Background: Evidence of fungal contamination of cannabis plants during drying has raised concerns... more Background: Evidence of fungal contamination of cannabis plants during drying has raised concerns of potential mycotoxin contamination of leaves and flowers and subsequent contamination of derived edible cannabis products. Methods are, therefore, needed for routine monitoring of cannabis to ensure consumer safety consistent with long-standing controls for mycotoxins such as aflatoxins and ochratoxin A (OTA) in foodstuffs. Objective: To generate preliminary validation data to demonstrate fitness-for-purpose of methods for aflatoxins and OTA in cannabis and cannabis products. Methods: Extraction of solid matrices with acetonitrile–water (75 + 25) and direct analysis of energy drinks after dilution. Extracts were either passed manually though an immunoaffinity column (IAC) containing antibodies to both aflatoxins and OTA or were analyzed sequentially using an automated system with in-line reusable immunoaffinity cartridges for aflatoxins or OTA. In both cases, analysis was by LC with f...
There is an urgent need for natural water reference materials certified for nutrients. In 1996, N... more There is an urgent need for natural water reference materials certified for nutrients. In 1996, NRC collected seawater for a proposed CRM at a depth of 200 m in the North Atlantic; this was immediately filtered through 0.05-µm cartridge filters into 50-L carboys. The water was later homogenized in the NRC laboratories in Ottawa and stabilized via gamma irradiation. Over six
In the field of mycotoxin analysis, substantial problems shown by high between-laboratory standar... more In the field of mycotoxin analysis, substantial problems shown by high between-laboratory standard deviations and noncomparable and nontraceable results have been caused by the lack of proper calibrants for external calibration. During a large-scale Standard Measurement and Testing project of the European Commission (EC) dealing with preparation and certification of reference materials for determination of the mycotoxin zearalenone (ZON) in maize, a ZON calibrant in acetonitrile was prepared and checked for purity, homogeneity, and stability. Before certification, on the basis of preparation, the calibrant was checked in a mini-interlaboratory study by UV spectrophotometric determination. The molar absorptivities of ZON in acetonitrile at 236, 274, and 314 nm were established, and as a main result, a common reference wavelength of 274 nm with molar absorptivity of 12623 +/- 111 L/mol x cm can be recommended for ZON in acetonitrile. A concentration and expanded uncertainty of the ZON...
In the field of mycotoxin analysis, substantial problems shown by high between-laboratory standar... more In the field of mycotoxin analysis, substantial problems shown by high between-laboratory standard deviations and noncomparable and nontraceable results have been caused by the lack of proper calibrants for external calibration. During a large-scale Standard Measurement and Testing project of the European Commission (EC) dealing with preparation and certification of reference materials for determination of the mycotoxin zearalenone (ZON) in maize, a ZON calibrant in acetonitrile was prepared and checked for purity, homogeneity, and stability. Before certification, on the basis of preparation, the calibrant was checked in a mini-interlaboratory study by UV spectrophotometric determination. The molar absorptivities of ZON in acetonitrile at 236, 274, and 314 nm were established, and as a main result, a common reference wavelength of 274 nm with molar absorptivity of 12623 +/- 111 L/mol x cm can be recommended for ZON in acetonitrile. A concentration and expanded uncertainty of the ZON...
In this study, laboratory experiments have addressed the acute toxicity of two common mycotoxins,... more In this study, laboratory experiments have addressed the acute toxicity of two common mycotoxins, deoxynivalenol (DON) and zearalenone (ZON), in a range of freshwater organisms (including rotifers Brachionus calyciflorus, insects Chironomus riparius (larvae), crustaceans Daphnia pulex and Thamnocephalus platyurus, cnidarians Hydra vulgaris, molluscs Lymnaea stagnalis (embryos) and Protozoa Tetrahymena thermophila). Acute EC50 values highlight crustaceans as the most sensitive organisms to DON, with T. platyurus having a 24 h EC50 of 0.14 and D. magna having a 48 h EC50 of 0.13 mg DON/L. During exposures to ZON, H. vulgaris and L. stagnalis embryos showed the highest sensitivity; mortality EC50 values were 1.1 (96 h) and 0.42 mg ZON/L (7 d), respectively. Combining these novel invertebrate toxicity results, along with recent published data for freshwater plant and fish toxicity for analysis of Species Sensitivity Distributions, provides freshwater HC5 values of 5.2 μg DON/L and 43 μg ZON/L, respectively. Using highest reported environmental concentrations and following REACH guidelines, risk ratios calculated here show the risk of ZON to freshwater organisms is low. In contrast, DON may periodically because for concern in streams subject to high agricultural run-off, likely during certain times of year where cereal crops are susceptible to higher fungal infections rates and may pose increased risks due to climate change.
