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

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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Citrinin Determination in Food and Food Supplements by LC-MS/MS: Development and Use of Reference Materials in an International Collaborative Study

Toxins ◽

10.3390/toxins13040245 ◽

2021 ◽

Vol 13 (4) ◽

pp. 245

Author(s):

Emmanuel K. Tangni ◽

François Van Hove ◽

Bart Huybrechts ◽

Julien Masquelier ◽

Karine Vandermeiren ◽

...

Keyword(s):

Reference Materials ◽

Ginkgo Biloba ◽

Raw Materials ◽

Collaborative Study ◽

Standard Operating Procedure ◽

Food Supplements ◽

Red Yeast Rice ◽

Proficiency Tests ◽

Red Yeast ◽

Food And Feed

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.

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Simultaneous Rapid Detection of Aflatoxin B1 and Ochratoxin A in Spices Using Lateral Flow Immuno-Chromatographic Assay

Foods ◽

10.3390/foods10112738 ◽

2021 ◽

Vol 10 (11) ◽

pp. 2738

Author(s):

Xue Zhao ◽

Xindi Jin ◽

Zhang Lin ◽

Qi Guo ◽

Bin Liu ◽

...

Keyword(s):

Ochratoxin A ◽

Aflatoxin B1 ◽

High Performance ◽

Simultaneous Detection ◽

High Sensitivity ◽

Strong Stability ◽

Test Strip ◽

Chromatography Analysis ◽

Relative Standard ◽

Strip Test

Spices are susceptible to contamination by aflatoxin B1 (AFB1) and ochratoxin A (OTA), which are both mycotoxins with high toxicity and carcinogenicity. In this study, we aimed to develop an immuno-chromatographic strip test for the simultaneous quantification of AFB1 and OTA in spices by spraying the coupled antigens AFB1–ovalbumin (AFB1–OVA) and OTA–ovalbumin (OTA–OVA) on a nitrocellulose membrane. The test strip had high sensitivity, good specificity, and strong stability. The detection limits of these two mycotoxins in Chinese prickly ash, pepper, chili, cinnamon, and aniseed were 5 μg/kg. The false positivity rate was 2%, and the false negativity rate was 0%. The maximum coefficient of variation was 4.28% between batches and 5.72% within batches. The average recovery rates of AFB1 and OTA in spices were 81.2–113.7% and 82.2–118.6%, respectively, and the relative standard deviation (RSD) was <10%. The actual sample detection was consistent with high performance liquid chromatography analysis results. Therefore, the immuno-chromatographic test strips developed in this study can be used for the on-site simultaneous detection of AFB1 and OTA in spices. This method would allow the relevant regulatory agencies to strengthen supervision in an effort to reduce the possible human health hazards of such contaminated spices.

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Postmarketing nutrivigilance safety profile: a line of dietary food supplements containing red yeast rice for dyslipidemia

Archives of Medical Science ◽

10.5114/aoms/133716 ◽

2021 ◽

Author(s):

Maciej Banach ◽

Niki Katsiki ◽

Gustavs Latkovskis ◽

Manfredi Rizzo ◽

Daniel Pella ◽

...

Keyword(s):

Adverse Events ◽

Adverse Reactions ◽

Case Reports ◽

Food Supplement ◽

Product Line ◽

Food Supplements ◽

Red Yeast Rice ◽

Red Yeast ◽

Consumer Safety ◽

Suspected Adverse Reactions

IntroductionIn the absence of a European standardized postmarketing food supplement surveillance system (nutrivigilance), some member states and companies have developed their own approaches to monitoring potential adverse reactions to secure a high level of product safety. This paper describes the use of a nutrivigilance system in monitoring the incidence of spontaneously reported suspected adverse reactions associated with food supplements containing red yeast rice (RYR).Material and methodsWe report the data from a widely used product marketed under the trademark Armolipid/Armolipid Plus. Postmarketing information was collected in a voluntary nutrivigilance system established by the manufacturing company (Meda Pharma SpA, a Viatris Company, Monza, Italy). From 1st October 2004 to 31st December 2019, this system captured cases of suspected adverse reactions spontaneously reported by consumers, healthcare professionals, health authorities, regardless of causality.ResultsThe total number of case reports received mentioning the RYR food supplement product line was 542, in which 855 adverse events (AEs) were reported. The total reporting rate of AEs was estimated to be 0.037% of 2,287,449 exposed consumers. Of the 542 cases, 21 (0.0009% of exposed consumers) included suspected serious adverse events (SAEs). After careful investigation, 6 cases (0.0003% of consumers exposed) and 6 AEs were assessed by the manufacturer as serious and potentially related to exposure to the above-mentioned RYR-based nutraceutical.ConclusionsThis nutrivigilance-derived data analysis clearly demonstrates a low prevalence of suspected adverse events associated with the red yeast rice product line. Consumer safety of food supplements could be generally improved by raising awareness of the importance of following the indications and warnings detailed in a food supplement’s labeling.

