper acetonitrile layer was taken for dispersive solid phase extraction (d-SPE) cleanup in a smaller PTFE centrifuge tube (15 mL) containing 100 mg of Bondesil-C18 sorbent and 300 mg of magnesium sulfate. The d-SPE tube was shaken by hand for 1 min and then centrifuged for 5 min (15 800g). An aliquot of the upper acetonitrile layer was microfiltered (0.2 μm PTFE microfilter, Alltech, United States) and transferred into a 2 mL PTFE autosampler vial (MicroSolv, United States). For solid samples with high amount of fat (preparations formed as encapsulated oily paste), the sample (1.00 g) was first shaken for 5 min with 3.00 mL of hexane to solubilize the samples. After this step, the procedure described above was followed (i.e., addition of 1% aqueous formic acid and acetonitrile, shaking, addition of inorganic salts, shaking again, and d-SPE cleanup with Bondesil-C18). Separation and Detection. Analyte separation and detection were performed using an U-HPLC-MS/MS system consisting of an ultrahigh performance liquid chromatograph (Acquity UPLC System, Waters, United States) coupled to a highly sensitive tandem mass spectrometer QTRAP 5500 (AB SCIEX, Canada). The chromato-

by more than one mycotoxin, mostly by AFs, T-2, and ZEN together.10 An extensive study investigating the content of 35 mycotoxins in traditional Chinese medicines found 83% of the 60 tested, commercially available supplements to be positive and contain mainly AFs, trichothecenes, and ochratoxins (OTs), usually at levels of units or tens of micrograms per kilogram.11 Different types of botanical-based dietary supplements were investigated by Di Mavungu et al., who analyzed set of samples containing mainly garlic, soy, radish, and maca.12 From the 23 investigated mycotoxins, only FBs and OTA were detected in 10% of samples. Mycotoxin contamination has been also evaluated in dietary supplements made from green coffee, red rice, and ginkgo biloba. The following mycotoxin concentration ranges were determined in a study testing 34 mycotoxins in 50 commercially available green coffee supplement products: OTA, 2.7−136.9 μg/kg; ochratoxin B (OTB), 3.5−20.2 μg/kg; fumonisin B1 (FB1), 110.0−415.0 μg/kg; and mycophenolic acid (MPA), 43.1−395.0 μg/kg.13 As concerns the red rice, Li et al. investigated the content of CIT in 109 samples, where 31 were positive, with CIT ranging from 16.6 to 5 253 μg/kg.14 A study by Martinez-Dominguez et al. focused on gikgo biloba-based dietary supplements and investigated a set of 9 samples for 10 mycotoxins.15 Six samples were contaminated with mycotoxins as follows: aflatoxin B1 (AFB1), 5−54 μg/kg; aflatoxin B2 (AFB2), 4−300 μg/kg; and T-2, 18− 20 μg/kg. The details about all of the mentioned studies are summarized in the Supporting Information Various conditions influencing the final quality of botanicalbased dietary supplements are thoroughly discussed in a study of Sanzini et al.16 In addition to potential contamination by mycotoxins, pesticide residues, and other environmental contaminants, other issues, such as addition of pharmacologically/psychotropically active compounds or adulteration with plants of different species, could potential threaten health of various herbal dietary supplements. The aim of this study was to assess mycotoxin contamination in a unique set of botanical-based dietary supplements using ultrahigh performance liquid chromatography coupled to tandem mass spectrometry (U-HPLC-MS/MS) for the analysis of 57 mycotoxins. The evaluated products included dietary supplements supporting liver function (based on milk thistle); reducing menopause effects (red clover, flax seed, and soya); and supporting health in general (green barley, nettle, goji berries, yucca, etc.). This pilot study provides interesting results about the cooccurrence of multiple toxins in these preparations and discusses the health risk associated with this phenomenon. Moreover, important questions related to the quality of the botanicals used in dietary supplements have been raised in this study, which could help to increase the quality requirements for the raw materials entering the dietary supplements production chain.

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MATERIALS AND METHODS

Analytical Standards and Chemicals. Analytical standards of mycotoxins 3- and 15-acetyldeoxynivalenol (3- and 15-ADON); beauvericin (BEA); DON, deoxynivalenol-3-glucoside (DON-3-Glc); diacetoxyscirpenol (DAS); enniatins A, A1, B, B1 (ENN-A, ENN-A1, ENN-B, ENN-B1); fumonisins B1, B2, B3 (FB1, FB2, FB3); fusarenon X (FUS-X); T-2; HT-2; neosolaniol (NEO); nivalenol (NIV); ZEN; α- and β-zearalenol (α-ZEL, β-ZEL); tentoxin (TEN); agroclavine (A-clavine); ergocornine (E-cornine); ergocorninine (Ecorninine); ergocristine (E-cristine); ergocristinine (E-cristinine); ergocryptine (E-cryptine); ergocryptinine (E-cryptinine); ergometrine 6634

