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Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20
Occurrence of enniatins and beauvericin in 60 Chinese medicinal herbs a
Ling Hu & Michael Rychlik a
ab
Analytical Food Chemistry, Technische Universität München, Freising, Germany
b
BIOANALYTIK Weihenstephan, ZIEL Research Center for Nutrition and Food Sciences, Technische Universität München, Freising, Germany Accepted author version posted online: 10 Apr 2014.Published online: 12 May 2014.
To cite this article: Ling Hu & Michael Rychlik (2014) Occurrence of enniatins and beauvericin in 60 Chinese medicinal herbs, Food Additives & Contaminants: Part A, 31:7, 1240-1245, DOI: 10.1080/19440049.2014.913813 To link to this article: http://dx.doi.org/10.1080/19440049.2014.913813
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Food Additives & Contaminants: Part A, 2014 Vol. 31, No. 7, 1240–1245, http://dx.doi.org/10.1080/19440049.2014.913813
Occurrence of enniatins and beauvericin in 60 Chinese medicinal herbs Ling Hua and Michael Rychlika,b* a Analytical Food Chemistry, Technische Universität München, Freising, Germany; bBIOANALYTIK Weihenstephan, ZIEL Research Center for Nutrition and Food Sciences, Technische Universität München, Freising, Germany
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(Received 19 February 2014; accepted 7 April 2014) A total of 60 Chinese medicinal herbs were examined for contamination of the emerging Fusarium mycotoxins enniatins (ENNs) A, A1, B, B1 and beauvericin (BEA). The herbs under study are commonly used in China as both medicines and food. The dried samples of herbs were randomly collected from traditional Chinese medicine stores in Zhejiang province, China. Sample preparation was achieved by methanol extraction, followed by a simple membrane filtration step; no tedious clean-ups were involved. ENNs A, A1, B, B1 and BEA were analysed by the recently developed stable isotope dilution assays, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). With limits of detection ranging between 0.8 and 1.2 µg kg–1 for the analytes under study, 25% of all analysed samples were contaminated with at least one of the ENNs and BEA. BEA was the most frequently detected toxin with a 20% incidence in all samples. The percentages of ENN-positive samples were lower: each single ENN was detected in 6.7–11.7% of all samples. Considering the total amounts of the five mycotoxins in single samples, values between 2.5 and 751 µg kg–1 were found. The mean total amount in positive samples was 126 µg kg–1. Regarding ginger, the frequent occurrence of ENNs and BEA in dried ginger could be confirmed in samples from Germany. However, in fresh ginger root the toxins were not detectable. This is the first report on the presence of ENNs and BEA in Chinese medicinal herbs. Keywords: enniatins; beauvericin; Fusarium; mycotoxin; Chinese medicinal herbs; ginger; stable isotope dilution assay
Introduction Enniatins (ENNs) and beauvericin (BEA) are emerging mycotoxins mainly produced by fungi of the Fusarium genus. They are structurally related cyclic hexadepsipeptides, consisting of three alternating hydroxyisovaleryl and N-methylamino acid residues (Hamill et al. 1969; Blais et al. 1992) (Figure 1). The most frequently reported ENNs as natural contaminants are ENNs A, A1, B and B1 (Mahnine et al. 2011). A wide range of biological activities of these mycotoxins have been reported. ENNs and BEA are toxic to insects (Grove & Pople 1980) and brine shrimp (Hamill et al. 1969; Tan et al. 2011). They also have inhibitory effects on sterol O-acyltransferase (also called acyl-CoA: cholesterol acyltransferase) (Tomoda et al. 1992) and, therefore, can interfere with lipid metabolism. Besides, they are toxic to several cell lines of human origin including hepatocellular carcinoma line Hep G2 and fibroblastlike foetal lung cell line MRC-5 (Ivanova et al. 2006). Furthermore, ENNs are also known to have phytotoxic (Burmeister & Plattner 1987) and antifungal activities (Pohanka et al. 2004), and BEA has been found to inhibit reversibly the L-type voltage-dependent Ca2+ current in a mouse neuroblastoma and rat glioma hybrid cell line, with an IC50 of 4 µM (Wu et al. 2002).
