Article

A quantitative UHPLC-MS/MS method for citrinin and ochratoxin A detection in food, feed and red yeast rice food supplements

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Abstract

Mycotoxins may cause deleterious effects (among others nephrogenic, hepatogenic, carcinogenic, teratogenic, neurogenic) in animals and humans, therefore they have been intensely studied and monitored over the years. For citrinin (CIT), a nephrotoxic mycotoxin, however, this has not yet been the case. According to the latest European Food Safety Authority report, a correct risk assessment of CIT was not possible due to the lack of occurrence data. Besides, traces of CIT or its metabolite, dehydrocitrinone are widely (in up to 90% of samples) present in human urine according to recent Belgian and German scientific reports, which might imply chronic exposure. Only recently, a European maximum limit has been set for CIT in cholesterol reducing food supplements including red yeast fermented rice (RYR). During production of RYR through fungal (among others Monascus purpureus) fermentation of rice other components, like CIT, as well as nephrotoxic ochratoxin A (OTA) may form. Consequently, the present work attempted develop to a robust and routinely applicable ultra-high performance liquid chromatographytandem mass spectrometry (UHPLC-MS/MS) method for the analysis of CIT and OTA in food, feed and in RYR food supplements. The method was successfully validated based on EU/657/2002 and EU/519/2014 in RYR food supplements and wheat flour, achieving respective limits of quantification (LOQ) for CIT of 0.4 μg/kg and 0.1 μg/kg and for OTA of 15 μg/kg and 0.4 μg/kg. The average between-day recoveries varied from 72 to 110% with relative standard deviations ≤16%. Single-day validation in rice, curry and apple matrices showed LOQs ranging from 0.3-1.0 μg/kg. Next, the occurrence of CIT/OTA was surveyed in 138 RYR, food and feed samples, proving the potential of this method for future data acquisition within a risk assessment framework specifically for CIT, while also gaining information about the (co-)occurrence of OTA in edible matrices.

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... Moreover, detection of mycotoxins can be achieved by various analytical methods, such as gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) in combination with fluorescence and/or UV detection or immunodiagnostic tools [12,13]. However, during the last 10 years, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the universal technique for mycotoxin analysis, and it has been widely applied for various matrices [14,15]. Recently, occurrence data of CIT and its biomarkers were obtained in a handful of monitoring studies performed hitherto in Europe [16,17]. ...
... Homogeneity and stability were evaluated using a previously published LC-MS/MS method [15]. This method was successfully validated to meet the criteria laid down in EC decision 2002/657/EC [23] and EU 519/2014 [24] in FS-RYR and wheat, achieving a validated LOQ of 2.5 µg/kg. ...
... The LC-MS/MS method [15] was applied for checking the CIT contents in the collected samples of wheat, RYR, GBL, FS-RYR, and FS-GBL. Briefly, a test portion (4.00 ± 0.02 g) was humidified with 10 mL of hydrochloric acid aqueous solution (water:glacial acetic acid, 99:1, v:v) and extracted with 20 mL of ethyl acetate/acetonitrile/glacial acetic acid (75:24:1, v:v:v) mixture for 60 min by shaking. ...
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The development of incurred reference materials containing citrinin (CIT) and their successful application in a method validation study (MVS) in order to harmonize CIT determination in food and food supplements are demonstrated. CIT-contaminated materials made of red yeast rice (RYR), wheat flour, and Ginkgo biloba leaves (GBL), as well as food supplements made of red yeast rice (FS-RYR) and Ginkgo biloba leaves (FS-GBL), were manufactured in-house via fungal cultivation on collected raw materials. The homogeneity and stability from randomly selected containers were verified according to the ISO 13528. CIT was found to be homogenously distributed and stable in all contaminated materials, with no significant degradation during the timescale of the MVS when storage was performed up to +4 °C. Next, an MVS was organized with eighteen international laboratories using the provided standard operating procedure and 12 test materials, including three RYRs (blank, < 50 µg/kg, < 2000 µg/kg), two wheat flours (blank, < 50 µg/kg), two GBL powders (blank, < 50 µg/kg), three FS-RYRs (blank, < 50 µg/kg, < 2000 µg/kg), and two FS-GBLs (blank, < 50 µg/kg). The results of seven CIT-incurred materials showed acceptable within-laboratory precision (RSDr) varying from 6.4% to 14.6% and between-laboratory precision (RSDR) varying from 10.2% to 37.3%. Evidenced by HorRat values < 2.0, the results of the collaborative trial demonstrated that the applied analytical method could be standardized. Furthermore, the appropriateness of producing CIT reference materials is an important step towards food and feed quality control systems and the organization of proficiency tests.
... Because CIT and OTA, both nephrotoxic contaminants, were commonly detected together in food [12,26] and were detected in urine samples from adults and children [7], it is important to investigate possible sources of intake of this mycotoxin. Therefore, although LC-MS/MS methods have already been -developed to detect CIT and OTA separately [25,27,28], the aim of this study was to develop and validate an easy and reliable method such as QuEChERS for the simultaneous analysis of CIT and OTA by LC-MS/MS in feed and a large range of complex foodstuffs. ...
... Other important ingredients were beet pulp, barley, soybean meal, rapeseed meal and several oils (linseed, sunflower, palm kernel, soybean). [28], using an overhead shaker (Agilitec, J. Toulemonde and Cie, Paris, France). Subsequently, 6.0 ± 0.2 g of magnesium sulfate and 1.5 ± 0.1 g of NaCl were added. ...
... These results are in accordance with previously reported methods for separate determination of CIT and OTA in food and feed by LC-MS/MS [28,42,43]. ...
Article
An ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-ESI+/−-MS/MS) method for the simultaneous analysis of citrinin (CIT) and ochratoxin A (OTA) in feed (chicken and pig) and food (cereal-based products, fruit, vegetable juices, nuts, seeds, herbs, spices, vegetarian and soy products, alcoholic beverages, baby food products and food supplements) was developed. The mycotoxins were extracted from these matrices using a QuEChERS-based extraction method without any further clean-up step. The samples were 5-fold concentrated. Final extracts were analyzed using a UPLC–MS/MS system and chromatographic separation was achieved by applying a gradient elution for a total run time of 10 min. Mycotoxins were quantified using an internal calibration via analyte/¹³C-labeled internal standard ratio. The developed method was validated according to the criteria described in Commission Regulation No. 401/2006/EC and Commission Decision No. 2002/657/EC. Specificity, linearity, apparent recovery, limit of detection and quantification, intraday and interday precision, measurement uncertainty, matrix effect, and extraction efficiency were the parameters studied. Finally, 90 Belgian chicken and pig feed samples were analyzed, revealing the simultaneous presence of CIT (<LOQ − 3.90 μg/kg) and OTA (<LOQ − 5.60 μg/kg) in more than 50% of these products.
... Citrinin is a polyketide secondary metabolite found in food and feed that is produced by several fungi, including M. purpureus (Bezeria da Rocha et al., 2014;Kiebooms et al., 2016). It is a mycotoxin known to cause kidney damage as well as disrupt metabolic processes in the liver . ...
... The EFSA has set a level of no concern for citrinin-caused nephrotoxicity at 0.2 μg/kg body weight per day, but it has acknowledged the need for more reliable data on the citrinin level in food and feed before it can undertake exposure studies and risk assessment (Marley et al., 2016). Different analytical methods have been developed for the quantification of citrinin in a variety of samples in the last few years, including LC-FDA (Marley et al., 2016), UHPLC-MS/ MS (Kiebooms et al., 2016), UHPLC-DAD-QToF-MS (Avula et al., 2014), LC-DAD/FLD-MS n , microspherebased flow cytometric immunoassays (MFCI) , and a multi-commutated fluorometric optosensor technology (Jimenez Lopez et al., 2014). The maximum allowed level of citrinin as a contaminant in RYR is 200 ppb in Japan, but the European Union has recommended a limit of 100 ppb Nigovic et al., 2013). ...
Article
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Red yeast rice (RYR), a Chinese traditional folk medicine produced by the fermentation of cooked rice kernels with a Monascaceae mold, Monascus purpureus, has long been used to treat blood circulation stasis, indigestion, diarrhea, and limb weakness in East Asian countries. This article provides a systematic review of the traditional uses, chemistry, biological activities, and toxicology of RYR to highlight its future prospects in the field of medicine. The literature reviewed for this article was obtained from the Web of Science, Elsevier, SciFinder, PubMed, CNKI, ScienceDirect, and Google Scholar, as well as Ph.D. and M.Sc. dissertations, published prior to July 2019. More than 101 chemical constituents have been isolated from RYR, mainly consisting of monacolins, pigments, organic acids, sterols, decalin derivatives, flavonoids, polysaccharides, and other compounds. Crude extracts of RYR, as well as its isolated compounds, possess broad pharmacological properties with hypolipidemic, anti-atherosclerotic, anti-cancer, neurocytoprotective, anti-osteoporotic, anti-fatigue, anti-diabetic, and anti-hypertensive activities. However, further studies are needed to characterize its diverse chemical constituents and the toxicological actions of the main bioactive compounds. New pharmacological trials addressing the overlooked traditional uses of RYR, such as in the treatment of indigestion and diarrhea, are required.
... This contaminant has been found in various food commodities, including maize wheat, rye, barley, oats, rice, cereal-based products, fruits and fruit juices, roasted nuts, oilseeds, spices, cheese, fermented sausages, and feedstuffs [2]. Also, CIT has been detected in rice fermented with the red yeast Monascus spp., which is used for food coloring and meat preservation, as well as a dietary supplement [3]. Although CIT has been found in a variety of foodstuffs, no regulation limits have been set except for a safe limit for red rice-based food supplements. ...
Article
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Citrinin (CIT), a polyketide mycotoxin produced by Penicillium, Aspergillus, and Monascus species, is a contaminant that has been found in various food commodities and was also detected in house dust. Several studies showed that CIT can impair the kidney, liver, heart, immune, and reproductive systems in animals by mechanisms so far not completely elucidated. In this study, we investigated the CIT mode of action on two human tumor cell lines, HepG2 (hepatocellular carcinoma) and A549 (lung adenocarcinoma). Cytotoxic concentrations were determined using an MTT proliferation assay. The genotoxic effect of sub-IC50 concentrations was investigated using the alkaline comet assay and the impact on the cell cycle using flow cytometry. Additionally, the CIT effect on the total amount and phosphorylation of two cell-cycle-checkpoint proteins, the serine/threonine kinase Chk2 and Fanconi anemia (FA) group D2 (FANCD2), was determined by the cell-based ELISA. The data were analyzed using GraphPad Prism statistical software. The CIT IC50 for HepG2 was 107.3 µM, and for A549, it was >250 µM. The results showed that sensitivity to CIT is cell-type dependent and that CIT in sub-IC50 and near IC50 induces significant DNA damage and cell-cycle arrest in the G2/M phase, which is related to the increase in total and phosphorylated Chk2 and FANCD2 checkpoint proteins in HepG2 and A549 cells.