DOI: 10.1021/acs.jafc.5b02105 J. Agric. Food Chem. 2015, 63, 6633−6643

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Journal of Agricultural and Food Chemistry Table 1. Validation Results of the Used QuEChERS-Based U-HPLC-MS/MS Multianalyte Methoda matrix analyte 15-acetyldeoxynivalenol 3-acetyldeoxynivalenol agroclavine aflatoxin B1 aflatoxin B2 aflatoxin G1 aflatoxin G2 altenuene alternariol alternariol-methyl ether beauvericin citrinin cyclopiazonic acid deoxynivalenol deoxynivalenol-3glucoside diacetoxyscirpenol enniatin A enniatin A1 enniatin B enniatin B1 ergocornine ergocorninine ergocristine ergocristinine ergocryptine ergocryptinine ergometrine ergosine ergosinine ergotamine ergotaminine fusarenon X fumonisin B1 fumonisin B2 fumonisin B3 gliotoxin HT-2 toxin meleagrin mycophenolic acid neosolaniol nivalenol ochratoxin A patulin paxilline penicillic acid penitrem A roquefortine C stachybotrylactam sterigmatocysin T-2 toxin tentoxin tenuazonic acid verrucarol verruculogen zearalenone α-zearalenol β-zearalenol a

milk thistle powder (no. 16)

oil-based milk thistle matrix (no. 9)

tea (no. 10, dry nettle)

recovery (%)

RSD (%)

LOQ (μg/kg)

SSE (%)

recovery (%)

RSD (%)

LOQ (μg/kg)

SSE (%)

recovery (%)

RSD (%)

LOQ (μg/kg)

SSE (%)

92 98 94 106 99 99 89 79 76 74 100 76 96 95 65

5.9 5.7 4.6 3.0 5.9 4.0 2.6 2.4 3.7 1.5 2.7 4.4 7.5 1.8 4.9

20 10 5 5 5 5 10 5 5 5 5 50 500 50 100

78 91 71 65 69 58 47 39 79 75 91 51 41 62 40

88 89 105 91 90 88 84 85 88 91 97 75 − 78 40

3.4 2.9 7.0 2.6 1.5 2.0 5.9 8.1 2.7 2.6 8.3 10.1 − 7.2 10.9

50 50 5 5 10 5 10 5 5 5 5 100 1000 50 100

101 96 82 72 85 69 71 48 81 74 105 65 45 65 47

82 82 82 97 93 96 97 84 91 88 97 85 − 83 44

7.3 7.3 7.3 4.4 9.0 7.3 10.2 10.6 4.3 7.5 6.4 7.1 − 6.8 7.2

50 50 10 5 10 5 20 10 5 5 5 250 1000 100 250

88 97 65 71 80 71 56 44 92 81 110 64 50 71 41

85 64 68 62 61 90 89 91 86 91 92 95 91 78 93 85 108 71 93 95 92 97 92 106 95 94 98 82 91 98 106 110 97 106 88 95 89 98 80 92 91 90

3.2 2.0 2.8 5.7 6.4 4.9 3.2 6.1 3.2 6.2 7.0 4.6 6.3 3.0 7.6 8.1 6.7 4.8 9.7 11 18 5.0 4.1 6.2 4.4 11 3.0 11 5.2 9.3 4.1 4.7 4.6 1.6 2.4 3.2 5.6 6.5 4.9 3.2 3.8 2.8

10 5 5 5 5 10 5 5 5 5 5 5 10 10 5 5 20 50 10 10 250 50 5 5 10 200 10 50 50 100 20 10 10 5 10 10 100 250 100 10 20 20

95 95 101 96 110 74 68 49 55 52 60 39 34 39 48 47 50 89 100 105 80 92 78 84 71 55 90 90 80 74 58 79 92 79 82 105 70 74 84 84 87 90

102 89 88 91 87 89 105 90 98 99 110 79 85 95 87 103 86 79 82 81 91 122 95 94 78 73 99 71 83 74 107 103 88 89 96 93 75 78 79 97 87 89

6.6 7.8 8.6 6.7 7.1 8.6 12.9 15.9 17.7 6.4 19.9 5.2 11.1 10.8 10.3 12.4 10.2 12.4 8.7 6.5 8.7 6.3 2.1 1.8 5.5 9.6 6.5 15.4 6.7 7.5 5.8 3.2 8.1 2.7 6.3 6.4 10.3 8.7 3.7 3.5 4.5 3.0

10 5 5 5 5 10 10 10 10 10 10 10 10 10 10 10 50 100 50 50 500 100 5 10 20 200 10 100 50 250 50 10 20 5 20 20 250 250 250 10 20 50

110 100 95 90 105 78 81 52 49 52 59 45 49 51 41 45 55 85 84 92 85 91 79 92 69 78 105 120 79 82 72 85 88 85 89 98 80 74 88 89 96 95

92 86 90 88 91 89 97 92 90 91 97 78 93 92 93 98 80 92 103 99 − 99 98 103 98 − 97 89 89 99 98 108 90 97 96 93 82 76 88 97 92 97

7.2 9.6 4.3 3.8 2.2 7.1 4.1 8.9 7.1 6.0 10.2 6.1 7.0 2.9 7.1 4.1 9.2 6.9 9.9 7.0 − 11.8 6.4 6.7 2.7 − 10.1 9.2 7.3 3.3 5.2 3.0 6.4 7.3 5.0 4.2 5.4 5.7 6.4 9.1 5.4 9.4