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Survey of Ochratoxin A in Freshly Harvested Durum and Hard Red Spring Wheat in the United States, 2011 and 2012

Journal of Food Protection ◽

10.4315/0362-028x.jfp-13-496 ◽

2014 ◽

Vol 77 (6) ◽

pp. 1005-1009 ◽

Cited By ~ 4

Author(s):

JULIE A. KURUC ◽

FRANK MANTHEY ◽

SENAY SIMSEK ◽

CHARLENE WOLF-HALL

Keyword(s):

Durum Wheat ◽

Ochratoxin A ◽

Great Plains ◽

High Performance ◽

The United States ◽

Cereal Grains ◽

Hard Red Spring Wheat ◽

The World ◽

Food And Feed ◽

The U.S

Ochratoxin A (OTA) is a toxin produced by some Penicillium and Aspergillus species around the world in a variety of food and feed, especially cereal grains, before harvest but primarily during storage. Durum and hard red spring (HRS) wheat samples were collected right after harvest as part of the U.S. regional crop quality survey in both 2011 (n = 560) and 2012 (n = 654) from the upper Great Plains. All samples were analyzed for OTA contamination using high-performance liquid chromatography with fluorescence detection. Overall, 2.1% of the samples were positive for OTA. In 2011, OTA was detected in 1.0% of the durum wheat samples but was not found in HRS wheat. In 2012, 8.3 and 1.4% of the durum and HRS wheat samples, respectively, were positive for OTA. Of the 25 samples that had detectable OTA, 3 samples (12%), all of which were durum wheat, had OTA that exceeded 5 ng/g.

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Analysis of Citrinin in Cereals, Red Yeast Rice Dietary Supplement, and Animal Feed by Immunoaffinity Column Cleanup and LC with Fluorescence Detection

Journal of AOAC International ◽

10.5740/jaoacint.16-0060 ◽

2016 ◽

Vol 99 (4) ◽

pp. 1025-1031 ◽

Cited By ~ 8

Author(s):

Elaine Marley ◽

Phyllis Brown ◽

Dave Leeman ◽

Carol Donnelly

Keyword(s):

Dietary Supplement ◽

Fluorescence Detection ◽

Animal Feed ◽

Red Yeast Rice ◽

Immunoaffinity Column ◽

Breakfast Cereal ◽

Method Performance ◽

Satisfactory Performance ◽

Red Yeast ◽

Food And Feed

Abstract The analysis of citrinin in various cereals (wheat, oats, maize, rice, and rye and multigrain breakfast cereal), red yeast rice (dietary supplement and traditional medicine), distillers dried grain with solubles, and barley (animal feed) was carried out using a citrinin immunoaffinity column (IAC) for sample cleanup before LC analysis with fluorescence detection (LC-fluorescence). To establish method performance characteristics, wheat was spiked with citrinin at levels of 10–200 μg/kg, whereas red yeast rice was spiked at levels of 100–3000 μg/kg. Methanol–water (75 + 25, v/v) was used for the extraction of cereals and animal feed, and extraction was with 100% methanol for red yeast rice. Cleanup used a commercial citrinin IAC, followed by LC-fluorescence (λex, 330 nm; λem, 500 nm). Recoveries ranged from 80 to 110%, with r from 0.7 to 4.3%. The LOQ for citrinin in both wheat and red yeast rice was 10 μg/kg, with an LOD of 3 μg/kg. Satisfactory performance was demonstrated in a proficiency testing exercise for a sample of maize contaminated with both citrinin and ochratoxin A. It was concluded that the commercial citrinin IAC was capable of providing an efficient and effective cleanup of complex food and feed matrixes to enable citrinin to be reliably determined with the specific LC-fluorescence system used.

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Multiresidue method for the simultaneous analysis of antibiotics and mycotoxins in feeds by ultra-high performance liquid chromatography coupled to tandem mass spectrometry

Acta Alimentaria ◽

10.1556/066.2020.00159 ◽

2021 ◽

Author(s):

Ü.İ. Konak ◽

H.A. Yatmaz ◽

Ş. Nilüfer ◽

T. Erkaymaz ◽

M. Certel

Keyword(s):