An interlaboratory study was performed on behalf of the UK Food Standards Agency to evaluate the ... more An interlaboratory study was performed on behalf of the UK Food Standards Agency to evaluate the effectiveness of an affinity column cleanup liquid chromatography (LC) method for the determination of zearalenone (ZON) in a variety of cereals and cereal products at proposed European regulatory limits. The test portion is extracted with acetonitrile:water. The sample extract is filtered, diluted, and applied to an affinity column. The column is washed, and ZON is eluted with acetonitrile. ZON is quantified by reversed-phase LC with fluorescence detection. Barley, wheat and maize flours, polenta, and a maize-based baby food naturally contaminated, spiked, and blank (very low level) were sent to 28 collaborators in 9 European countries and 1 collaborator in New Zealand. Participants were asked to spike test portions of all samples at a ZON concentration equivalent to 100 μg/kg. Average recoveries ranged from 91–111%. Based on results for 4 artificially contaminated samples (blind duplic...
Thirteen European laboratories experienced in the analysis of mycotoxins participated in an inter... more Thirteen European laboratories experienced in the analysis of mycotoxins participated in an intercomparison study within a European Commission-funded project. Goals of the study were to check the fitness for purpose of a small batch of gravimetrically prepared calibrants; to compare individually prepared calibrants with common calibrants; to check the feasibility of toxin mixtures as calibrant solutions; and to give recommendations on the production of future certified reference materials (CRMs) with regard to the nature of the calibrant and the means of certification. Each laboratory received ampules of each common calibrant containing single toxins [solution containing either deoxynivalenol (DON), 3-acetyl-DON (3-Ac-DON), nivalenol (NIV), or 15-acetyl-DON (15-Ac-DON)] and 3 ampules of toxinmixture (solutions of DON 3-Ac-DON NIV in acetonitrile) of known concentrations (about 20 g/mL). Ampules with single toxins (solution containing either DON, 3-Ac-DON, NIV, or 15-Ac-DON) and a to...
An interlaboratory study was performed on behalf of the UK Food Standards Agency to evaluate the ... more An interlaboratory study was performed on behalf of the UK Food Standards Agency to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatographic (LC) method for the determination of deoxynivalenol in a variety of cereals and cereal products at proposed European regulatory limits. The test portion was extracted with water. The sample extract was filtered and applied to an immunoaffinity column. After being washed with water, the deoxynivalenol was eluted with acetonitrile or methanol. Deoxynivalenol was quantitated by reversed-phase LC with UV determination. Samples of artificially contaminated wheat-flour, rice flour, oat flour, polenta, and a wheat based breakfast cereal, naturally contaminated wheat flour, and blank (very low level) samples of each matrix were sent to 13 collaborators in 7 European countries. Participants were asked to spike test portions of all samples at a range of deoxynivalenol concentrations equivalent to 200–2000 ng/g deoxynivalenol....
Cereal grains are commonly contaminated with Fusarium mycotoxins and their plant-derived masked m... more Cereal grains are commonly contaminated with Fusarium mycotoxins and their plant-derived masked metabolites. The fate of masked mycotoxins in the human gut is poorly understood. Here we assess the metabolism and transport of glucoside metabolites of common trichothecenes (deoxynivalenol, nivalenol, T-2 toxin) and zearalenone compounds (zearalenone, α- and β-zearalenol) in the human gut in vitro. Masked mycotoxins were incubated with artificial digestive juices and absorption was assessed in differentiated Caco-2/TC7 cells. Colonic metabolism was studied using fecal batch cultures from 5 donors and mycotoxins were detected using LC-MS/MS. All masked mycotoxins were stable under upper GI tract conditions and no absorption was observed. Free trichothecenes were absorbed intact whereas free zearalenone compounds were absorbed and metabolized to undetected compounds by Caco-2/TC7 cells. Human gut microbiota efficiently hydrolyzed all masked mycotoxins. Trichothecenes were fully recovered...