10 5 5 5 5 50 20 50 20 20 20 20 20 20 50 50 100 250 100 100 1000 100 5 10 20 1000 10 250 100 500 250 20 20 5 20 20 500 500 500 10 20 50

91 95 96 89 101 81 82 60 58 61 65 49 52 38 60 55 62 105 100 110 82 105 82 98 82 61 99 104 75 80 74 70 101 91 79 115 69 65 124 85 87 97

Analysis of spiked samples at 500 μg/kg was performed in 7 replicates. 6635

DOI: 10.1021/acs.jafc.5b02105 J. Agric. Food Chem. 2015, 63, 6633−6643

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Journal of Agricultural and Food Chemistry

Figure 1. Mean concentrations of mycotoxins (μg/kg) determined in the tested dietary supplements: (a) milk thistle-based supplements, n = 32; (b) supplements for menopause effects treatment, n = 9; (c) supplements for the general health improvement, n = 28. Maximum concentrations are noted in brackets. 1/2LOQ of analytes was used for samples without findings. Abbreviations used: 3-ADON, 3-acetyldeoxynivalenol; AME, alternariol-methyl ether; AOH, alternariol; BEA, beauvericin; DAS, diacetoxyscirpenol; DON, deoxynivalenol; ENN-A, enniatin A; ENN-A1, enniatin A1; ENN-B, enniatin B; ENN-B1, enniatin B1; FB1, fumonisin B1; FB2, fumonisin B2; FB3, fumonisin B3; FUS-X, fusarenon X; HT-2, HT-2 toxin; MPA, mycophenolic acid; NEO, neosolaniol; OTA, ochratoxin A; PAT, patulin; PEN-A, penitrem A; STER, sterigmatocystin; T-2, T-2 toxin; TeA, tenuazonic acid; TEN, tentoxin; ZEN, zearalenone. graphic separation was carried out with an Acquity UPLC HSS T3 column (100 × 2.1 mm i.d., 1.8 μm; Waters, United States). The working parameters of the separation−detection method were set according to Dzuman et al.:17 column temperature, 40 °C; injection volume, 2.0 μL; and autosampler temperature, 10 °C. The mobile phases were different for ESI(+) and ESI(−) analyses. ESI(+) employed 5 mM ammonium formate and 0.2% (v/v) formic acid in Milli-Q water and in MeOH as mobile phases A1 and B1, respectively. In ESI(−), 5 mM ammonium acetate in Milli-Q water was used as the mobile phase A2 and pure MeOH as B2. The gradient was different in ESI(−) and ESI(+). In ESI(+), the initial composition of mobile phases was 10% (v/v) of B1 at 0.35 mL/min followed by a linear change to 50% B1 in 1 min, a slower linear gradient from to 100% of B1 in 8.5 min followed, simultaneously with flow change to 0.55 mL/ min. The column was then washed for 2 min at 0.7 mL/min with 100% organic solvent and reconditioned for 2 min with 10% of B1 (0.35 mL/min). In ESI(−), the initial mobile phase composition was 10% of B2 at 0.35 mL/min followed by a linear change to 50% of B2 in 1 min. A linear gradient to 100% of B2 with linear increase of the flow

rate to 0.5 mL/min in 6.5 min was followed by a column wash with 100% of B2 for 2 min at 0.7 mL/min. The column was then reconditioned for 2 min using the starting composition of the mobile phases at 0.35 mL/min. The mass spectrometric detection was performed by QTRAP 5500 tandem mass spectrometer equipped with an electrospray (ESI) ion source operated in both positive and negative modes. The ESI(+) ion source parameters were as follows: needle voltage 4 500 V and turbo gas temperature, 500 °C. In ESI(−): needle voltage −4 500 V and turbo gas temperature 450 °C. Gas pressures were the same for both ESI(+) and ESI(−): curtain gas 241 kPa (35 psi), nebulizer and turbo gases 379 kPa (55 psi). Scheduled multiple reaction monitoring (MRM) methods were used in both ESI(+) and ESI(−) with the cycle time 0.6 and 1 s, respectively. Declustering potential (DP), collision energy (CE), collision cell exit potential (CXP), and particular MRM transitions of mycotoxins and all analyte-dependent parameters are summarized in the Supporting Information. Method Validation. For validation of the method performance characteristics (recoveries, repeatabilities, and limits of quantitation, 6636

DOI: 10.1021/acs.jafc.5b02105 J. Agric. Food Chem. 2015, 63, 6633−6643

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Journal of Agricultural and Food Chemistry

