Editorial

3 pages, 178 KiB

Open AccessEditorial

Introduction to the Toxins Special Issue on Improved Analytical Technologies for the Detection of Natural Toxins and Their Metabolites in Food

by Veronica Maria Teresa Lattanzio

Toxins 2020, 12(8), 467; https://doi.org/10.3390/toxins12080467 - 22 Jul 2020

Cited by 3 | Viewed by 1752

Abstract

Food, by nature, is a biological substrate and is therefore capable of supporting the growth of microbials that are potential producers of toxic compounds [...] Full article

(This article belongs to the Special Issue Improved Analytical Technologies for the Detection of Natural Toxins and Their Metabolites in Food)

Research

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34 pages, 1973 KiB

Open AccessEditor’s ChoiceArticle

Revisiting the Neuroblastoma Cell-Based Assay (CBA-N2a) for the Improved Detection of Marine Toxins Active on Voltage Gated Sodium Channels (VGSCs)

by Jérôme Viallon, Mireille Chinain and Hélène Taiana Darius

Toxins 2020, 12(5), 281; https://doi.org/10.3390/toxins12050281 - 27 Apr 2020

Cited by 33 | Viewed by 4683

Abstract

The neuroblastoma cell-based assay (CBA-N2a) is widely used for the detection of marine biotoxins in seafood products, yet a consensus protocol is still lacking. In this study, six key parameters of CBA-N2a were revisited: cell seeding densities, cell layer viability after 26 h [...] Read more.

The neuroblastoma cell-based assay (CBA-N2a) is widely used for the detection of marine biotoxins in seafood products, yet a consensus protocol is still lacking. In this study, six key parameters of CBA-N2a were revisited: cell seeding densities, cell layer viability after 26 h growth, MTT incubation time, Ouabain and Veratridine treatment and solvent and matrix effects. A step-by-step protocol was defined identifying five viability controls for the validation of CBA-N2a results. Specific detection of two voltage gated sodium channel activators, pacific ciguatoxin (P-CTX3C) and brevetoxin (PbTx3) and two inhibitors, saxitoxin (STX) and decarbamoylsaxitoxin (dc-STX) was achieved, with EC₅₀ values of 1.7 ± 0.35 pg/mL, 5.8 ± 0.9 ng/mL, 3 ± 0.5 ng/mL and 15.8 ± 3 ng/mL, respectively. When applied to the detection of ciguatoxin (CTX)-like toxicity in fish samples, limit of detection (LOD) and limit of quantification (LOQ) values were 0.031 ± 0.008 and 0.064 ± 0.016 ng P-CTX3C eq/g of flesh, respectively. Intra and inter-assays comparisons of viability controls, LOD, LOQ and toxicity in fish samples gave coefficients of variation (CVs) ranging from 3% to 29%. This improved test adaptable to either high throughput screening or composite toxicity estimation is a useful starting point for a standardization of the CBA-N2a in the field of marine toxin detection. Full article

(This article belongs to the Special Issue Improved Analytical Technologies for the Detection of Natural Toxins and Their Metabolites in Food)

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12 pages, 980 KiB

Open AccessEditor’s ChoiceArticle

Critical Comparison of Analytical Performances of Two Immunoassay Methods for Rapid Detection of Aflatoxin M₁ in Milk

by Ivan Pecorelli, Natascia Guarducci, Cristoph von Holst, Rita Bibi, Michelangelo Pascale, Biancamaria Ciasca, Antonio F. Logrieco and Veronica M. T. Lattanzio

Toxins 2020, 12(4), 270; https://doi.org/10.3390/toxins12040270 - 22 Apr 2020

Cited by 12 | Viewed by 3314

Abstract

Aflatoxin B₁ (AFB₁) is a secondary metabolite produced by some Aspergillus spp. fungi affecting many crops and feed materials. Aflatoxin M₁ (AFM₁), the 4-hydroxylated metabolite of AFB_1, is the main AFB₁-related compound present [...] Read more.