... Such urine biomarker data can be also used for calculating the mycotoxin's probable daily intake (PDI) since information on kinetics and urinary excretion rates in humans are now available, which then allows to assess risks by comparing the estimated PDIs for CIT to the 'level of no concern for nephrotoxicity' as provisional tolerable daily intake (pTDI) value [21]. This is of considerable interest, also in the light of co-occurrence with the nephrotoxic mycotoxin OTA in foods and in human fluids [4,11,[22][23][24]. However, biomonitoring data on CIT are limited, and only a few studies so far include children cohorts [20,23,25]. ...
Article
Full-text available
Citrinin (CIT), a mycotoxin known to exert nephrotoxicity, is a contaminant in food and feed. Since CIT contamination is not regularly analyzed, data on its occurrence and especially levels in food commodities are insufficient for conducting a conventional exposure assessment. Yet, human biomonitoring, i.e., an analysis of CIT and its metabolite dihydrocitrinone (DH-CIT) in urine samples allows to estimate exposure. This study investigated CIT exposure in young (2–14 years) and adult (24–61 years) residents of three federal states in Germany. A total of 179 urine samples from children and 142 from adults were collected and analyzed by a targeted LC-MS/MS based method for presence of CIT and DH-CIT. At least one of the biomarkers was detected and quantified in all urines, which indicated a widespread dietary exposure to the mycotoxin in Germany. Interestingly, the biomarker concentrations of CITtotal (sum of CIT and DH-CIT) were higher in children’s urine (range 0.05–7.62 ng/mL; median of 0.54 ng/mL) than in urines from adults (range 0.04–3.5 ng/mL; median 0.3 ng/mL). The biomarker levels (CITtotal) of individual urines served to calculate the probable daily CIT intake, for comparison to a value of 0.2 µg/kg bw/day defined as ‘level of no concern for nephrotoxicity’ by the European Food Safety Authority. The median exposure of German adults was 0.013 µg/kg b.w., with only one urine donor exceeding this provisional tolerable daily intake (pTDI) for CIT. The median exposure of children was 0.05 µg/kg bw per day (i.e., 25% of the pTDI); however, CIT exposure in 12 individuals (6.3% of our study group) exceeded the limit value, with a maximum intake of 0.46 µg/kg b.w. per day. In conclusion, these results show evidence for non-negligible exposure to CIT in some individuals in Germany, mainly in children. Therefore, further biomonitoring studies and investigations aimed to identify the major sources of CIT exposure in food commodities are required.
... In 1986, CIT was classified within the third group as a possible human carcinogen by the International Agency for Research on Cancer (IARC, 1986). Studies, especially in Belgium and Germany, have shown that CIT or its metabolite, dihydrocytrinone was found in almost 90% of human urine tests, which could result in chronic exposure of the human body to this mycotoxin (Kiebooms et al., 2016). ...
Article
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Citrinin (CIT) is a mycotoxin responsible for the contamination of many agricultural products, like wheat, barley, corn, rice and their products, as also other foodstuffs and feedstuffs used in human and animal nutrition. It is essentially produced by Penicillium citrinum, although it can also be biosynthesised from Penicillium expansum and Penicillium verrucosum and some species of Aspergillus and Monascus. However, several studies have shown that CIT is known for its genotoxic, hepatotoxic, fetotoxic and teratogenic properties. The aim of this study is to investigate the occurrence of CIT in wheat grain cultivated in Kosovo andAlbania. Given the fact that wheat flour is the most consumed product in Kosovo and Albania, it is necessary to analyse the CIT in wheat in these two countries. In total, 60 wheat samples were tested from Fusha e Kosovës (Kosovo), Myzeqeja (Albania) and Fusha e Maliqit (Albania), as places withthe highest wheat production. The enzyme- linked immunosorbent assay (ELISA) method was used to determine CIT concentrations. To identify moulds representing potential producers of CIT, traditional macroscopic and microscopic methods and the molecular PCR method of identification were implemented. CIT was detected in 96.6% and 86.6% of wheat grain samples collected in Kosovo and Albania, respectively. The maximum amount of CIT detected in wheat grain was 53.12 μg/ kg in Kosovo, and 45.74 μg/kg in Albania. The amount of CIT found in wheat grain is not comparable with the maximal limits (MLs), as the European legislation does not provide limits for this mycotoxin. However, since there is generally a lack of data about CIT in cereals in Kosovo and Albania, the results can serve as an indicator of wheat grain contamination in this part of the Balkan Peninsula.
... Among the food supplements, CIT was detected in two RYR samples, but in one sample, a concentration of 1,787 μg/kg was detected, which is approximately 17 times above the maximum level of 100 μg/kg. This high concentration is in accordance with previously reported values in literature (Ji et al., 2015;Kiebooms et al., 2016;López Sánchez et al., 2017;Ostry et al., 2013). For the other food categories, no maximum level exists in the European legislation. ...
Article
Exposure to mycotoxins is a worldwide problem. To ensure public health, it is imperative to characterize the risks related to these toxins. The present study aims to conduct a dietary exposure assessment of citrinin (CIT) and ochratoxin A (OTA) in the Belgian population using consumption data of a variety of foodstuffs. A total of 367 food samples from different food categories were collected in Belgian supermarkets and analysed for CIT and OTA using a validated liquid chromatography-tandem mass spectrometry method. Daily CIT and OTA exposure to the Belgian population was calculated based on the analytical results and food consumption data in three age categories (3–9, 10–17 and 18–64 years), obtained from a national food consumption survey. Furthermore, a risk characterization was performed for CIT, in which no intake values exceeded the tolerable daily intake (TDI) of 200 ng kg⁻¹ bw day⁻¹, indicating no health risk. However, a CIT intake level of 187 ng kg⁻¹ bw day⁻¹ was detected for children in the age category of 3–9 years in the worst case scenario for rice, indicating that rice consumption could contain a potential health hazard for young children. For OTA, a potential health risk was detected in several food categories (biscuits, croissants, rice, flour, meat imitates, herbs and spices) in the higher percentiles (P99) or at maximum found concentrations when calculating the margin of exposure (MoE) for neoplastic effects. An attempt to perform a cumulative health risk assessment for both toxins was done. Although a high number of uncertainties is involved, combined margin of exposure (MoET) values indicated a potential health risk related to the combined exposure to CIT and OTA. For the first time, our study demonstrated the potential health risks of CIT and OTA after individual and combined exposure, in particular related to rice consumption. Moreover, further research is recommended concerning multiple mycotoxin exposure in young children.
... These were inoculated and cultivated for 3 weeks on sterilized aliquots (wheat or rice grains). Incubation was stopped by autoclaving at 121 • C for 20 min and samples were thereafter dried at 50-60 • C for 48 h, ground and sieved (< 250 µm), homogenized and stored at −20 • C until analysis by LC-MS/MS [48]. Penicillium citrinum MUCL Toxins 2020, 12, 719 13 of 19 29781 was selected for CIT production in contaminated feed. ...
Article
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Citrinin (CIT) is a polyketide mycotoxin occurring in a variety of food and feedstuff, among which cereal grains are the most important contaminated source. Pigs and poultry are important livestock animals frequently exposed to mycotoxins, including CIT. Concerns are rising related to the toxic, and especially the potential nephrotoxic, properties of CIT. The purpose of this study was to clarify the histopathological effects on kidneys, liver, jejunum and duodenum of pigs, broiler chickens and laying hens receiving CIT contaminated feed. During 3 weeks, pigs (n = 16) were exposed to feed containing 1 mg CIT/kg feed or to control feed (n = 4), while 2 groups of broiler chickens and laying hens (n = 8 per group) received 0.1 mg CIT/kg feed (lower dose group) and 3 or 3.5 mg CIT/kg feed (higher dose group), respectively, or control feed (n = 4). CIT concentrations were quantified in plasma, kidneys, liver, muscle and eggs using a validated ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method. Kidneys, liver, duodenum and jejunum were evaluated histologically using light microscopy, while the kidneys were further examined using transmission electron microscopy (TEM). Histopathology did not reveal major abnormalities at the given contamination levels. However, a significant increase of swollen and degenerated mitochondria in renal cortical cells from all test groups were observed (p < 0.05). These observations could be related to oxidative stress, which is the major mechanism of CIT toxicity. Residues of CIT were detected in all collected tissues, except for muscle and egg white from layers in the lowest dose group, and egg white from layers in the highest dose group. CIT concentrations in plasma ranged between 0.1 (laying hens in lower dose group) and 20.8 ng/mL (pigs). In tissues, CIT concentrations ranged from 0.6 (muscle) to 20.3 µg/kg (liver) in pigs, while concentrations in chickens ranged from 0.1 (muscle) to 70.2 µg/kg (liver). Carry-over ratios from feed to edible tissues were between 0.1 and 2% in pigs, and between 0.1 and 6.9% in chickens, suggesting a low contribution of pig and poultry tissue-derived products towards the total dietary CIT intake for humans.
... Another minor change is related to citrinin that has been transferred to the negative ESI mode, as the related MRM transitions deriving from the deprotonated methanol adduct [M+CH 3 OH-H] − exhibited a drastically increased signal compared with the positive mode [22]. ...
Article
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This paper describes the validation of an LC-MS/MS-based method for the quantification of > 500 secondary microbial metabolites. Analytical performance parameters have been determined for seven food matrices using seven individual samples per matrix for spiking. Apparent recoveries ranged from 70 to 120% for 53–83% of all investigated analytes (depending on the matrix). This number increased to 84–94% if the recovery of extraction was considered. The comparison of the fraction of analytes for which the precision criterion of RSD ≤ 20% under repeatability conditions (for 7 replicates derived from different individual samples) and intermediate precision conditions (for 7 technical replicates from one sample), respectively, was met (85–97% vs. 93–94%) highlights the contribution of relative matrix effects to the method uncertainty. Statistical testing of apparent recoveries between pairs of matrices exhibited a significant difference for more than half of the analytes, while recoveries of the extraction showed a much better agreement. Apparent recoveries and matrix effects were found to be constant over 2–3 orders of magnitude of analyte concentrations in figs and maize, whereas the LOQs differed less than by a factor of 2 for 90% of the investigated compounds. Based on these findings, this paper discusses the applicability and practicability of current guidelines for multi-analyte method validation. Investigation of (apparent) recoveries near the LOQ seems to be insufficiently relevant to justify the enormous time-effort for manual inspection of the peaks of hundreds of analytes. Instead, more emphasis should be put on the investigation of relative matrix effects in the validation procedure. Graphical abstract
... Citrinin (CIT) (Figure 1a) a fungal metabolite produced by several fungal species of Aspergillus, Penicillium and Monascus, is one of the important mycotoxins that has been reported to contaminate rice, wheat, corn, barley and fruit juices (Čulig et al. 2017;Kiebooms et al. 2016;Pleadin et al. 2016). CIT is known to be nephrotoxic, hepatotoxic and carcinogenic to humans and animals. ...