Figure 2. Histogram showing co-occurrence of mycotoxins in analyzed samples (black, milk thistle-based supplements, n = 32; dark gray, supplements for menopause effects treatment, n = 9; light gray, supplements for general health improvement, n = 28). LOQs) we selected “blank” matrices with very low or no mycotoxin content, which also represented matrices with different chemical composition to verify the method rugedness. Based on these considerations, powdered milk thistle extract (sample 16), oil-based milk thistle preparation (sample 9), and dry nettle tea (sample 10) were used. The external matrix-matched calibration standards at levels 0.5, 1, 2, 5, 10, 25, 50, 100, and 200 μg/L (corresponding to 5−2 000 μg/kg in the sample) were prepared. The repeatability of the method expressed as a relative standard deviation (RSD) was assessed by analysis of spiked samples (mixed standard was added to the “blank” matrix before extraction at 500 μg/kg in seven replicates). LOQs were estimated as the lowest matrix-matched calibration standard levels which provided signal-to-noise ratio (S/N) higher than 10 at the quantifier and 3 at the qualifier ion transitions. For calculation of SSE (signal suppression/enhancement), matrix-matched calibration standards and solvent standards (acetonitrile) at 100 μg/L were used. Validation results are summarized in Table 1. Quantitation of Mycotoxins in Real Samples. Because medical plant-based extracts used for dietary supplements production represent very diverse matrices, also the matrix induced ionization suppression/ enhancement can be different for various mycotoxin/matrix combinations. To minimize potential bias in calculated results, the standard addition approach was chosen for quantitation, to compensate for matrix effects. The practical realization of the standard addition method was as follows. First, the set of samples was preliminarily screened and detected mycotoxins quantitated using generic matrix-matched standards (prepared using the “blank” matrix employed in the method validation). Then, the standard addition was performed by spiking the sample after extraction at three different concentration levels (referring to approximately 1 times, 3 times, and 5 times of the estimated concentration). A calibration curve was created by plotting the added concentrations (x-axis) against the peak areas measured in samples with standard addition after extraction (y-axis). The native extract (without any standard addition) was included in the regression using a concentration of zero. The analyte concentration in the sample was calculated from the linear regression formula according to eq 1, where the “k” is the calibration curve slope.

c (μg/kg) = Area(sample)/k

All the results were corrected for recovery calculated based on the standard added before and after the extraction procedure. Arithmetic means of mycotoxins concentrations (presented in Figure 1 and the Supporting Information) were calculated using determined mycotoxin concentrations and, in the case of no detection, 1/2 of LOQ value (micrograms per kilogram) for a given mycotoxin (LOQs derived from the milk thistle powder matrix were used for capsules with dried powder and for tablets; LOQs derived from nettle tea matrix were employed for herbal teas; and LOQs derived from the oil-based milk thistle matrix were utilized for capsules with oil-based content). Dietary Intake Calculation. Tolerable daily intake (TDI) values are set by the Health and Consumer Protection Directorate General of the European Commission for the following mycotoxins: 1 μg/kg body weight (bw) for DON, 0.1 μg/kg bw for the sum of HT-2 and T2, 0.25 μg/kg bw for ZEN, 2 μg/kg bw for sum of FB1 and FB2, 0.12 μg/kg bw for OTA, and 0.4 μg/kg bw for PAT.18 Overview of the above-mentioned TDIs is stated in Commission Regulation 1881/ 2006. For these mycotoxins, the percentage of TDI obtained by consumption of the tested dietary supplements was calculated. For the mycotoxin exposure calculation (in micrograms of the mycotoxin per kilogram of human bw), the average adult body weight (70 kg) and the recommended dosage of the particular dietary supplement declared by the manufacturer were considered.

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RESULTS AND DISCUSSION As dicussed in the Introduction, beside health-promoting biologically active components, also various contaminants, such as mycotoxins, may occur in botanical-based dietary supplements. Until now, only limitted information has been available on their presence. To get more comprehensive information on the mycotoxins pattern in this commodity, multianalyte method targeting altogether 57 toxic secondary fungal metabolites was employed for analysis of 69 samples obtained in the Czech and US market. Rather unexpectedly, almost 96% of all samples (66 of the 69) contained detectable mycotoxins. As shown in Figure 1 and the Supporting Information, 25 of 57 mycotoxins involved in the method scope were quantitated.

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DOI: 10.1021/acs.jafc.5b02105 J. Agric. Food Chem. 2015, 63, 6633−6643

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Journal of Agricultural and Food Chemistry

Figure 3. Concentration of mycotoxins (micrograms per kilogram) found in dietary supplements made from milk thistle: Fusarium mycotoxins for which the TDI has been established (a); other Fusarium mycotoxins (b); other Alternaria, Aspergillus, and Penicillium mycotoxins (c). Abbreviations used: 3-ADON, 3-acetyldeoxynivalenol; AME, alternariol-methyl ether; AOH, alternariol; BEA, beauvericin; DAS, diacetoxyscirpenol; DON, deoxynivalenol; ENN-A, enniatin A; ENN-A1, enniatin A1; ENN-B, enniatin B; ENN-B1, enniatin B1; FB3, fumonisin B3; FUS-X, fusarenon X; HT-2, HT-2 toxin; MPA, mycophenolic acid; NEO, neosolaniol; STER, sterigmatocystin; T-2, T-2 toxin; TDI, tolerable daily intake; TeA, tenuazonic acid; TEN, tentoxin; ZEN, zearalenone. 6638