Aflatoxin B₁ (AFB₁) is a secondary metabolite produced by some Aspergillus spp. fungi affecting many crops and feed materials. Aflatoxin M₁ (AFM₁), the 4-hydroxylated metabolite of AFB_1, is the main AFB₁-related compound present in milk, and it is categorized by the International Agency for Research on Cancer (IARC) as a “group 1 human carcinogen”. The aim of this work was to evaluate and compare the analytical performances of two commercial immunoassays widely applied for the detection of AFM₁ in milk, namely strip test immunoassay and enzyme linked immunosorbent assay (ELISA). Assay validation included samples at AFM₁ levels of 25, 50, 75 ng/kg and blank samples (AFM₁ < 0.5 ng/kg). With respect to a screening target concentration (STC) of 50 ng/kg the two assays showed cut-off values of 37.7 ng/kg and 47.5 ng/kg for strip test and ELISA, respectively, a false suspect rate for blanks <0.1% (for both assays) and a false negative rate for samples containing AFM₁ at levels higher than STC, of 0.4% (for both assays). The intermediate precision (RSD_ip) was <32% for the strip test and <15% for the ELISA. Method verification through long-term intra-laboratory quality control (QC) measurements confirmed the results from the validation study. Furthermore, a satisfactory correlation of the results obtained with both immunoassays and the AOAC Official Method 2000.08 was obtained for the analysis of cow milk samples naturally contaminated with AFM₁ at levels within “not detected” (< 0.5 ng/kg) and 50 ng/kg. Finally, the extension of the scope of the strip test method to goat and sheep milk was evaluated by applying the experimental design foreseen in the EU regulation. Full article

(This article belongs to the Special Issue Improved Analytical Technologies for the Detection of Natural Toxins and Their Metabolites in Food)

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16 pages, 1257 KiB

Open AccessArticle

Development and Validation of a UHPLC-ESI-MS/MS Method for Quantification of Oleandrin and Other Cardiac Glycosides and Evaluation of Their Levels in Herbs and Spices from the Belgian Market

by Svetlana V. Malysheva, Patrick P. J. Mulder and Julien Masquelier

Toxins 2020, 12(4), 243; https://doi.org/10.3390/toxins12040243 - 09 Apr 2020

Cited by 9 | Viewed by 3814

Abstract

Cardiac glycosides (CGs) are naturally occurring plant secondary metabolites that can be toxic to humans and animals. The aim of this work was to develop a targeted analytical method utilizing liquid chromatography—tandem mass spectrometry (LC-MS/MS) for quantification of these plant toxins in a [...] Read more.

Cardiac glycosides (CGs) are naturally occurring plant secondary metabolites that can be toxic to humans and animals. The aim of this work was to develop a targeted analytical method utilizing liquid chromatography—tandem mass spectrometry (LC-MS/MS) for quantification of these plant toxins in a herbal-based food and human urine. The method included oleandrin, digoxin, digitoxin, convallatoxin, and ouabain. Samples of culinary herbs were extracted with acetonitrile and cleaned using Oasis^® MAX solid-phase extraction (SPE), while samples of urine were diluted with acidified water and purified on Oasis^® HLB SPE cartridges. Limits of quantification were in the range of 1.5–15 ng/g for herbs and 0.025–1 ng/mL for urine. The mean recovery of the method complied with the acceptable range of 70–120% for most CGs, and relative standard deviations were at maximum 14% and 19% for repeatability and reproducibility, respectively. Method linearity was good with calculated R² values above 0.997. The expanded measurement uncertainty was estimated to be in the range of 7–37%. The LC-MS/MS method was used to examine 65 samples of culinary herbs and herb and spice mixtures collected in Belgium, from supermarkets and local stores. The samples were found to be free from the analyzed CGs. Full article

(This article belongs to the Special Issue Improved Analytical Technologies for the Detection of Natural Toxins and Their Metabolites in Food)

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10 pages, 2260 KiB

Open AccessEditor’s ChoiceArticle

Rapid, Sensitive, and Accurate Point-of-Care Detection of Lethal Amatoxins in Urine

by Candace S. Bever, Kenneth D. Swanson, Elizabeth I. Hamelin, Michael Filigenzi, Robert H. Poppenga, Jennifer Kaae, Luisa W. Cheng and Larry H. Stanker

Toxins 2020, 12(2), 123; https://doi.org/10.3390/toxins12020123 - 15 Feb 2020

Cited by 21 | Viewed by 10196

Abstract

Globally, mushroom poisonings cause about 100 human deaths each year, with thousands of people requiring medical assistance. Dogs are also susceptible to mushroom poisonings and require medical assistance. Cyclopeptides, and more specifically amanitins (or amatoxins, here), are the mushroom poison that causes the [...] Read more.