*Corresponding author. Email: [email protected] © 2014 Taylor & Francis
Due to their diverse toxic activities, ENNs and BEA have been drawing increasing attention. Their occurrence has been reported in cereals such as wheat, barley, maize and oats, as well as in cereal-based food. A high prevalence of ENNs and BEA was found in Norwegian grains in a survey by Uhlig et al. (2006), where 100% and 32% of the 228 grain samples (oats, barley and wheat) were contaminated with ENNs and BEA, respectively. In Denmark (Sørensen et al. 2008), 100% and 98% of the maize harvested in 2006 was contaminated with ENNs and BEA, respectively. In both reports, ENNs levels above 1000 µg kg–1 were found, and BEA levels above 100 µg kg–1 were detected. In later researches, even higher contamination levels of these mycotoxins have been reported. For instance, several hundreds of mg kg–1 of ENN A1 were found in cereals in Spain, with a maximum concentration of 814 mg kg–1 in a rice sample (Meca et al. 2010). Moreover, BEA levels of up to 11.8 mg kg–1 were detected (Meca et al. 2010). Similarly high concentrations were also found in breakfast cereals from Morocco (Mahnine et al. 2011). Although no maximum levels of ENNs and BEA have been regulated until now, their toxic effects in combination with high contamination levels could pose possible hazards to health. An EFSA opinion on the risks to human
Food Additives & Contaminants: Part A
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Figure 1.
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BEA: R1 = R2 = R3 = –CH2C6H5 ENN A: R1 = R2 = R3 = –CH(CH3)CH2CH3 ENN A1: R1 = R2 = –CH(CH3)CH2CH3, R3 = –CH(CH3)2 ENN B: R1 = R2 = R3 = –CH(CH3)2 ENN B1: R1 = R2 = –CH(CH3)2, R3 = –CH(CH3)CH2CH3
Chemical structures of ENNs A, A1, B, B1 and BEA.
and animal health related to ENNs and BEA in food and feed has thus been requested by the European Commission (EFSA 2010). In this study, 60 Chinese medicinal herbs used as both medicines and foods in China were selected to examine their contaminations with ENNs and BEA. These herbs, like other plants, could be infected with mycotoxin-producing fungi during growth, processing or storage if the conditions were favourable to fungi. Increasing research on mycotoxins in Chinese medicinal herbs has been carried out over the recent decade, including aflatoxins (Ip & Che 2006; Han et al. 2010), zearalenone and its analogues (Han et al. 2011), deoxynivalenol, nivalenol (Yue et al. 2010), fumonisins (Kong et al. 2012), and T-2 toxin (Yue et al. 2009), but none was concerned with ENNs and BEA up to now. The use of 15N3-labelled ENNs and BEA in stable isotope dilution assays developed in our previous study (Hu & Rychlik 2012) is especially beneficial for the analysis of various herbs. Because the matrices of different types of herbs are highly variable and complicated, external matrix calibration or standard addition would be tedious to overcome matrix effects frequently encountered in LC-MS/MS. In comparison, the use of stable isotopelabelled internal standards offers a better solution as they behave very similarly to the unlabelled analytes and can be distinguished from the latter due to mass differences (Rychlik & Asam 2008). The aim of this study is to determine for the first time the contamination levels of ENNs and BEA in 60 Chinese medicinal herbs using stable isotope dilution assays (SIDA).
Materials and methods Chemicals and reagents Methanol (MeOH) and acetonitrile (MeCN) of HPLC grade were purchased from Fisher Scientific (Pittsburgh, PA, USA). Water for HPLC was purified by a Milli-Q-system (Millipore, Bedford, MA, USA). BEA was obtained from AnaSpec (San Jose, CA, USA), ENN B was obtained from
Bioaustralis (Smithfield, NSW, Australia), and ENNs A, A1, B1 were purchased from Enzo Life Sciences (Lörrach, Germany). The internal standards [15N]3-ENN A, [15N]3-ENN A1, [15N]3-ENN B, [15N]3-ENN B1, and [15N]3-BEA were synthesised as reported recently (Hu & Rychlik 2012). Stock solutions of ENNs and BEA were dissolved in MeCN and kept in darkness at 4°C. Raw materials A total of 60 types of dried Chinese medicinal herbs were randomly purchased from traditional Chinese medicine stores in Zhejiang province, China. Additionally, six commercial ginger samples were obtained from Bavarian retail stores, of which five were dried to be used as spices or for herbal infusions and one of which was a fresh root. Their common names (pharmaceutical names) are as follows: Star anise (Fructus anisistellati), Hyacinth bean (Semen lablab album), Dahurian angelica root (Radix angelicae dahuricae), Ginkgo seed (Semen ginkgo), Lily bulb (Bulbus Lilii), Mint leaf (Herba menthae), Adzuki bean (Semen phaseoli), Colve (Flos caryophylli), Jack bean (Semen canavaliae), Finger citron (Fructus citri sarcodactylis), Fu-ling (Sclerotium