DOI: 10.1021/acs.jafc.5b02105 J. Agric. Food Chem. 2015, 63, 6633−6643

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Journal of Agricultural and Food Chemistry

hundreds of micrograms per kilogram. The mean concentration of TeA (143 μg/kg) exceeded the mean concentrations of other Alternaria mycotoxins 2-fold. Also, the Penicillium mycotoxins, PEN-A and MPA, were determined in the discussed supplements at average concentrations of 15 and 557 μg/kg, respectively. The highest concentration were detected for MPA (2 580 μg/kg, sample 6), ENN-B (1 230 μg/kg, sample 6), and TeA (1 200 μg/kg, sample 9). For detailed information about the sample contamination, see the Supporting Information. As in the case of other plants and agricultural commodities, also the soy, red clover, flax seed, and black cohosh can be affected by fungi in the field, as well as during the storage, when the appropriate agricultural and manufacturing practices are not followed. The most important factors probably include application of fungicides, air humidity, and temperature during the harvest and water activity during the storage. Other Dietary Supplements Intended for General Health Improvement. The last group of investigated samples included dietary supplements containing various antioxidants, based on a large number of different herbs and plants (green barley/wheat, nettle (Urtica dioica L.), goji (Lycium barbarum Mill.), yucca (Yucca spp. L.), boswellia (Boswellia serrata Roxb. ex Colebr.), acerola (Malpighia glabra L.), magnolia (Magnolia L.), guarana (Paullinia cupana Kunth ex H.B.K.), orange peel, turmeric root, black cohosh (Actaea racemosa L.), bacopa monnieri (Bacopa monnieri L.), Chinese jujube, and green tea leaves). From a total number of 28 samples, 25 of them were positive for at least one mycotoxin. Dietary supplements with a very low or no mycotoxin contamination were made of green wheat, green barley, acerola, and boswellia. Preparations based on nettle contained predominantly ENNs, BEA, AOH, TEN, and MPA at concentrations of tens up to hundreds of micrograms per kilogram (with the exception of nettle tea, sample 9, which was almost mycotoxin free). One sample of dried goji berry tea contained an extremely high concentration of TeA (6 780 μg/kg, sample 12), whereas no mycotoxins were detected in another sample of goji berry tea (sample 11). Regarding yucca-based preparations, only one sample from four investigated (sample 16) contained mycotoxins, ENNs (5−37 μg/kg) and Alternaria toxins (191−261 μg/kg). In the black cohosh sample (sample 18), rather high concentrations of FBs (110 and 101 μg/kg for FB1 and FB2, respectively), ZEN (45 μg/kg), and Alternaria toxins (123 and 129 μg/kg for AOH and AME, respectively) were determined. The main mycotoxins in an orange peel extract sample (sample 20) were FBs (141 and 191 μg/kg for FB1 and FB2, respectively). Turmeric powder samples (samples 24 and 25) contained mainly BEA (up to 215 μg/kg), FB2 (29 μg/kg), AME (27 μg/kg), ENN-B (5 μg/kg), NEO (8 μg/kg), and STE (11 μg/kg). In guarana bark powder (sample 26), BEA (21 μg/kg) and Alternaria toxins were found (169 and 106 μg/kg for AOH and AME, respectively). In the sample of Chinese jujube, magnolia bark, and green tea (samples No 23, 27, and 28, respectively), high concentrations of MPA were present (3 260, 1 380, and 304 μg/kg, respectively), and also very high levels of Alternaria toxins in the first two samples (samples 23 and 27). Magnolia bark (sample 27) contained 1 460 and 1 080 μg/kg of AOH and AME, and in Chinese jujube (sample 23), these two mycotoxins reached the concentration of 692 and 889 μg/kg, respectively. In bacopa monnieri-based sample (sample 21), 824 μg/kg of ZEN, 218 μg/kg of AME, and 956 μg/kg of OTA were determined.