Globally, mushroom poisonings cause about 100 human deaths each year, with thousands of people requiring medical assistance. Dogs are also susceptible to mushroom poisonings and require medical assistance. Cyclopeptides, and more specifically amanitins (or amatoxins, here), are the mushroom poison that causes the majority of these deaths. Current methods (predominantly chromatographic, as well as antibody-based) of detecting amatoxins are time-consuming and require expensive equipment. In this work, we demonstrate the utility of the lateral flow immunoassay (LFIA) for the rapid detection of amatoxins in urine samples. The LFIA detects as little as 10 ng/mL of α-amanitin (α-AMA) or γ-AMA, and 100 ng/mL of β-AMA in urine matrices. To demonstrate application of this LFIA for urine analysis, this study examined fortified human urine samples and urine collected from exposed dogs. Urine is sampled directly without the need for any pretreatment, detection from urine is completed in 10 min, and the results are read by eye, without the need for specialized equipment. Analysis of both fortified human urine samples and urine samples collected from intoxicated dogs using the LFIA correlated well with liquid chromatography–mass spectrometry (LC-MS) methods. Full article

(This article belongs to the Special Issue Improved Analytical Technologies for the Detection of Natural Toxins and Their Metabolites in Food)

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22 pages, 2633 KiB

Open AccessEditor’s ChoiceArticle

Analysis of Pyrrolizidine Alkaloids in Queensland Honey: Using Low Temperature Chromatography to Resolve Stereoisomers and Identify Botanical Sources by UHPLC-MS/MS

by Natasha L. Hungerford, Steve J. Carter, Shalona R. Anuj, Benjamin L. L. Tan, Darina Hnatko, Christopher L. Martin, Elipsha Sharma, Mukan Yin, Thao T. P. Nguyen, Kevin J. Melksham and Mary T. Fletcher

Toxins 2019, 11(12), 726; https://doi.org/10.3390/toxins11120726 - 11 Dec 2019

Cited by 24 | Viewed by 4693

Abstract

Pyrrolizidine alkaloids (PAs) are a diverse group of plant secondary metabolites with known varied toxicity. Consumption of 1,2-unsaturated PAs has been linked to acute and chronic liver damage, carcinogenicity and death, in livestock and humans, making their presence in food of concern to [...] Read more.

Pyrrolizidine alkaloids (PAs) are a diverse group of plant secondary metabolites with known varied toxicity. Consumption of 1,2-unsaturated PAs has been linked to acute and chronic liver damage, carcinogenicity and death, in livestock and humans, making their presence in food of concern to food regulators in Australia and internationally. In this survey, honey samples sourced from markets and shops in Queensland (Australia), were analysed by high-resolution Orbitrap UHPLC-MS/MS for 30 common PAs. Relationships between the occurrence of pyrrolizidine alkaloids and the botanical origin of the honey are essential as pyrrolizidine alkaloid contamination at up to 3300 ng/g were detected. In this study, the predominant alkaloids detected were isomeric PAs, lycopsamine, indicine and intermedine, exhibiting identical MS/MS spectra, along with lesser amounts of each of their N-oxides. Crucially, chromatographic UHPLC conditions were optimised by operation at low temperature (5 °C) to resolve these key isomeric PAs. Such separation of these isomers by UHPLC, enabled the relative proportions of these PAs present in honey to be compared to alkaloid levels in suspect source plants. Overall plant pyrrolizidine alkaloid profiles were compared to those found in honey samples to help identify the most important plants responsible for honey contamination. The native Australian vines of Parsonsia spp. are proposed as a likely contributor to high levels of lycopsamine in many of the honeys surveyed. Botanical origin information such as this, gained via low temperature chromatographic resolution of isomeric PAs, will be very valuable in identifying region of origin for honey samples. Full article

(This article belongs to the Special Issue Improved Analytical Technologies for the Detection of Natural Toxins and Their Metabolites in Food)

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11 pages, 1350 KiB

Open AccessArticle

A Rapid Extraction Method Combined with a Monoclonal Antibody-Based Immunoassay for the Detection of Amatoxins

by Candace S. Bever, Robert M. Hnasko, Luisa W. Cheng and Larry H. Stanker

Toxins 2019, 11(12), 724; https://doi.org/10.3390/toxins11120724 - 11 Dec 2019

Cited by 13 | Viewed by 4944

Abstract

Amatoxins (AMAs) are lethal toxins found in a variety of mushroom species. Detection methods are needed to determine the occurrence of AMAs in mushroom species suspected in mushroom poisonings. In this manuscript, we report the generation of novel monoclonal antibodies (mAbs, AMA9G3 and [...] Read more.