High Performance ◽

Safety Issue ◽

Animal Health ◽

Animal Origin ◽

Limit Of Quantification ◽

Multiresidue Method ◽

Animal Feeds ◽

Relative Standard ◽

Feed Samples

AbstractResidues in animal feeds and foods of animal origin have been important safety issue concerning both human and animal health. A multiresidue method for determination of eight mycotoxins and ten antibiotics was developed and validated in animal feeds by using QuEChERS (quick, easy, cheap, effective, rugged, and safe) extraction followed by UHPLC-MS/MS. Optimisation of UHPLC-MS/MS parameters was performed to achieve good separation and resolution. The method was validated according to the European Commission Decision 2002/657/EC. Matrix matched calibration curves showed good r2 (≥0.995) values, and limit of quantification (LOQ) values varied between 1.2 and 5.2 μg kg−1. Average recoveries ranged from 60 to 102% with relative standard deviations of 2.2 and 15.6% for all type of feed samples except for tetracyclines, lincomycin, tylosin, ochratoxin A, and fumonisin (B1 and B2).

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Development of a Magnetic Nanoparticles-Based Screen-Printed Electrodes (MNPs-SPEs) Biosensor for the Quantification of Ochratoxin A in Cereal and Feed Samples

Toxins ◽

10.3390/toxins10080317 ◽

2018 ◽

Vol 10 (8) ◽

pp. 317 ◽

Cited By ~ 8

Author(s):

Xian Zhang ◽

Zuohuan Wang ◽

Hui Xie ◽

Renjie Sun ◽

Tong Cao ◽

...

Keyword(s):

Magnetic Nanoparticles ◽

Ochratoxin A ◽

Limit Of Detection ◽

Screen Printed Electrodes ◽

Combined Use ◽

Highly Sensitive ◽

Relative Standard ◽

Liquid Chromatography Tandem Mass ◽

Feed Samples ◽

Screen Printed

A rapid and sensitive electrochemical biosensor based on magnetic nanoparticles and screen-printed electrodes (MNPs-SPEs sensor) was developed for the detection of ochratoxin A (OTA) in cereal and feed samples. Different types of magnetic nanoparticles-based ELISA (MNPs-ELISA) were optimized, and the signal detection, as well as sensitivity, was enhanced by the combined use of screen-printed electrodes (SPEs). Under the optimized conditions, the calibration curve of the MNPs-SPEs sensor was y = 0.3372x + 0.8324 (R2 = 0.9805). The linear range of detection and the detection limit were 0.01–0.82 ng/mL and 0.007 ng/mL, respectively. In addition, 50% inhibition (IC50) was detectable at 0.10 ng/mL. The limit of detection (LOD) of this MNPs-SPEs sensor in cereal and feed samples was 0.28 μg/kg. The recovery rates in spiked samples were between 78.7% and 113.5%, and the relative standard deviations (RSDs) were 3.6–9.8%, with the coefficient of variation lower than 15%. Parallel analysis of commercial samples (corn, wheat, and feedstuff) showed a good correlation between MNPs-SPEs sensor and liquid chromatography tandem mass spectrometry (LC/MS-MS). This new method provides a rapid, highly sensitive, and less time-consuming method to determine levels of ochratoxin A in cereal and feedstuff samples.

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Assessment of a New GC-MS/MS System for the Confirmatory Measurement of PCDD/Fs and (N)DL-PCBs in Food under EU Regulation

Foods ◽

10.3390/foods8080302 ◽

2019 ◽

Vol 8 (8) ◽

pp. 302

Author(s):

Flavio Antonio Franchina ◽

Eliane Lazzari ◽

George Scholl ◽

Jean-François Focant

Keyword(s):

Mass Spectrometry ◽

High Resolution Mass Spectrometry ◽

Multiple Reaction Monitoring ◽

Triple Quadrupole ◽

Eu Regulation ◽

Relative Standard ◽

Food And Feed ◽

Quantitative Results ◽

Feed Samples ◽

The Eu

Polychlorodibenzo-p-dioxins (PCDDs), polychloro-dibenzofurans (PCDFs), dioxin-like (DL), and non dioxin-like (NDL) polychlorinated biphenyls (PCBs) are currently regulated in food and feed within the European territory (EU 2017/644-771). The confirmatory methods of analysis for checking compliance with maximum levels (MLs) for these involve either the historically-established GC-magnetic sector high-resolution mass spectrometry (GC-HRMS) and, more recently, GC-triple quadrupole mass spectrometry operating in tandem mode (GC-QQQMS/MS). In this study, the performance of a novel triple quadrupole GC-QQQMS/MS system equipped with a programable temperature vaporization (PTV) injector was evaluated for the analysis of regulated PCDD/Fs and PCBs in food and feed. The MS analyzer was equipped with a titanium ionization chamber and a new short collision cell capable to accumulate and eject ions by means of very narrow pulses that allow to minimize the noise and to adapt accumulation times for sensitive multiple reaction monitoring (MRM). The analytical capability of the system was confronted by the strict requirements (selectivity, reproducibility, linearity, quant/qual MRM transitions, accuracy, robustness) set by the EU Regulation for a range of standards, quality control (QC) and food/feed samples. In this respect, the approach showed high precision (1.9–15% relative standard deviation (RSD) at low pg/µL) and accuracy (>80%, except for one hexa-CDD). The quantitative results were also compared to the most used GC-HRMS. In this case, comparable results in terms of single congener concentration basis and total toxic equivalent (TEQ) basis for PCDD/Fs and DL-PCBs were obtained for the QC samples analyzed.