The highest incidence and concentrations were observed for Fusarium mycotoxins (DON, HT-2/T-2, ZEN, and enniatins, ENNs); Alternaria mycotoxins (AOH, AME, TEN); and MPA. The comments on the contamination of particular sample groups are discussed in detail below. Milk Thistle-Based Dietary Supplements. Dietary supplements derived from the milk thistle were clearly the most contaminated samples from all three sample groups. As shown in Figure 1a and in the Supporting Information, in all of 32 tested samples at least one mycotoxin was present, and also the co-occurrence of more than one mycotoxin was very frequent as shown in Figure 2. The majority of the samples contained mycotoxins DON, ZEN, HT-2, and T-2. Moreover, a wide range of the newly emerging mycotoxins (other Fusarium mycotoxins such as 3-ADON, FUS-X, DAS; and NEO, ENNs, BEA; Alternaria mycotoxins such as AOH, AME, TEN; TeA; and several Penicillium and Aspergillus mycotoxins, from which the MPA was the most concentrated) were present. The frequency of occurrence of the highly abundant mycotoxins was 13−78% for trichothecenes, 78% for ZEN, 84−91% for ENNs, and 22−97% for Alternaria toxins. The average concentrations of trichothecene mycotoxins, ENNs, and Alternaria toxins were occurring typically in order of units of milligrams per kilogram (see Figure 3a−c and the Supporting Information). The highest concentration levels were observed for ENNs (2 340−10 900 μg/kg in samples 31 and 32), DON (2 890 μg/kg in sample 31), and AOH (4 560 μg/kg in sample 32). As mentioned in the Introduction, high levels of mycotoxins in dietary supplements can be associated with the use of lowquality raw materials for their production. The low quality of the raw material could relate to nonoptimal or unsuitable conditions during plant growing, harvesting, and storage. On the basis of the results of mycotoxins in milk thistle-based dietary supplements determined in our study, we contacted several Czech and European milk thistle producers to communicate the potential health risk and possible reasons for such high contamination of milk thistle-based products. As we have learned, the critical point influencing the spreading of fungi on the milk thistle heads and seeds is the humidity during harvesting. Wet and humid weather is needed during the harvest because, under these conditions, the milk thistle heads are closed, and losses of achens (seeds) are thereby prevented. After the harvest, the proper drying to 12% humidity should be performed. The weather during the harvest and the postharvest drying were assessed as the critical parameters in several studies.4,19,20 The truth is that only a small percentage of milk thistle intended for dietary supplements production is grown in Europe; the majority of the current commercial milk thistle production comes from Argentina and China. The European pharmaceutical companies or dietary supplements producers often purchase the “ready-to-use”, dried ethanolic extracts. Phytoestrogenic Dietary Supplements. Regarding the dietary supplements based on soy, red clover, flax seed, and black cohosh intended for menopause effects treatment, all of the nine examined samples were contaminated with at least one mycotoxin. The most frequently found mycotoxins included ENNs, Alternaria toxins, and MPA. Their incidence was 67− 78% for ENNs, 11−67% for Alternaria toxins, 67% for MPA. However, the average concentrations of Fusarium and Alternaria mycotoxins in these preparations were approximately one or two orders of magnitude lower than those found in milk thistle-based preparations. The average concentrations of T-2, ENNs, AOH, AME, and TEN were in order of tens to 6639

DOI: 10.1021/acs.jafc.5b02105 J. Agric. Food Chem. 2015, 63, 6633−6643

Article

Journal of Agricultural and Food Chemistry Table 2. Percent Values of TDIa for Particular Dietary Supplements main health effect declared liver treatment

sample 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

menopause effects treatment

general health improvement

26 27 28 29 30 31 32 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

recommended dosage 2 capsules 1 capsule 2 tea bags 1 tablet 1 tablet 14 g 1 capsule 2 capsules 1 capsule 3 small spoons 1 capsule 1 tablet 1 capsule 1 capsule 10 capsules not provided 1 capsule 2 spoons of seeds 1 tablet 1 tablet 1 tablet 1 small spoon of seeds 1 small spoon of seeds 1 small spoon of seeds 1 small spoon of seeds 2 capsules 2 capsules 2 capsules 2 capsules 2 capsules 2 capsules 2 capsules 2 tablets 2 tablets 1 capsule 2 capsules 2 tablets 2 tablets 2 capsules 1 tablet 3 tablets 10 g 10 g 4g 4g 4g 2 tea bags 6 tablets 1 capsule 10 g 1 tea bag 60 g 20 g 2 tablets 3 capsules 2 capsules 6 tablets

PMTDI SUMb FUMs (%)

TWIc OTA (%)

PMTDIb PAT (%)

m (g)

TDI DON (%)

TDI SUM T-2, HT-2 (%)

TDI ZEA (%)

1.1 0.4 4.0 0.7 0.9 14 0.6 1.9 0.4 19 1.4 1.0 0.5 0.7 3.8 0.5 0.4 19 0.4 0.3 0.9 6.0

2.3 0.3 − 1.6 − − − 0.7 − − − − − − 7.0 − 0.3 − 0.5 0.2 0.3 1.8

39.2 2.6 3.9 8.1 − − 1.3 7.0 − − 1.7 2.0 3.7 6.1 52.1 − 4.3 44.6 5.5 4.5 − 32.6

1.5 0.6 0.4 1.7 1.9 − 0.2 1.0 − − 2.4 − − 0.4 5.3 − 0.2 − 0.4 0.1 0.4 1.0

− − − − − − − − − − − − − − − − − − − − − −

− − − − − − − − − − − − − − − − − − − − − −

− − − − − − − − − − − − − − − − − − − − − −

6.0

2.3

57.3

2.3

−

−

−

6.0

1.3

74.8

1.6

−

−

−

6.0

2.5

37.9

3.8

−

−

−

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.1 1.2 0.4 1.0 1.2 1.8 0.9 0.7 1.5 10.0 10.0 4.0 4.0 4.0 6.0 3.0 0.5 50 3.0 60 20 1.0 1.5 1.0 3.0

− − 0.3 − 2.3 4.1 − − − − − − − − − − − − − − − − − − − − − − − − − −

2.6 2.6 5.1 6.8 30.4 48.5 − − − − 2.5 − − − − − − − − − − − − − − − − − − − − −

0.3 0.3 0.1 4.3 1.2 2.1 3.7 − 0.2 − 1.3 − 0.9 − − − − − − 0.1 − − 0.1 0.0 1.7 0.2 − − − − − −