... CIT is also known as unwanted contaminant in rice fermented with Monascus spp. (red yeast rice) which is used for food coloring and meat preservation in Asia and now widely marketed as a food supplement (DFG 2013;Liao et al. 2014;Kiebooms et al. 2016). CIT has been found along with ochratoxin A (OTA) in food and feed and their co-occurrence has raised concerns for human and animal health (Flajs and Peraica 2009;Ostry et al. 2013). ...
Article
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The mycotoxin citrinin (CIT) deserves attention due to its known toxic effects in mammalian species and a widespread occurrence in food commodities, often along with ochratoxin A, another nephrotoxic mycotoxin. Human exposure, a key element in assessing risks related to these food contaminants, depends upon mycotoxin levels in food and on food consumption. Yet, data available for CIT levels in food are insufficient for reliable intake estimates. Now biomonitoring, i.e., analysis of parent compound and/or metabolites in human specimen (blood, urine, breast milk), is increasingly used to investigate mycotoxin exposure. Biomonitoring requires sensitive methods for determining biomarkers of exposure, combined with kinetic data to conclude on the absorbed internal dose in an individual. Recent advances in LC–MS/MS-based analytical techniques have facilitated biomonitoring studies on the occurrence of CIT biomarkers in body fluids, mainly in urine samples. This review compiles evidence on human exposure to CIT in different countries, on CIT kinetics in humans, and on biomarker-based CIT intake estimates. Human CIT exposures are discussed in light of an intake value defined as ‘level of no concern for nephrotoxicity’ by the European Food Safety Agency, and some uncertainties in the toxicological data base. Further studies on CIT, including biomarker-based studies are warranted along with regular food surveys for this mycotoxin to protect consumers against undesirable health effects.
... Mainly for its health benefits by lowering cholesterol levels, there is an increasing interest for RYR around the world (Kalaivani, Sabitha, Kalaiselvan, & Rajasekaran, 2010). In RYR, the levels of monacolin K, which is formed during the fermentation process and inhibits HMG-CoA reductase (involved in cholesterol synthesis), are crucial to achieve the desirable functional properties, but since fungal activity is also associated with the production of CIT, there is a demand for the optimization of the process (Kalaivani et al., 2010;Kiebooms, Huybrechts, Thiry, Tangni, & Callebaut, 2016;Liao, Chen, Lin, Chiueh, & Shih, 2014). The selection of Monascus strains and the application of mutagenesis techniques to increase the monacolin K/CIT ratio produced during fermentation are being explored (Kanpiengjai, Mahanwan, Pengnoi, Lumyong, & Khanongnuch, 2018;Tsukahara, Shinzato, Tamaki, Namihira, & Matsui, 2009). ...
Article
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Rice is part of many people's diet around the world, being the main energy source in some regions. Although fewer reports exist on the occurrence of mycotoxins in rice compared to other cereals, fungal contamination and the associated production of toxic metabolites, even at lower occurrence levels compared to other crops, are of concern because of the high consumption of rice in many countries. Due to the diversity of fungi that may contaminate the rice food chain, the co‐occurrence of mycotoxins is frequent. Specific strategies to overcome these problems may be applied at the preharvest part of the crop chain, while assuring good practices at harvest and postharvest stages, since different fungi may find suitable conditions to grow at the various stages of the production chain. Therefore, the aim of this review is to present the state‐of‐the‐art knowledge on such strategies in an integrated way, from the field to the final products, to reduce mycotoxin contamination in rice.
... Eight positive samples were analyzed by UHPLC-MS/MS in the European Union Referent Laboratory (EURL) for mycotoxins in the Veterinary and Agrochemical Research Center VAR-CODA-CERVA, Brussels, Belgium (Kiebooms et al., 2016). OTA toxin was detected by this lab in only one sample. ...
Article
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To assess food safety associated with the occurrence of ochratoxin A (OTA) residues in different kinds of flours, a survey was carried out during 2016–2017 in 5 municipalities of the Prishtina region (Prishtina, Gllogovc, Lipjan, Fushë Kosova, and Podujevë). In the present study, a total of 120 flour samples were collected, consisting of domestic and imported samples. The analytical method used in our study to measure the occurrence and concentration range of OTA in the samples was competitive enzyme-linked immunosorbent assay (ELISA) method. A method based on UHPLC-MS/MS was used to confirm the presence of ochratoxin A in samples in which the toxin was detected by ELISA. Out of all the samples examined, nine were contaminated with OTA. The frequency of OTA contamination in 2016 was 5 out of 75 samples (6.7%), and in 2017, 4 out of 45 samples examined (8.9%) were contaminated. OTA was detected in four wheat flour samples (9%), in four maize flour samples (14.8%) and in one rye flour sample (8.3%). The level of OTA ranged from 0.26 to 0.85 μg kg-1 in the four positive wheat samples, from 0.77 to 2.75 μg kg-1 in the four positive maize samples and the level was 0.77 μg kg-1 in the one positive rye sample. None of the contaminated samples exceeded the maximum levels (ML) of 3 μg kg-1, set by to the European Union Regulation and Kosovo Food Codex.
... Several analytical methods have been developed for the determination of CIT including thin-layer chromatography, high-performance liquid chromatography (Lhotska, Satinsky, Havlikova, & Solich, 2016), liquid chromatography tandem mass spectrometry (Ji et al., 2015), (Kiebooms, Huybrechts, Thiry, Tangni, & Callebaut, 2016), gas chromatographymass spectrometer (Shu & Lin, 2002), ultra-high-performance liquid chromatography and fluorescence detection (Huertas-Perez, Arroyo-Manzanares, Garcia-Campana, & Gamiz-Gracia, 2015), and high-performance liquid chromatography with UV diode array (Urraca et al., 2016). In addition, novel sensors have been developed as quartz crystal microbalance sensor (Fang, Liu, Yang, & Wang, 2016) and molecular imprinted electrochemical sensor (Atar, Yola, & Eren, 2016). ...
Article
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A sensitive and specific anti-citrinin (anti-CIT) monoclonal antibody 1F2 was obtained following immunization and cell fusion. An indirect competitive enzyme-linked immunosorbent assay was developed with a 50% inhibitory concentration of 0.761 ng/mL and a limit of detection of 0.089 ng/mL. The recovery rates for CIT-spiked cereals (maize, wheat, and rice) ranged from 112% to 123%. A lateral-flow immunochromatographic assay was developed for both semi-quantitative and quantitative detection. With CIT-spiked cereals, the visual limit of detection was 8 ng/g and the cut-off value was 40 ng/g (semi-quantitative analysis with naked-eye detection). Using a strip scan reader, the calculated limit of detection was 1.28–1.8 ng/g for different CIT-spiked cereals. The recovery rates ranged from 110% to 127%. Therefore, both methods were effective for CIT detection and suitable for on-site detection and rapid screening of samples.
... Due to confidentiality issues, the identification of the samples in Table 18 is omitted. The levels of citrinin were in accordance with finding in other publications (Liao et al., 2014;Kiebooms et al., 2016). Although it was labelled that the origin of the samples was Europe, it is not clear whether the raw material came originally from Europe or it was processed here. ...
... CIT is also known as unwanted contaminant in rice fermented with Monascus spp. (red yeast rice) which is used for food coloring and meat preservation in Asia and now widely marketed as a food supplement (DFG 2013;Liao et al. 2014;Kiebooms et al. 2016). CIT has been found along with ochratoxin A (OTA) in food and feed and their co-occurrence has raised concerns for human and animal health (Flajs and Peraica 2009;Ostry et al. 2013). ...
Article
Citrinin (CIT) is a mycotoxin produced by several fungi of the genera Penicillium, Aspergillus and Monascus. CIT is nephrotoxic, and can co-occur with ochratoxin A (OTA), another nephrotoxic mycotoxin contaminant in food commodities [1]. A survey in Bangladesh documented the presence of OTA in maize [2]; but no study has been carried out on CIT contamination in foods or feed in Bangladesh. Since biological monitoring provides the best approach to assess human exposure to contaminants from various sources and by all routes, it was the aim of this study to analyze CIT and its metabolite dihydrocitrinone (HO-CIT) in urine samples collected in Bangladesh. Urines were collected from inhabitants of a rural (n=32) and an urban (n=37) area in Bangladesh during May 2013. A sensitive method [3] was used for clean up of urines by immunoaffinity column and subsequent LC/MS-MS analysis of the extracts. The limits of detection (LOD) were 0.02 ng/mL and 0.05 ng/mL urine for CIT and HO-CIT. CIT and HO-CIT were detectable in 94% and 73% of all urine samples. Urinary levels of CIT and its metabolite did not show significant correlations with age, sex and body mass index of the donors. But, excretion of CIT and its metabolite (total) was significantly higher in rural people (LOD – 7.9 ng/mL) than in urban people (LOD – 0.8 ng/mL) in Bangladesh, indicative of differences in mycotoxin exposure. Most of the people in the rural cohort of Bangladesh are farmers or farm workers involved in grain production while urban people are office workers or students. Food habits also differ between the rural and the urban cohort. It can be concluded that contaminated food commodities are major contributors for CIT exposure in humans in Bangladesh. Analysis of other mycotoxins in the two cohorts is underway and the results of biomonitoring will be compared to those in urine samples from German volunteers.