poriae cocos), Chinese raspberry (Fructus rubi), Licorice root (Radix glycyrrhizae), Goji berry (Fructus lycii), Kudzuvine root (Radix puerariae), Pricklyash peel (Pericarpium zanthoxyli), Lotus leaf (Folium nelumbinis), Black sesame (Semen sesami nigrum), Black pepper (Fructus piperis nigri), Hemp fruit (Fructus cannabis), Sophora flower (Flos sophorae), Wrinkled giant hyssop herb (Herba agastaches), Sicklepod (Semen cassiae), Honeysuckle flower (Flos lonicerae), Ginger (Rhizoma zingiberis recens), Chrysanthemum flower (Flos chrysanthemi), Tangerine peel (Pericarpium citri reticulatae), Red tangerine peel (Exocarpium citri rubrum), Chicory (Herba cichorii), Platycodon root (Radix platycodi), Kombu (Thallus laminariae), Longan pulp (Arillus longan), Luohanguo (Fructus momordicae), Lotus seed (Semen nelumbinis), Common purslane (Herba portulacae), Chinese quince fruit (Fructus chaenomelis), Boat-fruited sterculia seed (Semen sterculiae lychnophorae), Dandelion herb (Herba
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L. Hu and M. Rychlik
taraxaci), Gordon euryale seed (Semen euryales), nutmeg (Semen myristicae), Cassia bark (Cortex cinnamomi), Chinese yam (Radix dioscoreae oppositae), Hawthorn fruit (Fructus crataegi), Seabuckthorn berry (Fructus hippophae), Mulberry leaf (Folium mori), Mulberry fruit (Fructus mori), Spina date seed (Semen ziziphi spinosae), Peach kernel (Semen persicae), Smoked plum (Fructus mume), Fennel (Fructus foeniculi), Bitter apricot seed (Semen armeniacae amarum), Cogongrass rhizome (Rhizoma imperatae), Reed rhizome (Rhizoma phragmitis), Longstamen onion bulb (Bulbus allii macrostemi), Bitter cardamom (Fructus alpiniae oxyphyllae), Heartleaf houttuynia herb (Herba houttuyniae), Coix seed (Semen coicis), Chinese date (Fructus zizyphi), Cape jasmine fruit (Fructus gardeniae), Perilla leaf (Folium perillae), and Perilla seed (Semen perillae). Sample extraction All samples (approximately 100 g each) were ground into fine particles using a laboratory mill Chuangli CLF-30B (Wenling, China) equipped with different types of blades suitable for normal and viscous herbs and homogenised before extraction. A total of 1 g of each sample was weighed and spiked with 10 ng (100 µl of a 100 ng ml–1 solution in MeCN) of each of the labelled internal standards. After evaporation of the solvent, 10 ml of MeOH was added to each sample. The suspension was homogenised with a mixer (Hong-Hua, SK-1, Jiangsu, China) and extracted for 1.5 h on a shaker (160 rpm). Afterwards, the samples were centrifuged at 4800 rpm for 10 min, and filtered through a 0.22 µm membrane filter prior to HPLC. For samples that exceeded the upper linear range (10) of calibration curves, a second extraction was carried out, in which 1 g of the sample was spiked with 100 ng (100 µl of a 1 μg ml–1 solution in MeCN) of each of the labelled internal standards, and the following steps were the same as mentioned above.
in 3 min, and equilibrated for 4 min before next injection. Flow rate was kept at 0.2 ml min–1, and injection volume was 10 µl. As the retention times of ENNs and BEA were between 13 and 19 min, the effluent from the column was directed to the mass spectrometer from 11 to 21 min (expanded in case of retention time shifts) and to the waste for the rest of the run by a switching valve. Analyst 1.5 software (Applied Biosystems Inc., Foster City, CA, USA) was used for data acquisition and processing.
Method validation Due to the variable and complex components of the Chinese medicinal herbs, a mixture of four blank samples was used for method validation. To be representative of all the analysed samples, the blank surrogate mixture was made from lotus leaf (representative for leaf), chrysanthemum flower (representative for flower), kudzuvine root (representative for root), and black sesame (representative for seed), all of which were free of ENNs and BEA. Method validation was performed analogously to that reported recently (Hu & Rychlik 2012). Briefly, to draw calibration curves, 15N-labelled standards (S) were mixed with unlabelled analytes (A) in molar ratios between 0.1 and 10, and, after LC-MS/MS measurements, response curves were obtained from molar ratios between A and S versus peak area ratios between A and S, using linear regression. The contents of analytes were calculated from the response curves. For the determination of LODs, LOQs as well as for recoveries, 1 g of the mixture was spiked with unlabelled ENNs and BEA at four different levels (2, 5, 15 and 20 µg kg–1) in triplicate and analysed by LC-MS/MS. LODs and LOQs were calculated according to the method of Vogelgesang & Hädrich (1998). Intraday precision was determined with five measurements within the same day; interday precision was determined with triplicate measurements in three different days within 2 weeks.