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A Dilute-and-Shoot UHPLC-MS/MS Isotope Dilution Method for Simultaneous Determination and Confirmation of Eleven Mycotoxins in Dried Distillers Grains with Solubles

Journal of AOAC International ◽

10.1093/jaoacint/qsab111 ◽

2021 ◽

Author(s):

Cristina B Nochetto ◽

Li Hui

Keyword(s):

Ochratoxin A ◽

High Performance ◽

Animal Feed ◽

Animal Health ◽

Fumonisin B1 ◽

Isotope Dilution Method ◽

Dilution Method ◽

Distillers Grains With Solubles ◽

Distiller's Grains ◽

Relative Standard

Abstract Background Natural contamination of mycotoxins in dried distiller’s grains with solubles (DDGS) as a mainstream animal feed ingredient poses risk to animal health. Objective A regulatory method was needed for the agency to simultaneously detect eleven mycotoxins of high regulatory priority in DDGS. Methods Ten grams of DDGS sample were extracted twice with acetonitrile/water under mildly acidic condition. Two aliquots from the combined crude extract were taken and processed separately: (1) diluted 400-fold with solvent for analysis of deoxynivalenol and fumonisins B1 and B2; (2) pH adjusted to 7.5, then diluted 15.7-fold for analysis of aflatoxins B1, B2, G1, G2, ochratoxin A, zearalenone, and T-2 and HT-2 toxins. Uniformly-labelled 13C-isotopologues of these mycotoxins were added as internal standards to the diluted extracts for quantitative analysis by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS). Results. The linear quantitation ranges (µg/kg) were: aflatoxin B1, B2, G1, and G2, 1.57 to 105; zearalenone, 16.3 to 1090; T-2 toxin, 3.14 to 208; HT-2 toxin, 48.2 to 3220; ochratoxin A, 0.47 to 31.4; deoxynivalenol, 240 to 16000; fumonisin B1 and B2, 320 to 21200. Accuracies for these analytes at each of three fortification levels range from 70.7% to 100%, with corresponding relative standard deviations between 1.4% to 10.5%. True recoveries were all higher than 83%. Conclusions This method was successfully validated to meet the agency’s performance guidelines for regulatory methods. Highlights This method is easy, quick and robust to simultaneously quantify and confirm presence of eleven regulated mycotoxins in DDGS.

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Determination of Citrinin in Red Yeast Rice by High Performance Liquid Chromatography with Immuno-Affinity Column

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.1073-1076.353 ◽

2014 ◽

Vol 1073-1076 ◽

pp. 353-356

Author(s):

Wei Meng ◽

Li Xin Zhu ◽

Xue Mei Guo ◽

Kai Hong Li ◽

Ren Rong Liu

Keyword(s):

Monoclonal Antibody ◽

High Performance Liquid Chromatography ◽

Liquid Chromatography ◽

High Performance ◽

Reversed Phase ◽

Affinity Column ◽

Red Yeast Rice ◽

Fluorescence Excitation ◽

Red Yeast

To prepare immune-affinity column (IAC) of citrinin, CNBr activated Sepharose 4FF was used as a carrier to couple with monoclonal antibody against citrinin. A reversed-phase high performance liquid chromatography (HPLC) with fluorescence detection method was used to determine the samples elution after the IAC cleaning up. The fluorescence excitation and emission maxima of citrinin were 331 and 500 nm, respectively. Citrinin is determined by HPLC with a retention time of 6.5 min (flow rate of 1 mL/min) with acetonitrile-H2O (45:55, v/v, pH 2.0). The linear range is 1-500 ng/mL. The column capacity is 0.35 μg when using 0.5 mL CNBr activated Sepharose 4FF and 500 μg monoclonal antibody against citrinin. Average recoveries of citrinin from red yeast rice spiked at levels of 0.1-0.6 mg/kg range from 74.2% to 87.12%. 16 samples of red yeast rice were determined by HPLC with immune-affinity column. The results show 12 samples contain citrinin, and concentrations of citrinin of red yeast rice are 100.6~443.6 μg/kg. The IAC method is found to be highly effective, sensitive and convenient for isolating the target analyte from samples.

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