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Pork-derived products can contribute to the overall ochratoxin A (OTA) intake via carry-over from contaminated feed or via mould spoilage of meat products (salami, dry-cured ham, sausage). An analytical method using liquid chromatography coupled with mass spectrometry (LC-MS/MS) was developed and validated in accordance with the specifications laid down by European Commission. It offered quantification limits of 0.2 for kidney, liver and 0.4 μg/kg for black sausage. Spiking experiments of blank samples at 5–10 μg/kg showed recoveries ranging from 88 to 101%, 89 to 97% and 80 to 85% for kidney, liver and black sausage, respectively. The respective intra-laboratory repeatabilities ranged between 9.8–11.1%, 9.4–14.4% and 9.7–14.2%, and extended measurement uncertainties MU(k = 2) were 33%, 35% and 43% for kidney, liver and black sausage. Next, the validated method was applied to kidney (110), liver (20) and black sausage (20) samples collected in Belgium in the period 2012–2019. Neither liver nor black sausage samples were contaminated with OTA. Kidney samples (37.3%) were OTA contaminated at the mean level of 0.22 ± 0.25 μg/kg (up to 1.91 μg/kg). These data combined with the offal consumption in the Belgian population revealed average daily OTA exposures ranged from 0.167 and 0.319 ng/kg bw for 3 age groups (3–9, 10–17 and 18–64 years). Taking into account, the OTA non-neoplastic and neoplastic effects, risk characterization assessed via the margin of exposure for reference endpoints revealed no potential health risk for the consumers. As the presence of low OTA content in foods together with other mycotoxins or derivatives may interactively potentiate its toxicity, monitoring of OTA and its metabolites in meat and meat by-products is advised.
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A novel surface-plasmon-coupled chemiluminescent immunosensor was developed for the detection of multiplex mycotoxins with ultrahigh sensitivity, high throughput and simplicity. The immunosensor was constructed by immobilizing carboxyl modified silver nanoparticles (AgNPs) and bovine serum albumin combined antigens sequentially on the glass chip modified with amino groups. The optical properties of AgNPs could amplify the chemiluminescence (CL) generated on the chip through the surface plasmon resonance phenomenon. Using a competitive immunoassay, the CL signals from all the sensing sites on the chip were collected simultaneously by a charge-coupled device for ultrasensitive analysis of multiple mycotoxins. Citrinin, aflatoxin B1 and ochratoxins A were selected as model analytes. Under optimal conditions, the surface-plasmon-coupled chemiluminescent immunosensor-based method presented wide linear ranges over 4 orders of magnitude and much lower limits of detection than previous work. The assay results of mycotoxins in red yeast rice samples using the proposed method were in good agreement with that using the liquid chromatography-mass spectrometry method. Its high selectivity, high throughput, acceptable stability and accuracy showed broad prospects in mycotoxin monitoring and evaluation of safety on Traditional Chinese Medicine.
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As data on food contamination with the mycotoxin citrinin (CIT) are scarce, a recently developed method for biomarker analysis (Blaszkewicz et al. in Arch Toxicol 87:1087-1094, 2013) was applied to investigate CIT exposure of German adults. CIT and its human metabolite dihydrocitrinone (HO-CIT) were determined in urine samples from a group of 50 healthy adults (n = 27 females and n = 23 males). After cleanup by immunoaffinity (CitriTest(®)) columns, extracts were analyzed by LC-MS/MS. The mycotoxin and its major metabolite HO-CIT were detected in 82 and 84 % of all urine samples, at concentrations ranging from 0.02 (limit of detection, LOD) to 0.08 ng/mL for CIT, and 0.05 (LOD) to 0.51 ng/mL for HO-CIT. Median urine analyte levels in the cohort were 0.03 (CIT) and 0.06 ng/mL (OH-CIT) or adjusted to creatinine 20.2 ng/g crea (CIT) and 60.9 ng/g crea (HO-CIT), respectively. Except for higher urinary CIT levels in males, differences between subgroups were not significant. This first biomarker analysis indicates widespread and variable exposure to CIT in German adults, and conversion of ingested mycotoxin to its less toxic metabolite HO-CIT, which may serve as biomarker of exposure in addition to the parent compound.
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A direct, fast and sensitive LC-MS/MS method was developed to measure biomarkers for mycotoxin exposure in human urine. In total, 32 biomarkers were quantitatively or semi-quantitatively measured in 32 urine samples of Belgian volunteers using two injections. All urine samples contained deoxynivalenol-15-glucuronide, the major detoxification metabolite of deoxynivalenol, in the ng/mL range. Also deoxynivalenol-3-glucuronide and de-epoxy-deoxynivalenol-glucuronide were present in, respectively, 90 and 25 % of the samples, while deoxynivalenol was detected in 60 % of the samples, in lower concentrations. Deoxynivalenol glucuronides were the major biomarkers for deoxynivalenol exposure. Ochratoxin A was detected in 70 % of the samples in pg/mL. Citrinin and/or dihydrocitrinone were detected in 90 % of the samples, also in concentrations of pg/mL. The presence of ochratoxin A and citrinin was confirmed by a second method using sample cleanup by immunoaffinity columns, followed by LC-MS/MS. Our data show that humans are much more exposed to citrinin than realized before and suggest further work on citrinin exposure in relation with ochratoxin A exposure, as both mycotoxins are nephrotoxic.
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Citrinin (CIT) and ochratoxin A (OTA) are mycotoxins produced by several species of the genera Aspergillus, Penicillium and Monascus. Both can be present as contaminants in various food commodities and in animal feed. The occurrence and toxicity of OTA and human exposure have been intensively studied, but for CIT such data are scarce by comparison. Recently, dihydrocitrinone (DH-CIT) was detected as main metabolite of CIT in human urine, and co-occurrence of CIT and OTA was shown in human blood plasma (Blaszkewicz et al. in Arch Toxicol 87:1087-1094, 2013). In light of these new findings, we have now investigated the toxicity of the metabolite DH-CIT in comparison with CIT and analysed the effects of mixtures of CIT and OTA in vitro. The cytotoxic potency of DH-CIT (IC50 of 320/200 μM) was distinctly lower compared with CIT (IC50 of 70/62 μM) after treatment of V79 cells for 24 and 48 h. Whereas CIT induced a concentration-dependent increase in micronucleus frequencies at concentrations ≥30 μM, DH-CIT showed no genotoxic effect up to 300 μM. Thus, conversion of CIT to DH-CIT in humans can be regarded as a detoxification step. Mixtures of CIT and OTA exerted additive effects in cytotoxicity assays. The effect of CIT and OTA mixtures on induction of micronuclei varied dependent on the used concentrations between additive for low μM concentrations and more-than-additive for high μM concentrations. Effects on cell cycle were mostly triggered by OTA when both mycotoxins were used in combination. The implications of our and related in vitro studies are discussed with respect to in vivo concentrations of CIT and OTA, which are found in animals and in humans.
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Citrinin is a toxic product usually produced during the Monascus fermentation. The presence of citrinin in xuezhikang capsule has been a concern due to its ingredient which is derived from monascus-fermented rice. A rapid and sensitive RP-HPLC method with fluorescence detection at λex = 331 nm and λem = 500 nm for analysis of citrinin in Monascus-fermented products was developed to analyze citrinin in Monascus-fermented products. The chromatography was performed with mobile phase containing acidified water and acetonitrile. The calibration curve was linear (r = 0.9999) over a range of 0.0107- 0.537 μg/mL. The limit of detection (LOD) and the limit of quantitation (LOQ) were 0.187 ng/mL and 0.6 ng/mL respectively. The analysis of xuezhikang capsules using the developed method suggested that the product does not contain detectable citrinin and the result has been further confirmed using independent LC-MS/MS analysis. The proposed method has also been applied to analyze 11 samples of other Monascus-fermented products. The results suggested that there were no detectable citrinin in 4 of the 11 samples, however citrinin with the levels between 0.10-594 ng/kg has been detected in the other 7 samples. It indicates that the proposed method can also be applied to carry out the quantitative detection of citrinin for other Monascus-fermented products.
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Ochratoxin A (OTA) is a nephrotoxic mycotoxin with carcinogenic properties. Its presence was detected in various foodstuffs all over the world but with significantly higher frequency and concentrations in areas with endemic nephropathy (EN). Even though food is often contaminated with more than one mycotoxin, earlier studies focused on the occurrence and toxicology of only OTA. Only a limited number of surveys showed that OTA co-occurs in food with mycotoxins (citrinin-CIT, penicilic acid, fumonisin B1-FB1, aflatoxins-AF) which exert nephrotoxic, carcinogenic or carcinogen-promoting activity. This review summarises the findings on OTA and its co-occurrence with the mentioned mycotoxins in food as well as experimental data on their combined toxicity. Most of the tested mycotoxin mixtures involving OTA produced additive or synergistic effects in experimental models suggesting that these combinations represent a significant health hazard. Special attention should be given to mixtures that include carcinogenic and cancer-promoting mycotoxins.
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A survey was made of the literature concerning the occurrence and incidence of mycotoxic nephropathy in pigs and chicks in different countries. Various etiological factors contributing to the development of the disease were considered. The main nephrotoxic fungi as well as the specific conditions for their growth and toxins production were briefly described. A survey was made about the most frequent nephrotoxic fungal contaminants in various feedstuffs from plant origin. In addition, their natural quantities and importance for development of mycotoxic porcine/chick nephropathy (MPN/MCN) are also explored. In addition, a survey was made of the feedstuffs representing the most favorable environment for nephrotoxic fungal growth as well as the most favorable storehouse conditions for this fungal growth were shortly described. The significance of some underestimated fungal species, which can provoke kidney damage, was studied. The importance of joint mycotoxin interaction and newly identified fungal metabolites in the complex etiology of mycotoxic nephropathy ranged in some countries is deeply investigated. The toxicity of the low contamination levels of some combinations of mycotoxins often administered by pigs and chicks in the practice was carefully studied.
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Citrinin (CIT), produced by several Penicillium, Aspergillus, and Monascus species, has been detected as contaminant in feeds, grains, and other food commodities. CIT can co-occur with ochratoxin A (OTA), a mycotoxin also known for its nephrotoxicity, and this raises concern regarding possible combined effects. But, in contrast to OTA, data on CIT contamination in foods for human consumption are scarce, and CIT biomonitoring has not been conducted so far due a lack of suitable methods for human specimen. Thus, it was the aim of the present study to develop sensitive methods for the analysis of CIT in human blood and urine to investigate human exposure. To this end, we assessed different methods of sample preparation and instrumental analysis for these matrices. Clean-up of blood plasma by protein precipitation followed by LC-MS/MS-based analysis allowed robust detection of CIT (LOD 0.07 ng/mL, LOQ 0.15 ng/mL). For urine, sample clean-up by an immunoaffinity column (CitriTest(®)) proved to be clearly superior to SPE with RP(18) material for subsequent analysis by LC-MS/MS. For CIT and its metabolite dihydrocitrinone (HO-CIT), the LOD and LOQ determined by external calibration curves in matrix were 0.02 and 0.05 ng/mL for CIT, and those for HO-CIT were 0.05 and 0.1 ng/mL urine. The newly developed method was applied in a small pilot study: CIT was present in all plasma samples from 8 German adults, at concentrations ranging from 0.11 to 0.26 ng/mL. The molar (nM) concentrations of CIT are similar to those measured for OTA in these samples as a result of dietary mycotoxin intake. CIT was detected in 8/10 urines (from 4 adults and 6 infants) in a range of 0.16-0.79 ng/mL, and HO-CIT was present in 5/10 samples at similar concentrations. Thus, CIT is excreted in urine as parent compound and also as metabolite. These first results in humans point to the need for further studies on CIT exposure.