Mycotoxin analysis Analysis of ENNs and BEA was carried out by LC-MS/ MS. A Shimadzu LC-20A Prominence LC system (Shimadzu, Kyoto, Japan) was coupled to a hybrid triple–quadrupole/linear ion-trap mass spectrometer (API 4000 QTrap; Applied Biosystems Inc., Foster City, CA, USA) and operated in positive ESI and MRM mode. MS parameters were identical with those of our previous study (Hu & Rychlik 2012). Separation was achieved by a YMC-Pack ProC18 column (150 × 3.0 mm i.d., 3 µm particle size; YMC Europe GmbH, Dinslaken, Germany). The starting mobile phase MeCN/H2O (80:20, v/v) was maintained constant for 5 min, then linearly raised to 100% MeCN in 10 min, held for 3 min before returning to the starting conditions
Results and discussion Method validation The SIDA presented here revealed good linearity with good correlation coefficients (R2 = 0.992–0.998) from the calibration curves. Mean recoveries of the blank mixture sample spiked with 15N3-labelled ENNs and BEA at levels of 2–20 µg kg–1 were 93–109% for ENNs and 93–101% for BEA, with RSDs of 2.1–8.7% and 2.5– 5.6% for ENNs and BEA, respectively. LODs were 0.8 (ENN A), 1.1 (ENN A1), 1.0 (ENN B), 1.1 (ENN B1), and 1.2 (BEA) µg kg–1. LOQs were 2.5 (ENN A), 3.4 (ENN A1), 2.8 (ENN B), 3.2 (ENN B1), and 3.7 µg kg–1 (BEA). Inter-day (n = 3) precision calculated as
Food Additives & Contaminants: Part A coefficients of variation was 2.8–7.1% for ENNs and 5.8% for BEA; intra-day (n = 5) precision given in coefficients of variation was 3.5–6.3% for ENNs and 4.7% for BEA. Samples with contamination levels above the LODs were considered to be positive; concentrations between the LOD and the LOQ were considered as the mean of the LOD and the LOQ.
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Occurrence of ENNs and BEA in medicinal herbs Among all 60 medicinal herbs under study, 15 samples were found to be contaminated with at least one of the ENNs and BEA, thus resulting in a frequency of contamination of 25%. The contamination levels of ENN and BEA in positive samples are detailed in Table 1. The mean concentrations of positive samples were 28.9, 28.4, 32.0, 3.9 and 33.0 µg kg–1 for ENN A, ENN A1, ENN B, ENN B1 and BEA, respectively. Maximum levels of the single toxins were 355 µg kg–1 (ENN A), 253 µg kg–1 (ENN A1), 290 µg kg–1 (ENN B), 40.2 µg kg–1 (ENN B1), and 125 µg kg–1 (BEA). Ginger revealed the highest levels of ENNs A and ENN A1; cogongrass rhizome contained the highest levels of Table 1.
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ENN B and BEA; and the highest level of ENN B1 was found in long-stamen onion bulb. BEA was the most frequently detected single mycotoxin in this study, with 12 positive samples, i.e. 20% of all examined samples. The incidences of ENNs were lower, with ENN B being most prevalent and detectable in seven samples of all, i.e. 11.7% and ENN A1 and B1 being least prevalent in four samples, i.e. 6.7% of all samples. As ginger is also a commonly used herb in Europe, we analysed six further ginger samples obtained from Bavarian stores. The results (Table 2) of the dried samples underlined the common occurrence of the toxins under study in ginger, whereas in the Bavarian samples BEA was the predominant toxin and the ENNs did not reach the contents found in the Chinese sample. Interestingly, the fresh ginger root did not contain detectable amounts of ENNs and BEA, which points to the assumption that toxin contamination or production occurs during drying of the herb. Considering the total amounts of the five mycotoxins in the single samples of all herbs, values between 2.5 and 751 µg kg–1 were found. The mean total amount in positive samples was 126 µg kg–1. The maximum total amount of the five mycotoxins again was found in ginger, and the
Concentrations (μg kg–1) of ENNs and BEA in positive samples.