− − − − − − 0.01 − − 0.01 − − − − − − − − − − − − − − − − − − − − − −

− − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − −

− − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − −

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Journal of Agricultural and Food Chemistry Table 2. continued main health effect declared

17 18 19 20 21 22 23 24 25 26 27 28 a

recommended dosage

sample 2 3 3 2 4 2 2 2 2 2 2 2

capsules tablets tablets tablets tablets tablets tablets tablets tablets tablets tablets tea bags

m (g)

TDI DON (%)

1.0 1.5 1.5 1 2 1 1 1 1 1 1 6

− − − − − − − − − − − −

TDI SUM T-2, HT-2 (%) − − − − − − − − − − − −

TDI ZEA (%)

PMTDI SUMb FUMs (%)

TWIc OTA (%)

PMTDIb PAT (%)

− 0.3 − − − 4.7 − 0.1 − − − −

− 0.2 0.01 0.2 0.04 − − − − − − −

− − − − − 11.4 − − − − − −

− − − − − − − − − − 1.4 −

Values are calculated for a 70 kg person. bProvisional maximum TDI. cTolerable weekly intake.

that they often dramatically exceeded even the highest maximum limits established for other food commodities. For example, in the bacopa-based tablets, 956 μg/kg of OTA was found (see the Supporting Information). The highest maximum regulatory limit for OTA, which is set for the liquorice extract, is 80 μg/kg.25 Therefore, the bacopa-based sample exceeded this level more than 10-fold. The other example can be given for the sum of HT-2 and T-2. The legislatively recommended maximum concentration level in cereals intended for human consumption is 200 μg/kg.27 As shown in the Supporting Information, the sum of HT-2 and T-2 in 56% of the investigated milk thistle-based preparations exceeded this value up to 17-times. Of course, this comparison of the mycotoxin concentrations determined in dietary supplements with the maximum limits set for other food commodities is meant as approximate only because the maximum limits consider not only the toxicity but also the quantities of the food which is usually consumed. The best way to express the risk associated with the mycotoxins occurrence in this commodity is to calculate the daily exposure based on the recommended dosage of the particular dietary supplement and compare the calculated value with the tolerable daily intake (TDI) given by the regulations.18 This exposure assessment is discussed in detail in the paragraph below. Exposure Assessment. To assess possible health risk associated with mycotoxins occurrence, the mycotoxin daily intake, calculated based on the recommended dosage of the respective dietary supplement, should be considered. Table 2 provides the information about fulfilling the TDI (in %) for mycotoxins, for which the TDI level (in micrograms per kilogram of body weight) have been set.18 The highest consumer exposure to mycotoxins occurs through the milk thistle-based preparations. Administration of the dose recommended by the manufacturers (usually, two capsules or tablets can cause fulfilling of up to approximately 50% of the TDI for the sum of HT-2 and T-2 (e.g., samples 1, 15, 18, 23, and 24 fulfilled the TDI of HT-2 and T-2 from 39, 52, 45, 57, and 75%, respectively). Considering the fact that the milk thistle preparations are mainly used by people who suffer from a liver disease, such high intake of immunotoxic, genotoxic, and hepatotoxic mycotoxins from the “health beneficial” preparations are of concern. Moreover, when taking into account high concentrations of mycotoxins co-occurring in cocktails, the toxicological impact might be even more serious. On the basis of the results obtained from the analysis of mycotoxins in the set of botanical-based dietary supplements,

There are probably similar reasons for the mycotoxin contamination of raw materials used for production of these dietary supplements as in the previously described cases (i.e., weather conditions in the field and agricultural and storage management). The recommendation on keeping the good agricultural and manufacturing practices should be commonly applied. Co-occurrence of Mycotoxins. We would like to emphasize that most of the samples were contaminated with multiple mycotoxins, i.e., more than one mycotoxin occurred in one sample. Figure 2 shows a histogram of multiply contaminated samples and demonstrates the relationship between the number of co-occurring mycotoxins and the dietary supplement type. For example, approximately 58% of milk thistle-based samples contained a “cocktail” of more than 12 mycotoxins together. On the other hand, a low number of co-occurring mycotoxins was determined for supplements made from green barley/wheat, nettle, goji, yucca, acerola, magnolia, guarana, orange peel, turmeric, black cohosh, bacopa monnieri, Chinese jujube, green tea leaf, and boswellia. Quite a high number of co-occurring mycotoxins was observed for supplements made from red clover, soy, flax, and black cohosh (preparations containing phytoestrogens); 22% of samples contained 4−7 mycotoxins, 33% of samples contained 8−11 mycotoxins, and 10.5% of samples contained more than 12 mycotoxins together in one sample. Interestingly, cooccurrence of mycotoxins representing various toxin classes was very common. The most frequent combination was Fusarium mycotoxins ENNs, HT-2/T-2, and Alternaria toxins (AOH, AME, TEN). The next very frequent combination was ENNs and Alternaria toxins (AOH, AME, TEN) and MPA. As discussed in several previous studies, the toxic effects resulting from the co-occurrence of multiple mycotoxins can be both additive and even synergistic, i.e., the overall toxicity might be even higher than the sum of individual toxicities.21−23 From this point of view, the co-occurrence of mycotoxins may pose an underestimated toxicological hazard because the impact of mycotoxin “cocktails” on human health is currently unknown. Mycotoxins and Legislation. As regards mycotoxins included in the EU legislation, OTA, PAT, DON, ZEA, FBs, HT-2, and T-2 were found in many samples in various combinations (for details see the discussion above).18,24−27 AFs were not detected in the investigated samples. There are no maximum limits established for mycotoxins in dietary supplements in the EU. Nevertheless, when considering the concentrations determined in our study, we can point out 6641