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Toxicological Properties of Citrinin Citrinin (CTN) is a nephrotoxic mycotoxin produced by several fungal strains belonging to the genera Penicillium, Aspergillus , and Monascus. It contaminates various commodities of plant origin, cereals in particular, and is usually found together with another nephrotoxic mycotoxin, ochratoxin A (OTA). These two mycotoxins are believed to be involved in the aetiology of endemic nephropathy. In addition to nephrotoxicity, CTN is also embryocidal and fetotoxic. The genotoxic properties of CTN have been demonstrated with the micronuleus test (MN), but not with single-cell gel electrophoresis. The mechanism of CTN toxicity is not fully understood, especially not whether CTN toxicity and genotoxicity are the consequence of oxidative stress or of increased permeability of mitochondrial membranes. CTN requires complex cellular biotransformation to exert mutagenicity. Compared with other mycotoxins, CTN contamination of food and feed is rather scarce. However, it is reasonable to believe that humans are much more frequently exposed to CTN than generally accepted, because it is produced by the same moulds as OTA, which is a common contaminant of human food all over the world. At present, there are no specific regulations either in Croatia or in the European Union concerning CTN in any kind of commodity.
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Red yeast rice is a Chinese fermented rice product (Monascus purpureus) that some have claimed improves blood circulation by decreasing cholesterol and triglyceride levels in humans. The supplement contains naturally occurring monacolin K, the active ingredient found in Merck's prescription agent lovastatin (Mevacor). Lovastatin is associated with various adverse effects such as myopathy and abnormal liver function test results, which can lead to serious problems if patients are not monitored and treated. The inclusion of lovastatin in red yeast rice and the lack of dietary supplement regulation by the FDA raise safety concerns for health care professionals as well as for patients. Studies have shown that red yeast rice products can be beneficial in lowering serum cholesterol levels, but they are not without risk. Furthermore, product uniformity, purity, labeling, and safety cannot be guaranteed.
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We examined the cholesterol-lowering effects of a proprietary Chinese red-yeast-rice supplement in an American population consuming a diet similar to the American Heart Association Step I diet using a double-blind, placebo-controlled, prospectively randomized 12-wk controlled trial at a university research center. We evaluated the lipid-lowering effects of this red-yeast-rice dietary supplement in US adults separate from effects of diet alone. Eighty-three healthy subjects (46 men and 37 women aged 34-78 y) with hyperlipidemia [total cholesterol, 5.28-8.74 mmol/L (204-338 mg/dL); LDL cholesterol, 3.31-7.16 mmol/L (128-277 mg/dL); triacylglycerol, 0.62-2.78 mmol/L (55-246 mg/dL); and HDL cholesterol 0.78-2.46 mmol/L (30-95 mg/dL)] who were not being treated with lipid-lowering drugs participated. Subjects were treated with red yeast rice (2.4 g/d) or placebo and instructed to consume a diet providing 30% of energy from fat, <10% from saturated fat, and <300 mg cholesterol daily. Main outcome measures were total cholesterol, total triacylglycerol, and HDL and LDL cholesterol measured at weeks 8, 9, 11, and 12. Total cholesterol concentrations decreased significantly between baseline and 8 wk in the red-yeast-rice-treated group compared with the placebo-treated group [(x+/-SD) 6.57+/-0.93 mmol/L (254+/-36 mg/dL) to 5.38+/-0.80 mmol/L (208+/-31 mg/dL); P < 0.001]. LDL cholesterol and total triacylglycerol were also reduced with the supplement. HDL cholesterol did not change significantly. Red yeast rice significantly reduces total cholesterol, LDL cholesterol, and total triacylglycerol concentrations compared with placebo and provides a new, novel, food-based approach to lowering cholesterol in the general population.
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Ochratoxin A (OTA) is an ubiquitous nephrotoxic and carcinogenic mycotoxin considered to be involved in the aetiology of Balkan endemic nephropathy (BEN). The occurrence of this human fatal disease that appears in regions of Bosnia and Herzegovina, Bulgaria, Croatia, Rumania, and Serbia and Monte Negro correlates with very high incidence of otherwise rare urothelial tumours of the renal pelvis and ureters. Although OTA was found more frequently and/or in higher concentration in food and blood of inhabitants in regions with BEN than in other regions, the involvement of OTA in the development of BEN is still open. Patients with chronic renal insufficiency treated with dialysis have higher blood OTA concentrations than healthy persons, and OTA concentration does not decrease with such treatment.
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Very often, the accuracy of quantitative analytical methods for the determination of mycotoxins by liquid chromatography (LC)-mass spectrometry (MS) and LC-MS/MS is limited by matrix effects during the ionization process in the MS source. Stable isotope labeled standards are best suited to correct for matrix effects and to improve both the trueness and the precision of analytical methods employing LC-MS and LC-MS/MS. This paper describes the successful use of fully 13C isotope labeled deoxynivalenol [(13C15)DON] as an internal standard (IS) for the accurate determination of DON in maize and wheat by LC electrospray ionization MS/MS. To show the full potential of (13C15)DON as IS, maize and wheat extracts were analyzed without further cleanup. Subsequent to calibration for the LC-MS end determination, DON was quantified in matrix reference materials (wheat and maize). Without consideration of the IS, apparent recoveries of DON were 29+/-6% (n=7) for wheat and 37+/-5% (n=7) for maize. However, the determination of DON in the reference materials yielded 95+/-3% (wheat) and 99+/-3% (maize) when (13C15)DON was used as an IS for data evaluation.
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The powerful combination of liquid chromatography and mass spectrometry (MS) is often limited by matrix effects during ionization in the MS ion source. The use of fully isotope-substituted (13C15)-deoxynivalenol ((13C15)-DON) as an internal standard (IS) corrects matrix effects and improves the accuracy of analytical methods using mass spectrometry for the quantitative determination of the Fusarium mycotoxin deoxynivalenol (DON). The IS was characterized with respect to its chromatographic purity by liquid chromatography-ultraviolet light and its isotope distribution by time-of-flight mass spectrometry. Its low-energy collision-induced dissociation behaviour was compared with DON. Moreover, this work describes the successful application of (13C15)-DON as IS for the determination of DON in maize using high-performance liquid chromatography (HPLC) electrospray (ESI) with tandem mass spectrometry. The results demonstrate that the IS can successfully correct for fluctuations during extraction and clean-up of the sample as well as the ionization of DON in the MS ion source. Random variations in ionization affect the IS in the same way as the analyte. Recoveries for DON in maize of 76% +/- 1.9% (external calibration) or 101% +/- 2.4% (internal calibration) were reached, respectively, after sample clean-up.
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A multi-analyte method for the liquid chromatography-tandem mass spectrometric determination of mycotoxins in crude grain extracts without clean-up has been applied to the analysis of spelt, rice and barley. Method performance characteristics were determined after spiking blank samples at multiple levels and were found to be comparable for all investigated matrices as regards linearity (linear calibration functions were obtained for all analyte/matrix combinations except for moniliformin), precision (coefficient of variations <6%) and sensitivity. Matrix-induced signal suppression/enhancement was studied in detail and varied significantly between the investigated matrices, as well as between individual samples (relative standard deviation was as high as 40% within three rice varieties) and individual toxins. It was concluded that a reliable quantitative analysis using matrix-matched calibration requires careful consideration of the model matrix, which should match the investigated samples as close as possible.
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To evaluate the gap between food-based dietary guidelines (FBDG) and the usual food consumption in Belgium. Information on food intake was collected with two non-consecutive 24 h recalls, using the validated software package EPIC-SOFT in combination with a self-administered FFQ. Habitual food intake was estimated by the Nusser method. Physical activity was evaluated according to the International Physical Activity Questionnaire. A representative sample of the Belgian population aged 15 years and older was randomly selected from the National Register using a multistage stratified procedure. Dietary information was obtained from 3245 individuals. Food intakes deviated significantly from the recommendations. In particular, fruit (118 g/d) and vegetable (138 g/d) consumption and intake of dairy and Ca-enriched soya products (159 g/d) were inadequate. Consumption of energy-dense, nutrient-poor foods (soft drinks, alcohol and snacks) was excessive (481 g/d). There were important age and gender differences. Fruit, vegetable and spreadable fat consumption was lowest, while consumption of dairy, starchy and energy-dense, nutrient-poor foods was highest among the youngest age group. Men consumed more animal and starchy foods than women, who consumed more fruits. There were only slight differences by education level. Food intakes differed substantially from the FBDG. Improvement of the Belgian food pattern, in particular among the youngest age group, is necessary for a better prevention of diet-related diseases. In addition, continuous or regular monitoring is crucial to permit trend analyses and to plan effective education or intervention strategies.
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The European Food Safety Authority (EFSA) was asked by the European Commission to deliver a scientific opinion on the health risks from citrinin in food and feed. Citrinin is a mycotoxin produced by several species of the genera Aspergillus, Penicillium and Monascus and occurs mainly in stored grains. The available occurrence data were not adequate to carry out a dietary exposure assessment. Citrinin is nephrotoxic and a no-observed-adverse-effect level (NOAEL) of 20 µg/kg body weight (b.w.) per day was identified from a 90-day study in rats. Due to the limitations and uncertainties in the database, the derivation of a health-based guidance value was not considered appropriate but a level of no concern for nephrotoxicity of 0.2 µg/kg b.w. per day was determined. Based on the available data a concern for genotoxicity and carcinogenicity could not be excluded at the level of no concern for nephrotoxicity. In the absence of adequate exposure data, characterisation of the risk of citrinin as a food contaminant was based on the estimate of the citrinin concentrations in grains and grain-based products that would result in an exposure equal to the level of no concern for nephrotoxicity. For high consuming toddlers, other children and adults this citrinin concentration is between 9 and 53 µg citrinin/kg and between 19 and 100 µg citrinin/kg for average consumers, respectively. For animals, risk characterisation was based on the estimate of the citrinin concentration in grains that would result in exceedance of the NOAEL of 20 µg/kg b.w. per day for pigs, which ranged between 640 and 1 173 µg/kg. The CONTAM Panel concluded that the impact of uncertainties on the risk assessment is large, and more data regarding the toxicity and the occurrence of citrinin in food and feed in Europe are needed to enable refinement of the risk assessment.