Name Mint leaf Licorice root Black pepper Wrinkled giant hyssop herb Ginger Platycodon root Chinese quince fruit Mulberry leaf Spina date seed Smoked plum Bitter apricot seed Cogongrass rhizome Longstamen onion bulb Bitter cardamom Heartleaf houttuynia herb
ENN A
ENN A1
ENN B
ENN B1
BEA
Totala
Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20
Occurrence of enniatins and beauvericin in 60 Chinese medicinal herbs a
Ling Hu & Michael Rychlik a
ab
Analytical Food Chemistry, Technische Universität München, Freising, Germany
b
BIOANALYTIK Weihenstephan, ZIEL Research Center for Nutrition and Food Sciences, Technische Universität München, Freising, Germany Accepted author version posted online: 10 Apr 2014.Published online: 12 May 2014.
To cite this article: Ling Hu & Michael Rychlik (2014) Occurrence of enniatins and beauvericin in 60 Chinese medicinal herbs, Food Additives & Contaminants: Part A, 31:7, 1240-1245, DOI: 10.1080/19440049.2014.913813 To link to this article: http://dx.doi.org/10.1080/19440049.2014.913813
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Food Additives & Contaminants: Part A, 2014 Vol. 31, No. 7, 1240–1245, http://dx.doi.org/10.1080/19440049.2014.913813
Occurrence of enniatins and beauvericin in 60 Chinese medicinal herbs Ling Hua and Michael Rychlika,b* a Analytical Food Chemistry, Technische Universität München, Freising, Germany; bBIOANALYTIK Weihenstephan, ZIEL Research Center for Nutrition and Food Sciences, Technische Universität München, Freising, Germany
Downloaded by [University of Winnipeg] at 08:14 08 September 2014
(Received 19 February 2014; accepted 7 April 2014) A total of 60 Chinese medicinal herbs were examined for contamination of the emerging Fusarium mycotoxins enniatins (ENNs) A, A1, B, B1 and beauvericin (BEA). The herbs under study are commonly used in China as both medicines and food. The dried samples of herbs were randomly collected from traditional Chinese medicine stores in Zhejiang province, China. Sample preparation was achieved by methanol extraction, followed by a simple membrane filtration step; no tedious clean-ups were involved. ENNs A, A1, B, B1 and BEA were analysed by the recently developed stable isotope dilution assays, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). With limits of detection ranging between 0.8 and 1.2 µg kg–1 for the analytes under study, 25% of all analysed samples were contaminated with at least one of the ENNs and BEA. BEA was the most frequently detected toxin with a 20% incidence in all samples. The percentages of ENN-positive samples were lower: each single ENN was detected in 6.7–11.7% of all samples. Considering the total amounts of the five mycotoxins in single samples, values between 2.5 and 751 µg kg–1 were found. The mean total amount in positive samples was 126 µg kg–1. Regarding ginger, the frequent occurrence of ENNs and BEA in dried ginger could be confirmed in samples from Germany. However, in fresh ginger root the toxins were not detectable. This is the first report on the presence of ENNs and BEA in Chinese medicinal herbs. Keywords: enniatins; beauvericin; Fusarium; mycotoxin; Chinese medicinal herbs; ginger; stable isotope dilution assay
Introduction Enniatins (ENNs) and beauvericin (BEA) are emerging mycotoxins mainly produced by fungi of the Fusarium genus. They are structurally related cyclic hexadepsipeptides, consisting of three alternating hydroxyisovaleryl and N-methylamino acid residues (Hamill et al. 1969; Blais et al. 1992) (Figure 1). The most frequently reported ENNs as natural contaminants are ENNs A, A1, B and B1 (Mahnine et al. 2011). A wide range of biological activities of these mycotoxins have been reported. ENNs and BEA are toxic to insects (Grove & Pople 1980) and brine shrimp (Hamill et al. 1969; Tan et al. 2011). They also have inhibitory effects on sterol O-acyltransferase (also called acyl-CoA: cholesterol acyltransferase) (Tomoda et al. 1992) and, therefore, can interfere with lipid metabolism. Besides, they are toxic to several cell lines of human origin including hepatocellular carcinoma line Hep G2 and fibroblastlike foetal lung cell line MRC-5 (Ivanova et al. 2006). Furthermore, ENNs are also known to have phytotoxic (Burmeister & Plattner 1987) and antifungal activities (Pohanka et al. 2004), and BEA has been found to inhibit reversibly the L-type voltage-dependent Ca2+ current in a mouse neuroblastoma and rat glioma hybrid cell line, with an IC50 of 4 µM (Wu et al. 2002).