DOI: 10.1021/acs.jafc.5b02105 J. Agric. Food Chem. 2015, 63, 6633−6643

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Journal of Agricultural and Food Chemistry

E-corninine, ergocorninine; E-cristine, ergocristine; E-cristinine, ergocristinine; E-cryptine, ergocryptine; E-cryptinine, ergocryptinine; E-metrine, ergometrine; E-sine, ergosine; E-sinine, ergosinine; E-tamine, ergotamine; E-taminine, ergotaminine; FB1/B2/B3, fumonisin B1/B2/B3 FBs, fumonisins; FUS-X, fusarenon X; GLIO, gliotoxin; HT-2, HT-2 toxin; LOQ, limit of quantitation; MEL, meleagrin; MPA, mycophenolic acid; MRM, multiple reaction monitoring; MS/MS, tandem mass spectrometry; NEO, neosolaniol; NIV, nivalenol; OTA, ochratoxin A; OTB, ochratoxin B; OTs, ochratoxins; PAT, patulin; PAX, paxilline; PEN-Ac, penicillic acid; PEN-A, penitrem A; PTFE, polytetrafluorethylene; QuEChERS, quick, easy, cheap, effective, rugged, safe; ROQ-C, roquefortine C; RSD, relative standard deviation; S/N, signal-to-noise; SSE, signal suppression/enhancement; STACH, stachybotrylactam; STER, sterigmatocystin; T-2, T-2 toxin; TDI, tolerable daily intake; TEN, tentoxin; TeA, tenuazonic acid; U-HPLC, ultrahigh performance liquid chromatography; VER, verrucarol; Verruc, verruculogen; ZEN, zearalenone; α-ZEL, alphazearalenol; β-ZEL, beta-zearalenol

we can conclude that the preparations made from milk thistle were of the highest contamination; mycotoxin concentrations in the sum reached up to milligrams per kilogram. Effective communication of this topic with dietary supplement manufacturers and increased requirements on the quality of botanical-based raw material would be very helpful. The toxicological impact of many emerging mycotoxins is still unknown (especially when present in cocktails), and research focused on this topic should be performed. Nevertheless, considering the 75% fulfillment of the TDI for HT-2 and T-2 toxins at the recommended dosage of milk thistle preparations, it is obvious that the health risk problem should not be marginalized (especially for people who suffer from a liver disease).

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ASSOCIATED CONTENT

S Supporting Information *

Tables S1−S5: (S1) overview of studies dealing with analysis of mycotoxins in dietary supplements, (S2) description of the analyzed samples, (S3) analyte-dependent MS/MS parameters in the used U-HPLC-MS/MS method, (S4) concentrations of the mycotoxins in the sample groups, and (S5) concentrations of the detected mycotoxins in individual samples. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b02105.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +420 220 443 142. Fax: +420 220 443 184. University of Chemistry and Technology, Prague, Faculty of Food and Biochemical Technology, Department of Food Analysis and Nutrition, Technicka 3, Prague, Czech Republic, 166 28. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial support of the European Union Seventh Framework Programme (EP7/CSA/KBBE.2010.2.6-01) under Grant Agreement 266061, Safe Food for Europe − Coordination of research activities and dissemination of research results of EC funded research on food safety (FOODSEG), is gratefully acknowledged. The work was also supported by “Operational Program Prague − Competitivness” (CZ.2.16/3.1.00/22197), “National Program of Sustainability” (NPU I (LO) MSMT − 34870/2013), and Ministry of Education, Youth and Sports of the Czech Republic (Project AMVIS LH11059).

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ABBREVIATIONS USED 15-ADON, 15-acetyldeoxynivalenol; 3-ADON, 3-acetyldeoxynivalenol; A-clavine, agroclavine; AFB1/B2/G1/G2, aflatoxin B1/B2/G1/G2; AFs, aflatoxins; AME, alternariol-methyl ether; AOH, alternariol; ATE, altenuene; BEA, beauvericin; CE, collision energy; CIT, citrinin; CPA, cyclopiazonic acid; CXP, collision cell exit potential; DAS, diacetoxyscirpenol; DON, deoxynivalenol; DON-3-Glc, deoxynivalenol-3-glucoside; DP, declustering potential; d-SPE, dispersive solid phase extraction; EC, European Commission; EFSA, European Food Safety Authority; ENN-A, enniatin A; ENN-A1, enniatin A1; ENN-B, enniatin B; ENN-B1, enniatin B1; ENNs, enniatins; EP, entrance potential; ESI, electrospray; E-cornine, ergocornine; 6642

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