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Mycotoxins are important food contaminants responsible for health effects such as cancer, nephrotoxicity, hepatotoxicity or immunosuppression. The assessment of mycotoxin exposure is often based on calculations combining mycotoxin occurrence data in food with population data on food consumption. Because of limitations inherent to that approach, the direct measurement of biomarkers of exposure in biological fluids has been proposed as a suitable alternative to perform an accurate mycotoxin exposure assessment at individual level. For this reason, the BIOMYCO study was designed to assess mycotoxin exposure in Belgian adults and children using urinary biomarkers of exposure. Morning urine was gathered in a representative part of the Belgian population according to a standardised study protocol, whereby 155 children (3–12 years old) and 239 adults (19–65 years old) were selected based on random cluster sampling. These urine samples were analysed for the presence of 33 potential biomarkers with focus on aflatoxins, citrinin (CIT), fumonisins, trichothecenes, ochratoxin A (OTA), zearalenone and their metabolites using two validated LC–MS/MS methods.
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Ochratoxin A (OTA) and citrinin (CTN) are the most commonly co-occurring mycotoxins in a wide variety of food and feed commodities. The major target organ of these toxins is kidney but liver could also be a target organ. The combined toxicity of these two toxins in kidney cells has been studied but not in liver cell. In this study HepG2 cells were exposed to OTA and CTN, alone and in combination, with a view to compare the molecular and cellular mechanisms underlying OTA, CTN and OTA+CTN hepatotoxicity. OTA and CTN alone as well as in combination affected the viability of HepG2 cells in a dose-dependent manner. OTA+CTN, at a dose of 20% of IC50 of each, produced effect almost similar to that produced by either of the toxins at its IC50 concentration, indicating that the two toxins in combination act synergistically. The cytotoxicity of OTA+CTN on hepatocytes is mediated by increased level of intracellular ROS followed/accompanied by DNA strand breaks and mitochondria-mediated intrinsic apoptosis. Co-treatment of vitamin E (Vit E) with OTA, CTN and OTA+CTN reduced the levels of ROS and the cytotoxicity. But the genotoxic effect of OTA and OTA+CTN was not completely alleviated by Vit E treatment whereas the DNA damage as caused by CTN when treated alone was obviated, indicating that OTA induces DNA damage directly whereas CTN induces ROS-mediated DNA damage and OTA+CTN combination induces DNA damage not exclusively relying on but influenced by ROS generation. Taken together, these findings indicate that OTA and CTN in combination affect hepatocytes at very low concentrations and, thereby, pose a potential threat to public and animal health. Copyright © 2015. Published by Elsevier Ltd.
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Background: Red yeast rice is an herbal supplement that decreases low-density lipoprotein (LDL) cholesterol level. Objective: To evaluate the effectiveness and tolerability of red yeast rice and therapeutic lifestyle change to treat dyslipidemia in patients who cannot tolerate statin therapy. Design: Randomized, controlled trial. Setting: Community-based cardiology practice. Patients: 62 patients with dyslipidemia and history of discontinuation of statin therapy due to myalgias. Intervention: Patients were assigned by random allocation software to receive red yeast rice, 1800 mg (31 patients), or placebo (31 patients) twice daily for 24 weeks. All patients were concomitantly enrolled in a 12-week therapeutic lifestyle change program. Measurements: Primary outcome was LDL cholesterol level, measured at baseline, week 12, and week 24. Secondary outcomes included total cholesterol, high-density lipoprotein (HDL) cholesterol, triglyceride, liver enzyme, and creatinine phosphokinase (CPK) levels; weight; and Brief Pain Inventory score. Results: In the red yeast rice group, LDL cholesterol decreased by 1.11 mmol/L (43 mg/dL) from baseline at week 12 and by 0.90 mmol/L (35 mg/dL) at week 24. In the placebo group, LDL cholesterol decreased by 0.28 mmol/L (11 mg/dL) at week 12 and by 0.39 mmol/L (15 mg/dL) at week 24. Low-density lipoprotein cholesterol level was significantly lower in the red yeast rice group than in the placebo group at both weeks 12 (P < 0.001) and 24 (P = 0.011). Significant treatment effects were also observed for total cholesterol level at weeks 12 (P < 0.001) and 24 (P = 0.016). Levels of HDL cholesterol, triglyceride, liver enzyme, or CPK; weight loss; and pain severity scores did not significantly differ between groups at either week 12 or week 24. Limitation: The study was small, was single-site, was of short duration, and focused on laboratory measures. Conclusion: Red yeast rice and therapeutic lifestyle change decrease LDL cholesterol level without increasing CPK or pain levels and may be a treatment option for dyslipidemic patients who cannot tolerate statin therapy.
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Penicillium verrucosum is a fungus that can produce ochratoxin A and citrinin, two structurally related nephrotoxic mycotoxins. P. verrucosum usually occurs on wheat but can occasionally also be found in NaCl rich habitats such as salted cheeses or olives, indicating that this fungus can adapt to different environments. The ratio of ochratoxin A to citrinin produced by P. verrucosum is shifted to one of either mycotoxin at the expense of the other dependent on the environmental conditions. High NaCl concentrations shift secondary metabolite biosynthesis towards ochratoxin A production. P. verrucosum copes with NaCl stress by increased ochratoxin A biosynthesis, ensuring chloride homeostasis. Ochratoxin A carries chlorine in its molecule and can excrete chlorine from the cell. It was further shown that the regulation of ochratoxin A by high NaCl conditions is mediated by the HOG MAP kinase signal transduction pathway. Here it is shown that high oxidative stress conditions, evoked for example by increasing concentrations of Cu2 + cations in the growth medium, shift secondary metabolite biosynthesis of P. verrucosum from ochratoxin A to citrinin. The production of citrinin normalizes the oxidative status of the fungal cell under oxidative stress conditions leading to an adaptation to these environmental conditions and protects against increased oxidative stress caused by increased Cu2 + concentrations. Moreover citrinin also protects against light of short wavelength, which may also increase the oxidative status of the environment. The biosynthesis of citrinin is apparently regulated by a cAMP/PKA signaling pathway, because increasing amounts of external cAMP reduce citrinin biosynthesis in a concentration dependent manner. These conditions lead to the cross-regulation of the ochratoxin A/citrinin secondary metabolite pair and support the adaptation of P. verrucosum to different environments.
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Red yeast rice (RYR) is prepared by fermenting rice with various strains of the yeast Monascus spp of the Aspergillaceae family. Depending on the Monascus strains and the fermentation conditions, the products may contain monacolins, pigments and citrinin as secondary metabolites. Authentic and commercial RYR samples were analyzed using UHPLC-DAD-QToF-MS for monacolins, pigments and citrinin. A separation by UHPLC was achieved using a reversed-phase column and a gradient of water/acetonitrile each containing formic acid as the mobile phase. Accurate mass QToF spectrometry was used to distinguish isobaric monacolins. Principle component analysis (PCA), a chemometric technique was used to discriminate between authentic RYR, commercial RYR raw materials and dietary supplements. Three authentic RYR samples, 31 commercial RYR raw materials and 14 RYR dietary supplements were analyzed. Monacolin K content in 600mg of authentic RYR samples ranged from 1.2mg to 1.38mg. Amounts of monacolin K in dietary supplements labeled as containing 600mg of RYR varied more than 40-fold from 0.03mg to 2.18mg. Monacolin K content of dietary supplements labeled as containing 1200mg RYR varied more than 20-fold from 0.22mg to 5.23mg. In addition to large variations in quantity of monacolin K found in dietary supplements, RYR dietary supplements contained ratios of monacolins that differed significantly from authentic samples. The results indicated that RYR commercial products are of variable quality and the analytical method is suitable for quality control testing of a variety of RYR products.
Article
Mycotoxins are toxic metabolites produced by fungal species that commonly contaminate staple foods and feeds. They represent an unavoidable problem due to their presence in globally consumed cereals such as rice, maize and wheat. Most mycotoxins are immunosuppressive agents and some are carcinogens, hepatotoxins, nephrotoxins, and neurotoxins. Worldwide trends envision a stricter control of mycotoxins, however, the changing global environment may not be the ideal setting to control and reduce the exposure to these toxins. Although new technologies allow us to inspect the multi-mycotoxin presence in foods, new sources of exposure, gaps in knowledge of mycotoxins interactions, appearance of "emergent" mycotoxins and elucidation of consequent health effects can complicate their control even more. While humans are adapting to cope with environmental changes, such as food scarcity, decreased food quality, mycotoxin regulations, crop production and seasonality, and other climate related modifications, fungal species are also adapting and increased cases of mycotoxin adverse health effects are likely to occur in the future. To guarantee access to quality food for all, we need a way to balance global mycotoxin standards with the realistic feasibility of reaching them, considering limitations of producers and designing strategies to reduce mycotoxin exposure based on sound research.
Article
Citrinin is a toxic metabolite produced by several filamentous fungi of the genera Penicillium, Aspergillus and Monascus, which has been encountered as a natural contaminant in grains, foods, feedstuffs, as well as biological fluids. This mycotoxin is hepato-nephrotoxic and implicated in disease outbreaks in animals and humans. Some analytical systems have been developed for its detection and quantification. The purpose of this paper is to review physicochemical properties, qualitative and quantitative analytical methods of citrinin, evaluate advantages and disadvantages of various analytical techniques, and bring forward some constructive suggestions on establishment of international criteria for quality control of products contaminated with citrinin by comparing chromatographic properties, sample pre-treatment, recovery rate and detection limit of citrinin among various analytical methods. This paper concentrates the most important achievements on the analytical methods of citrinin published from 1980 to early 2004.