*Corresponding author. Email: [email protected] © 2014 Taylor & Francis
Due to their diverse toxic activities, ENNs and BEA have been drawing increasing attention. Their occurrence has been reported in cereals such as wheat, barley, maize and oats, as well as in cereal-based food. A high prevalence of ENNs and BEA was found in Norwegian grains in a survey by Uhlig et al. (2006), where 100% and 32% of the 228 grain samples (oats, barley and wheat) were contaminated with ENNs and BEA, respectively. In Denmark (Sørensen et al. 2008), 100% and 98% of the maize harvested in 2006 was contaminated with ENNs and BEA, respectively. In both reports, ENNs levels above 1000 µg kg–1 were found, and BEA levels above 100 µg kg–1 were detected. In later researches, even higher contamination levels of these mycotoxins have been reported. For instance, several hundreds of mg kg–1 of ENN A1 were found in cereals in Spain, with a maximum concentration of 814 mg kg–1 in a rice sample (Meca et al. 2010). Moreover, BEA levels of up to 11.8 mg kg–1 were detected (Meca et al. 2010). Similarly high concentrations were also found in breakfast cereals from Morocco (Mahnine et al. 2011). Although no maximum levels of ENNs and BEA have been regulated until now, their toxic effects in combination with high contamination levels could pose possible hazards to health. An EFSA opinion on the risks to human
Food Additives & Contaminants: Part A
O R1
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Figure 1.
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BEA: R1 = R2 = R3 = –CH2C6H5 ENN A: R1 = R2 = R3 = –CH(CH3)CH2CH3 ENN A1: R1 = R2 = –CH(CH3)CH2CH3, R3 = –CH(CH3)2 ENN B: R1 = R2 = R3 = –CH(CH3)2 ENN B1: R1 = R2 = –CH(CH3)2, R3 = –CH(CH3)CH2CH3
Chemical structures of ENNs A, A1, B, B1 and BEA.
and animal health related to ENNs and BEA in food and feed has thus been requested by the European Commission (EFSA 2010). In this study, 60 Chinese medicinal herbs used as both medicines and foods in China were selected to examine their contaminations with ENNs and BEA. These herbs, like other plants, could be infected with mycotoxin-producing fungi during growth, processing or storage if the conditions were favourable to fungi. Increasing research on mycotoxins in Chinese medicinal herbs has been carried out over the recent decade, including aflatoxins (Ip & Che 2006; Han et al. 2010), zearalenone and its analogues (Han et al. 2011), deoxynivalenol, nivalenol (Yue et al. 2010), fumonisins (Kong et al. 2012), and T-2 toxin (Yue et al. 2009), but none was concerned with ENNs and BEA up to now. The use of 15N3-labelled ENNs and BEA in stable isotope dilution assays developed in our previous study (Hu & Rychlik 2012) is especially beneficial for the analysis of various herbs. Because the matrices of different types of herbs are highly variable and complicated, external matrix calibration or standard addition would be tedious to overcome matrix effects frequently encountered in LC-MS/MS. In comparison, the use of stable isotopelabelled internal standards offers a better solution as they behave very similarly to the unlabelled analytes and can be distinguished from the latter due to mass differences (Rychlik & Asam 2008). The aim of this study is to determine for the first time the contamination levels of ENNs and BEA in 60 Chinese medicinal herbs using stable isotope dilution assays (SIDA).
Materials and methods Chemicals and reagents Methanol (MeOH) and acetonitrile (MeCN) of HPLC grade were purchased from Fisher Scientific (Pittsburgh, PA, USA). Water for HPLC was purified by a Milli-Q-system (Millipore, Bedford, MA, USA). BEA was obtained from AnaSpec (San Jose, CA, USA), ENN B was obtained from
Bioaustralis (Smithfield, NSW, Australia), and ENNs A, A1, B1 were purchased from Enzo Life Sciences (Lörrach, Germany). The internal standards [15N]3-ENN A, [15N]3-ENN A1, [15N]3-ENN B, [15N]3-ENN B1, and [15N]3-BEA were synthesised as reported recently (Hu & Rychlik 2012). Stock solutions of ENNs and BEA were dissolved in MeCN and kept in darkness at 4°C. Raw materials A total of 60 types of dried Chinese medicinal herbs were randomly purchased from traditional Chinese medicine stores in Zhejiang province, China. Additionally, six commercial ginger samples were obtained from Bavarian retail stores, of which five were dried to be used as spices or for herbal infusions and one of which was a fresh root. Their common names (pharmaceutical names) are as follows: Star anise (Fructus anisistellati), Hyacinth bean (Semen lablab album), Dahurian angelica root (Radix angelicae dahuricae), Ginkgo seed (Semen ginkgo), Lily bulb (Bulbus Lilii), Mint leaf (Herba menthae), Adzuki bean (Semen phaseoli), Colve (Flos caryophylli), Jack bean (Semen canavaliae), Finger citron (Fructus citri sarcodactylis), Fu-ling (Sclerotium poriae cocos), Chinese raspberry (Fructus rubi), Licorice root (Radix glycyrrhizae), Goji berry (Fructus lycii), Kudzuvine root (Radix puerariae), Pricklyash peel (Pericarpium zanthoxyli), Lotus leaf (Folium nelumbinis), Black sesame (Semen sesami nigrum), Black pepper (Fructus piperis nigri), Hemp fruit (Fructus cannabis), Sophora flower (Flos sophorae), Wrinkled giant hyssop herb (Herba agastaches), Sicklepod (Semen cassiae), Honeysuckle flower (Flos lonicerae), Ginger (Rhizoma zingiberis recens), Chrysanthemum flower (Flos chrysanthemi), Tangerine peel (Pericarpium citri reticulatae), Red tangerine peel (Exocarpium citri rubrum), Chicory (Herba cichorii), Platycodon root (Radix platycodi), Kombu (Thallus laminariae), Longan pulp (Arillus longan), Luohanguo (Fructus momordicae), Lotus seed (Semen nelumbinis), Common purslane (Herba portulacae), Chinese quince fruit (Fructus chaenomelis), Boat-fruited sterculia seed (Semen sterculiae lychnophorae), Dandelion herb (Herba
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taraxaci), Gordon euryale seed (Semen euryales), nutmeg (Semen myristicae), Cassia bark (Cortex cinnamomi), Chinese yam (Radix dioscoreae oppositae), Hawthorn fruit (Fructus crataegi), Seabuckthorn berry (Fructus hippophae), Mulberry leaf (Folium mori), Mulberry fruit (Fructus mori), Spina date seed (Semen ziziphi spinosae), Peach kernel (Semen persicae), Smoked plum (Fructus mume), Fennel (Fructus foeniculi), Bitter apricot seed (Semen armeniacae amarum), Cogongrass rhizome (Rhizoma imperatae), Reed rhizome (Rhizoma phragmitis), Longstamen onion bulb (Bulbus allii macrostemi), Bitter cardamom (Fructus alpiniae oxyphyllae), Heartleaf houttuynia herb (Herba houttuyniae), Coix seed (Semen coicis), Chinese date (Fructus zizyphi), Cape jasmine fruit (Fructus gardeniae), Perilla leaf (Folium perillae), and Perilla seed (Semen perillae). Sample extraction All samples (approximately 100 g each) were ground into fine particles using a laboratory mill Chuangli CLF-30B (Wenling, China) equipped with different types of blades suitable for normal and viscous herbs and homogenised before extraction. A total of 1 g of each sample was weighed and spiked with 10 ng (100 µl of a 100 ng ml–1 solution in MeCN) of each of the labelled internal standards. After evaporation of the solvent, 10 ml of MeOH was added to each sample. The suspension was homogenised with a mixer (Hong-Hua, SK-1, Jiangsu, China) and extracted for 1.5 h on a shaker (160 rpm). Afterwards, the samples were centrifuged at 4800 rpm for 10 min, and filtered through a 0.22 µm membrane filter prior to HPLC. For samples that exceeded the upper linear range (10) of calibration curves, a second extraction was carried out, in which 1 g of the sample was spiked with 100 ng (100 µl of a 1 μg ml–1 solution in MeCN) of each of the labelled internal standards, and the following steps were the same as mentioned above.
in 3 min, and equilibrated for 4 min before next injection. Flow rate was kept at 0.2 ml min–1, and injection volume was 10 µl. As the retention times of ENNs and BEA were between 13 and 19 min, the effluent from the column was directed to the mass spectrometer from 11 to 21 min (expanded in case of retention time shifts) and to the waste for the rest of the run by a switching valve. Analyst 1.5 software (Applied Biosystems Inc., Foster City, CA, USA) was used for data acquisition and processing.
Method validation Due to the variable and complex components of the Chinese medicinal herbs, a mixture of four blank samples was used for method validation. To be representative of all the analysed samples, the blank surrogate mixture was made from lotus leaf (representative for leaf), chrysanthemum flower (representative for flower), kudzuvine root (representative for root), and black sesame (representative for seed), all of which were free of ENNs and BEA. Method validation was performed analogously to that reported recently (Hu & Rychlik 2012). Briefly, to draw calibration curves, 15N-labelled standards (S) were mixed with unlabelled analytes (A) in molar ratios between 0.1 and 10, and, after LC-MS/MS measurements, response curves were obtained from molar ratios between A and S versus peak area ratios between A and S, using linear regression. The contents of analytes were calculated from the response curves. For the determination of LODs, LOQs as well as for recoveries, 1 g of the mixture was spiked with unlabelled ENNs and BEA at four different levels (2, 5, 15 and 20 µg kg–1) in triplicate and analysed by LC-MS/MS. LODs and LOQs were calculated according to the method of Vogelgesang & Hädrich (1998). Intraday precision was determined with five measurements within the same day; interday precision was determined with triplicate measurements in three different days within 2 weeks.