Amatoxins (AMAs) are lethal toxins found in a variety of mushroom species. Detection methods are needed to determine the occurrence of AMAs in mushroom species suspected in mushroom poisonings. In this manuscript, we report the generation of novel monoclonal antibodies (mAbs, AMA9G3 and AMA9C12) and the development of a competitive, enzyme-linked immunosorbent assay (cELISA) that is sensitive at 1 ng mL⁻¹ and shows selectivity for α-amanitin (α-AMA) and γ-amanitin (γ-AMA), and less for β-amanitin (β-AMA). In order to decrease the overall time needed for analysis, the extraction procedure for mushrooms was also simplified. A rapid (1 min) extraction procedure of AMAs using solvents as simple as water alone was successfully demonstrated using Amanita mushrooms. Together, the extraction method and the mAb-based ELISA represent a simple and rapid method that readily detects AMAs extracted from mushroom samples. Full article

(This article belongs to the Special Issue Improved Analytical Technologies for the Detection of Natural Toxins and Their Metabolites in Food)

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13 pages, 950 KiB

Open AccessArticle

Development and Characterization of Monoclonal Antibodies for the Mycotoxin Citreoviridin

by Chris M. Maragos, Yosuke Uchiyama, Naoki Kobayashi, Fumichika Kominato and Yoshiko Sugita-Konishi

Toxins 2019, 11(11), 630; https://doi.org/10.3390/toxins11110630 - 30 Oct 2019

Cited by 3 | Viewed by 3163

Abstract

Citreoviridin (CTV) in an inhibitor of mitochondrial ATPase that has been isolated from molded yellow rice and linked to the human disease Shoshin-kakke (acute cardiac beriberi). The disease results from a deficiency of thiamine, however, purified CTV can reproduce the symptoms in experimental [...] Read more.

Citreoviridin (CTV) in an inhibitor of mitochondrial ATPase that has been isolated from molded yellow rice and linked to the human disease Shoshin-kakke (acute cardiac beriberi). The disease results from a deficiency of thiamine, however, purified CTV can reproduce the symptoms in experimental animals. The link between CTV and Shoshin-kakke has been difficult to resolve, in part because cases of the disease are rare. In addition to rice, CTV has been found in maize, pecan nuts, and wheat products. A method to screen for CTV and its geometric isomer, iso-CTV, in commodities was developed, based upon the isolation of two novel monoclonal antibodies (mAb). In an antigen-immobilized competitive enzyme-linked immunosorbent assay format (CI-ELISA), the observed IC₅₀s for CTV were 11 ng/mL and 18 ng/mL (mAbs 2-2 and 2-4, respectively). The assays were relatively tolerant to methanol and acetonitrile, which allowed their application to the detection of CTV in spiked polished white rice. For quantification, a standard mixture of CTV and iso-CTV was used, along with matrix matched calibration. The dynamic range of the ELISA using mAb 2-4 was equivalent to 0.23 to 2.22 mg/kg in rice. Recoveries over the range of 0.36 to 7.23 mg/kg averaged 97 ± 10%. The results suggest that the mAb 2-4-based immunoassay can be applied to the screening of white rice for CTV. Both mAbs were also observed to significantly enhance the fluorescence of the toxin. Full article

(This article belongs to the Special Issue Improved Analytical Technologies for the Detection of Natural Toxins and Their Metabolites in Food)

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Review

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23 pages, 4732 KiB

Open AccessEditor’s ChoiceReview

Two-Dimensional Layered Nanomaterial-Based Electrochemical Biosensors for Detecting Microbial Toxins

by Zhuheng Li, Xiaotong Li, Minghong Jian, Girma Selale Geleta and Zhenxin Wang

Toxins 2020, 12(1), 20; https://doi.org/10.3390/toxins12010020 - 31 Dec 2019

Cited by 25 | Viewed by 5118

Abstract

Toxin detection is an important issue in numerous fields, such as agriculture/food safety, environmental monitoring, and homeland security. During the past two decades, nanotechnology has been extensively used to develop various biosensors for achieving fast, sensitive, selective and on-site analysis of toxins. In [...] Read more.

Toxin detection is an important issue in numerous fields, such as agriculture/food safety, environmental monitoring, and homeland security. During the past two decades, nanotechnology has been extensively used to develop various biosensors for achieving fast, sensitive, selective and on-site analysis of toxins. In particular, the two dimensional layered (2D) nanomaterials (such as graphene and transition metal dichalcogenides (TMDs)) and their nanocomposites have been employed as label and/or biosensing transducers to construct electrochemical biosensors for cost-effective detection of toxins with high sensitivity and specificity. This is because the 2D nanomaterials have good electrical conductivity and a large surface area with plenty of active groups for conjugating 2D nanomaterials with the antibodies and/or aptamers of the targeted toxins. Herein, we summarize recent developments in the application of 2D nanomaterial-based electrochemical biosensors for detecting toxins with a particular focus on microbial toxins including bacterial toxins, fungal toxins and algal toxins. The integration of 2D nanomaterials with some existing antibody/aptamer technologies into electrochemical biosensors has led to an unprecedented impact on improving the assaying performance of microbial toxins, and has shown great promise in public health and environmental protection. Full article

(This article belongs to the Special Issue Improved Analytical Technologies for the Detection of Natural Toxins and Their Metabolites in Food)

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