Article
  Mycotoxin toxicity occurs at very low concentrations, therefore sensitive and reliable methods for their detection are required. Consequently, sampling and analysis of mycotoxins is of critical importance because failure to achieve a suitable verified analysis can lead to unacceptable consignments being accepted or satisfactory shipments unnecessarily rejected. The general mycotoxin analyses carried out in laboratories are still based on physicochemical methods, which are continually improved. Further research in mycotoxin analysis has been established in such techniques as screening methods with TLC, GC, HPLC, and LC–MS. In some areas of mycotoxin method development, immunoaffinity columns and multifunctional columns are good choices as cleanup methods. They are appropriate to displace conventional liquid–liquid partitioning or column chromatography cleanup. On the other hand, the need for rapid yes/no decisions for exported or imported products has led to a number of new screening methods, mainly, rapid and easy-to-use test kits based on immuno-analytical principles. In view of the fact that analytical methods for detecting mycotoxins have become more prevalent, sensitive, and specific, surveillance of foods for mycotoxin contamination has become more commonplace. Reliability of methods and well-defined performance characteristics are essential for method validation. This article covers some of the latest activities and progress in qualitative and quantitative mycotoxin analysis.
Article
Crops may be contaminated by mycotoxins which can persist in the final products. Forty-five breakfast cereals were collected in French supermarkets. Ochratoxin A (OTA) and citrinin (CIT) were simultaneously extracted by a new method based on solvent partition validated in-house. The recoveries were over 80% for CIT and OTA. Fumonisin B1 (FB1) was analysed by an IUPAC method. The recoveries for FB1 ranged from 50% to 70%, depending on the matrix. The losses were located at the step of immunoaffinity clean-up.OTA was detected in 69% of the samples; 20% of them were above the EU limit of 3 μg/kg. Twenty percent contained CIT (1.5–42 μg/kg). FBs were detected, not only in cornflakes, but also in products containing oats or rice, in the range 1–1110 μg/kg. Some samples were contaminated by all three mycotoxins.
Article
Red yeast rice (RYR) is a widely available dietary supplement used by millions of patients as an alternative therapy for hyperlipidemia. It contains 14 active compounds called monacolins that inhibit hepatic cholesterol synthesis. Although studies have suggested that some formulations of RYR may be effective and safe for lipid lowering, monacolin levels are not standardized among marketed products and are generally not published on labels. We evaluated monacolin levels in 12 commercial RYR formulations and tested for citrinin, a mycotoxin that is nephrotoxic in animals. Each formulation of RYR was labeled "600 mg/capsule" of active product. Analyses for monacolins and citrinin were performed between August 2006 and June 2008 using high-performance liquid chromatography with mass spectroscopy-mass spectroscopy detection. Laboratory analyses of RYR products were conducted by ConsumerLab.com, White Plains, New York. There was marked variability in the 12 RYR products in total monacolins (0.31-11.15 mg/capsule), monacolin K (lovastatin) (0.10-10.09 mg/capsule), and monacolin KA (0.00-2.30 mg/capsule). Four products had elevated levels of citrinin. We found striking variability in monacolin content in 12 proprietary RYR products and the presence of citrinin in one-third of the formulations tested. Although RYR may have potential as an alternative lipid-lowering agent, our findings suggest the need for improved standardization of RYR products and product labeling. Until this occurs, physicians should be cautious in recommending RYR to their patients for the treatment of hyperlipidemia and primary and secondary prevention of cardiovascular disease.
Article
Currently, no consensus has been reached regarding the management of hyperlipidemia in patients who develop statin-associated myalgia (SAM). Many statin-intolerant patients use alternative lipid-lowering therapies, including red yeast rice. The present trial evaluated the tolerability of red yeast rice versus pravastatin in patients unable to tolerate other statins because of myalgia. The study was conducted in a community-based setting in Philadelphia, Pennsylvania. A total of 43 adults with dyslipidemia and a history of statin discontinuation because of myalgia were randomly assigned to red yeast rice 2,400 mg twice daily or pravastatin 20 mg twice daily for 12 weeks. All subjects were concomitantly enrolled in a 12-week therapeutic lifestyle change program. The primary outcomes included the incidence of treatment discontinuation because of myalgia and a daily pain severity score. The secondary outcomes were muscle strength and plasma lipids. The incidence of withdrawal from medication owing to myalgia was 5% (1 of 21) in the red yeast rice group and 9% (2 of 22) in the pravastatin group (p = 0.99). The mean pain severity did not differ significantly between the 2 groups. No difference was found in muscle strength between the 2 groups at week 4 (p = 0.61), week 8 (p = 0.81), or week 12 (p = 0.82). The low-density lipoprotein cholesterol level decreased 30% in the red yeast rice group and 27% in the pravastatin group. In conclusion, red yeast rice was tolerated as well as pravastatin and achieved a comparable reduction of low-density lipoprotein cholesterol in a population previously intolerant to statins.
Article
Red yeast rice is an herbal supplement that decreases low-density lipoprotein (LDL) cholesterol level. To evaluate the effectiveness and tolerability of red yeast rice and therapeutic lifestyle change to treat dyslipidemia in patients who cannot tolerate statin therapy. Randomized, controlled trial. Community-based cardiology practice. 62 patients with dyslipidemia and history of discontinuation of statin therapy due to myalgias. Patients were assigned by random allocation software to receive red yeast rice, 1800 mg (31 patients), or placebo (31 patients) twice daily for 24 weeks. All patients were concomitantly enrolled in a 12-week therapeutic lifestyle change program. Primary outcome was LDL cholesterol level, measured at baseline, week 12, and week 24. Secondary outcomes included total cholesterol, high-density lipoprotein (HDL) cholesterol, triglyceride, liver enzyme, and creatinine phosphokinase (CPK) levels; weight; and Brief Pain Inventory score. In the red yeast rice group, LDL cholesterol decreased by 1.11 mmol/L (43 mg/dL) from baseline at week 12 and by 0.90 mmol/L (35 mg/dL) at week 24. In the placebo group, LDL cholesterol decreased by 0.28 mmol/L (11 mg/dL) at week 12 and by 0.39 mmol/L (15 mg/dL) at week 24. Low-density lipoprotein cholesterol level was significantly lower in the red yeast rice group than in the placebo group at both weeks 12 (P < 0.001) and 24 (P = 0.011). Significant treatment effects were also observed for total cholesterol level at weeks 12 (P < 0.001) and 24 (P = 0.016). Levels of HDL cholesterol, triglyceride, liver enzyme, or CPK; weight loss; and pain severity scores did not significantly differ between groups at either week 12 or week 24. The study was small, was single-site, was of short duration, and focused on laboratory measures. Red yeast rice and therapeutic lifestyle change decrease LDL cholesterol level without increasing CPK or pain levels and may be a treatment option for dyslipidemic patients who cannot tolerate statin therapy.
Article
A convenient method for the production and analysis of citrinin in corn is described. Up to 2.964 g of citrinin can be produced by Penicillium citrinum per kg of corn by harvesting on day 21 or later. The analysis method has a lower detection limit of 0.25 ppm. Heating citrinin-contaminated corn destroys citrinin but may produce another toxin instead.
Article
Citrinin, a nephrotoxic mycotoxin, has been of growing importance also for the "International Agency for Research on Cancer", ever since its presumable role in the occurrence of Balcan endemic nephropathy (BEN) was discussed at the congress on "Mycotoxins, Endemic Nepthropathy and Urinary Tract Tumours" held in Lyon in June 1991 (12). In late 1991, citrinin was therefore also included in the list of toxins to be examined by the screening subcommittees on natural toxins of the International Live Science Institute, European Branch.
Article
A survey of the nephrotoxic mycotoxins ochratoxin and citrinin in cereals used as feed for bacon pigs was conducted, employing thin-layer chromatography techniques, in districts of Denmark where the incidence rate of mycotoxic porcine nephropathy was high. A total of 58 per cent of the samples contained ochratoxin A and 9 per cent contained citrinin, always together with ochratoxin A. The detection of the two mycotoxins was confirmed by derivative formation and, for selected samples, by nuclear magnetic resonance spectroscopy (NMR). The maximal concentration of ochratoxin A and citrinin was 27.5 ppm and 2 ppm, respectively. About half of the contaminated samples contained more than 200 ppb, which is the level at which development of porcine nephropathy is possible. This is the first report which elucidates the association of ochratoxin A and citrinin and a naturally occurring disease in domestic animals.
Article
Cereal samples were collected in 1998 from Bulgarian villages without [control village (C), n = 20] or with [endemic villages (E); E1, n = 21; E2, n = 30; E3, n = 23] a history of Balkan endemic nephropathy (BEN). Sampling included foods (wheat, corn) and feeds (barley, oats, wheat bran). Analysis of ochratoxin A and citrinin was done by enzyme immunoassays (EIA), with detection limits of 0.5 and 5 ng/g, respectively. Ochratoxin A-positive results were confirmed by HPLC after immunoaffinity chromatography. Highest toxin levels were found in wheat, wheat bran, and oats. For ochratoxin A, the percentages of positives were 35% (C), 29% (E1), 30% (E2), and 47% (E3), the mean/median values of positives were 1.5/1.3 ng/g (C), 11/1.6 ng/g (E1), 18/1.6 ng/g (E2), and 3.5/1.5 ng/g (E3). For citrinin, 5.0% (C), 14% (E1), 3.3% (E2), and 13% (E3) were positive, and the mean/median values were 6.1/6.1 ng/g (C), 180/83 ng/g (E1), 10/10 ng/g (E2), and 84/20 ng/g (E3). Highest concentrations of ochratoxin (maximum = 140 ng/g) and citrinin (maximum = 420 ng/g) were found in samples from endemic villages. Co-contamination with ochratoxin A and citrinin was found for one sample (14% of positives) from village C and for six samples (22% of positives) from villages E1-E3. Citrinin levels in these samples were 2-200 times higher than those of ochratoxin A.
Article
Detailed analyses were undertaken of the natural constituents of red yeast rice, a traditional Chinese medicine and food known for centuries to improve blood circulation. Preparation of red yeast rice following ancient methods by fermenting the fungal strain Monascus purpureus Went on moist and sterile rice indicated the presence of a group of metabolites belonging to the monacolin family of polyketides, together with fatty acids, and trace elements. The presence of these compounds may explain in part the cholesterol-lowering ability associated with this traditional Chinese food.