Mycotoxin analysis Analysis of ENNs and BEA was carried out by LC-MS/ MS. A Shimadzu LC-20A Prominence LC system (Shimadzu, Kyoto, Japan) was coupled to a hybrid triple–quadrupole/linear ion-trap mass spectrometer (API 4000 QTrap; Applied Biosystems Inc., Foster City, CA, USA) and operated in positive ESI and MRM mode. MS parameters were identical with those of our previous study (Hu & Rychlik 2012). Separation was achieved by a YMC-Pack ProC18 column (150 × 3.0 mm i.d., 3 µm particle size; YMC Europe GmbH, Dinslaken, Germany). The starting mobile phase MeCN/H2O (80:20, v/v) was maintained constant for 5 min, then linearly raised to 100% MeCN in 10 min, held for 3 min before returning to the starting conditions
Results and discussion Method validation The SIDA presented here revealed good linearity with good correlation coefficients (R2 = 0.992–0.998) from the calibration curves. Mean recoveries of the blank mixture sample spiked with 15N3-labelled ENNs and BEA at levels of 2–20 µg kg–1 were 93–109% for ENNs and 93–101% for BEA, with RSDs of 2.1–8.7% and 2.5– 5.6% for ENNs and BEA, respectively. LODs were 0.8 (ENN A), 1.1 (ENN A1), 1.0 (ENN B), 1.1 (ENN B1), and 1.2 (BEA) µg kg–1. LOQs were 2.5 (ENN A), 3.4 (ENN A1), 2.8 (ENN B), 3.2 (ENN B1), and 3.7 µg kg–1 (BEA). Inter-day (n = 3) precision calculated as
Food Additives & Contaminants: Part A coefficients of variation was 2.8–7.1% for ENNs and 5.8% for BEA; intra-day (n = 5) precision given in coefficients of variation was 3.5–6.3% for ENNs and 4.7% for BEA. Samples with contamination levels above the LODs were considered to be positive; concentrations between the LOD and the LOQ were considered as the mean of the LOD and the LOQ.
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Occurrence of ENNs and BEA in medicinal herbs Among all 60 medicinal herbs under study, 15 samples were found to be contaminated with at least one of the ENNs and BEA, thus resulting in a frequency of contamination of 25%. The contamination levels of ENN and BEA in positive samples are detailed in Table 1. The mean concentrations of positive samples were 28.9, 28.4, 32.0, 3.9 and 33.0 µg kg–1 for ENN A, ENN A1, ENN B, ENN B1 and BEA, respectively. Maximum levels of the single toxins were 355 µg kg–1 (ENN A), 253 µg kg–1 (ENN A1), 290 µg kg–1 (ENN B), 40.2 µg kg–1 (ENN B1), and 125 µg kg–1 (BEA). Ginger revealed the highest levels of ENNs A and ENN A1; cogongrass rhizome contained the highest levels of Table 1.
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ENN B and BEA; and the highest level of ENN B1 was found in long-stamen onion bulb. BEA was the most frequently detected single mycotoxin in this study, with 12 positive samples, i.e. 20% of all examined samples. The incidences of ENNs were lower, with ENN B being most prevalent and detectable in seven samples of all, i.e. 11.7% and ENN A1 and B1 being least prevalent in four samples, i.e. 6.7% of all samples. As ginger is also a commonly used herb in Europe, we analysed six further ginger samples obtained from Bavarian stores. The results (Table 2) of the dried samples underlined the common occurrence of the toxins under study in ginger, whereas in the Bavarian samples BEA was the predominant toxin and the ENNs did not reach the contents found in the Chinese sample. Interestingly, the fresh ginger root did not contain detectable amounts of ENNs and BEA, which points to the assumption that toxin contamination or production occurs during drying of the herb. Considering the total amounts of the five mycotoxins in the single samples of all herbs, values between 2.5 and 751 µg kg–1 were found. The mean total amount in positive samples was 126 µg kg–1. The maximum total amount of the five mycotoxins again was found in ginger, and the
Concentrations (μg kg–1) of ENNs and BEA in positive samples.
Name Mint leaf Licorice root Black pepper Wrinkled giant hyssop herb Ginger Platycodon root Chinese quince fruit Mulberry leaf Spina date seed Smoked plum Bitter apricot seed Cogongrass rhizome Longstamen onion bulb Bitter cardamom Heartleaf houttuynia herb
ENN A
ENN A1
ENN B
ENN B1
BEA
Totala