Article
Some strains of Chinese red yeast rice, when prepared by solid fermentation, produce compounds called monacolins that inhibit cholesterol production. When used as a dietary supplement to achieve and maintain healthy cholesterol levels, Chinese red yeast rice has significant potential to reduce health care costs and contribute to public health by reducing heart disease risk in individuals with moderate elevations of circulating cholesterol levels. Whereas one proprietary strain of Chinese red yeast rice has been demonstrated to lower cholesterol levels significantly in clinical trials, other strains being sold as Chinese red yeast rice dietary supplements have not undergone similar evaluation. In order to determine whether the results of a clinical trial conducted with one strain of Chinese red yeast rice could be generalized to other preparations of Chinese red yeast rice, nine different commercially available dietary supplements were purchased tested for chemical constituents. Monacolins were measured by high performance liquid chromatography (HPLC) that separates the various monacolins in Chinese red yeast rice. Citrinin concentration, a toxic fermentation byproduct, was measured by radioimmunoassay. Total monacolin content varied from 0% to 0.58% w/w and only 1 of 9 preparations had the full complement of 10 monacolin compounds. Citrinin was found at measurable concentrations in 7 of the 9 preparations. The findings from clinical trials demonstrating significant and clinically relevant cholesterol reduction using a defined Chinese red yeast rice preparation containing 10 different monacolins cannot be generalized to preparations that do not contain the same levels and profile of monacolins. Standardized manufacturing practices should be established for Chinese red yeast rice sold as a dietary supplement in order ensure equivalence of content of active ingredients in preparations being sold to the public and to limit the production of unwanted byproducts of fermentation such as citrinin. In common with other botanical dietary supplements, the full potential of this product will not be realized until standards for production and labeling of Chinese red yeast rice are further developed.
Article
A stable isotope dilution assay (SIDA) was developed for quantification of the mycotoxin ochratoxin A (OTA) by using [2H5]-OTA as internal standard. The synthesis of labelled OTA was accomplished by acid hydrolysis of unlabelled OTA and subsequent coupling one of the products, ochratoxin alpha, to [2H5]-L-phenylalanine. The mycotoxin was quantified in foods by LC-tandem MS after extraction with buffers containing [2H5]-OTA and clean-up by immuno affinity chromatography or by solid phase extraction on silica. The method showed a sufficient sensitivity with a low detection and quantification limit of 0.5 and 1.4 microg/kg, respectively, and good precision in inter-assay studies showing a CV (n = 3) of 3.6%. The analysis of certified reference materials resulted in a low bias of 2.1% from the certified values and revealed excellent accuracy of the new method. To prove the suitability of SIDA. OTA was quantified in a number of food samples and resulted mainly in not detectable OTA contents. However, three samples of raisins exceeded the legal limit of 10 microg/kg and highlighted the need for further controlling the contamination with the mycotoxin.
Article
The assessment of the relationship between species and mycotoxins production has proven to be very difficult. The modern literature is cluttered with examples of species purported to make particular mycotoxins, but where the association is incorrect. In some cases, mycotoxins have even been named based on an erroneous association with a particular species: verruculogen, viridicatumtoxin and rubratoxin come to mind. As time has gone on, and more and more compounds have been described, lists of species-mycotoxin associations have become so large, and the inaccuracies in them so widespread in acceptance, that determining true associations has become very difficult. It does not need to be emphasised how important it is that these associations be known accurately. The possible presence of mycotoxigenic fungi in foods, and rational decisions on the status of foods suspected to contain mycotoxins, are ever present problems in the food industry around the world. In defining mycotoxins, we exclude fungal metabolites which are active against bacteria, protozoa, and lower animals including insects.
Article
Trichothecenes are secondary metabolites produced by several fungi of the Fusarium genus during their growth period. They inhibit protein biosynthesis in eukaryotic cells resulting in numerous toxic effects such as diarrhea, vomiting, and gastro-intestinal inflammation. Considering its occurrence in food and feedstuff, deoxynivalenol (DON) is one of the most important trichothecenes. We report the synthesis of stable isotope labeled 15-d(1)-deoxynivalenol (15-d(1)-DON) from its natural precursor 3-acetyldeoxynivalenol (3-AcDON) as starting material. Furthermore, a method for the analysis of DON and 3-AcDON using HPLC-MS/MS with stable isotope labeled 15-d(1)-DON and 3-d(3)-AcDON as internal standards has been developed. In total, 18 cereal product samples were analyzed with contamination levels ranging from 10-301 microg/kg for DON and 5-14 microg/kg for 3-AcDON. This is the first report of an isotope dilution MS method for the analysis of type B-trichothecenes.
Article
Several studies implicated mycotoxins, in endemic kidney disease geographically limited to Balkan region (Balkan endemic nephropathy (BEN)). In Bulgaria, much higher prevalence of ochratoxin A (OTA), exceeding 2 microg/L, was observed in the blood of affected population. OTA is found more often in the urine of people living in BEN-endemic villages. To confirm and quantify exposure to OTA in Vratza district, we followed up OTA intake for 1 month, OTA in blood and urine from healthy (20-30 years old) volunteers, from two villages with high risk for BEN disease. Food samples were collected daily, blood and urine at the beginning of each week. Relations between increasing OTA intake, blood concentration and elimination of OTA in urine have been studied in rats. Average weekly intake of OTA varies from 1.9 to 206 ng/kg body weight, twice tolerable weekly intake recommended by JECFA. OTA blood concentrations are in the same range as previously reported in this region with concentrations reaching 10 microg/L. Weekly OTA food intake is not directly correlated with blood and urine concentrations. Biomarkers of biological effects such as DNA adducts were detected in patients affected by urinary tract tumours (UTT) and in rat study. All these plead for the implication of OTA, in BEN and UTT.
Article
The first stable isotope dilution assay (SIDA) for the simultaneous quantitation of the most abundant type A trichothecenes in foods and feeds was developed. Synthesis of carbon-13-labeled T2-toxin, HT2-toxin, diacetoxyscirpenol, and monoacetoxyscirpenol was accomplished by [13C2]-acetylation of T2-triol and scirpentriol, respectively. Scirpentriol was prepared from diacetoxyscirpenol by complete alkaline hydrolysis and subsequently was converted to [13C6]-triacetoxyscirpentriol by peracetylation with [13C4]-acetic anhydride. The latter compound was selectively hydrolyzed using ammonium hydroxide to give [13C4]-diacetoxyscirpenol and [13C2]-monoacetoxyscirpenol in reasonable yields. Analogously, [13C6]-T2-triacetate was prepared from T2-triol and subjected to controlled hydrolysis to yield [13C4]-T2-toxin and [13C2]-HT2-toxin. All synthesized products were characterized by NMR and MS experiments. Using the prepared isotopically labeled standards, SIDAs were developed for the quantitation of type A trichothecenes in food and feeds. The mycotoxins were quantified by LC-single and tandem MS after cleanup on multifunctional columns. The method revealed good sensitivity with low detection and quantification limits along with excellent recovery and good precision in interassay studies. Food samples were analyzed using the developed SIDA and showed substantial contamination of oat products with T2-toxin and HT2-toxin. Diacetoxyscirpenol was detected on potatoes, whereas monoacetoxyscirpenol was not present in the analyzed samples.
Article
To find out whether ochratoxin A (OTA), citrinin (CIT), aristolochic acids (AA) are etiologic agents of Balkan endemic nephropathy (BEN) or Chinese herbal nephrotoxicity, and associated urinary tract tumor (UTT), we have compared (i) in human kidney cell culture, the DNA adduct formation and persistence of OTA/CIT and AA adducts (ii) analyzed DNA adduct in several tumors from human kidney suspected to be exposed to either OTA and CIT, or AAs (iii) analyzed OTA, CIT, and AA in food. In kidney cell cultures, formation of specific OTA-DNA adduct and AA-DNA adduct were detected in the same range (around 10 adducts/10(9) nucleotides) and were time- and dose-dependent. After 2 days all disappeared. DNA adduct related to OTA and CIT are found in human kidney tissues from Balkans, France, and Belgium whereas no DNA adducts related to AA could be found in any tumors of BEN patients from Croatia, Bulgaria, or Serbia. No DNA adduct was found in kidney biopsy or necropsy of the French women suspected to be exposed to AA. OTA and CIT are more frequently found in rural area. AA was never detected. All these plead for implication of mycotoxins, especially OTA, in BEN and UTT.
Occurrence of the mycotoxin citrinin and its metabolite dihydrocitrinone in urines of German adults Synthesis of four carbon-13-labeled type A trichothecene mycotoxins and their application as internal standards in stable isotope dilution assays
  • N Ali
  • M Blaszkewicz
  • G H Degen
  • S Asam
  • M Rychlik
Ali, N., Blaszkewicz, M. and Degen, G.H., 2015. Occurrence of the mycotoxin citrinin and its metabolite dihydrocitrinone in urines of German adults. Archives of Toxicology 89: 573-578. Asam, S. and Rychlik, M., 2006. Synthesis of four carbon-13-labeled type A trichothecene mycotoxins and their application as internal standards in stable isotope dilution assays. Journal of Agricultural and Food Chemistry 54: 6535-6546.
Assessment of dietary intake of ochratoxin A by the population of EU member states. Reports on tasks for scientific cooperation
  • M Miraglia
  • C Brera
Miraglia, M. and Brera, C., 2002. Assessment of dietary intake of ochratoxin A by the population of EU member states. Reports on tasks for scientific cooperation. Reports of experts participating in SCOOP Task 3(7). Directorate-General Health and Consumer Protection, Brussels, Belgium. Available at: http://tinyurl.com/ hkefqsd.
  • H K Frank
Frank, H.K., 1992. Citrinin. Zeitschrift für Ernährungswissenschaft 31: 164-177.
Commission Regulation (EU) No. 212/2014 amending regulation No 1881/2006 as regards maximum levels of the contaminant citrinin in food supplements based rice fermented with red yeast Monascus purpureus
European Commission (EC), 2014a. Commission Regulation (EU) No. 212/2014 amending regulation No 1881/2006 as regards maximum levels of the contaminant citrinin in food supplements based rice fermented with red yeast Monascus purpureus. Official Journal of the European Union L 67: 3-4.
Mycotoxins and their health effects on human and animal health Methods for analysis of citrinin in human blood and urine
  • E M Bezerra Da Rocha
  • F Da Chagas Oliveira Freire
  • F E Feitosa Maia
  • Florindo Guedesc
  • M I Rondina
Bezerra da Rocha, E.M., Da Chagas Oliveira Freire, F., Feitosa Maia, F.E., Florindo Guedesc, M.I. and Rondina, D., 2014. Mycotoxins and their health effects on human and animal health. Food Control 36: 159-165. Blaszkewicz, M., Munoz, K. and Degen, G.H., 2013. Methods for analysis of citrinin in human blood and urine. Archives of Toxicology 87: 1087-1094.
Commission Decision EC implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results
European Commission (EC), 2002. Commission Decision 2002/657/ EC implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Official Journal of the European Communities L 221: 8-36.