A method is reported for the analysis of sterigmatocystin in various food and feed matrices using... more A method is reported for the analysis of sterigmatocystin in various food and feed matrices using a commercial sterigmatocystin immunoaffinity column (IAC) for sample clean-up prior to HPLC analysis by UV with mass spectrometric detection (LC-MS/MS). Cereals (wheat, oats, rye, maize and rice), sunflower seeds and animal feed were spiked with sterigmatocystin at levels from 0.75 to 50 µg kg(-1) to establish method performance. Using acetonitrile/water extraction followed by IAC clean-up, and analysis by HPLC with detection at 325 nm, recoveries ranged from 68-106%, with repeatability from 4.2 to 17.5%. The limit of quantification with UV detection in these matrices was 1.5 µg kg(-1). For the analysis of beer and cheese the sample preparation prior to IAC clean-up was changed to accommodate the different properties of the matrix, prior to analysis by LC-MS/MS. For beer and cheese spiked at 5.0 µg kg(-1) the recoveries were 94% and 104%, and precision (RSDs) was 1.9% and 2.9% respectively. The limits of quantification by LC-MS/MS in beer and cheese were 0.02 µg kg-1 and 0.6 µg kg(-1) respectively. The sterigmatocystin IAC was demonstrated to provide an efficient clean-up of various matrices to enable this mycotoxin to be determined by either HPLC with UV detection or LC-MS/MS.
This review highlights developments in mycotoxin analysis and sampling over a period between mid-... more This review highlights developments in mycotoxin analysis and sampling over a period between mid-2011 and mid- 2012. It covers the major mycotoxins aflatoxins, Alternaria toxins, ergot alkaloids, fumonisins, ochratoxin, patulin, trichothecenes, and zearalenone. A section on mycotoxins in botanicals and spices is also included. Methods for mycotoxin determination continue to be developed using a wide range of analytical systems ranging from rapid immunochemical-based methods to the latest advances in mass spectrometry. This review follows the format of previous reviews in this series (i.e. sections on individual mycotoxins), but due to the rapid spread and developments in the field of multimycotoxin methods by liquid chromatography-tandem mass spectrometry, a separate section has been devoted to advances in this area of research.
An automated HPLC method for the simultaneous detection of aflatoxins (AF) and ochratoxin A (OA) ... more An automated HPLC method for the simultaneous detection of aflatoxins (AF) and ochratoxin A (OA) has been developed. The method uses an immunoaffinity column containing antibodies specific to both AF and OA. The samples were extracted with an acetonitrile/water mixture and diluted with phosphate buffer saline (PBS). The aqueous extracts were then transferred to an ASPEC HPLC system for automated
A study has been carried out to assess appropriate statistical models for use in evaluating retai... more A study has been carried out to assess appropriate statistical models for use in evaluating retail sampling plans for the determination of mycotoxins in food. A compound gamma model was found to be a suitable fit. A simulation model based on the compound gamma model was used to produce operating characteristic curves for a range of parameters relevant to retail sampling. The model was also used to estimate the minimum number of increments necessary to minimize the overall measurement uncertainty. Simulation results showed that measurements based on retail samples (for which the maximum number of increments is constrained by cost) may produce fit-for-purpose results for the measurement of ochratoxin A in dried fruit, but are unlikely to do so for the measurement of aflatoxin B1 in pistachio nuts. In order to produce a more accurate simulation, further work is required to determine the degree of heterogeneity associated with batches of food products. With appropriate parameterization in terms of physical and biological characteristics, the systems developed in this study could be applied to other analyte/matrix combinations.
Background: Evidence of fungal contamination of cannabis plants during drying has raised concerns... more Background: Evidence of fungal contamination of cannabis plants during drying has raised concerns of potential mycotoxin contamination of leaves and flowers and subsequent contamination of derived edible cannabis products. Methods are, therefore, needed for routine monitoring of cannabis to ensure consumer safety consistent with long-standing controls for mycotoxins such as aflatoxins and ochratoxin A (OTA) in foodstuffs. Objective: To generate preliminary validation data to demonstrate fitness-for-purpose of methods for aflatoxins and OTA in cannabis and cannabis products. Methods: Extraction of solid matrices with acetonitrile–water (75 + 25) and direct analysis of energy drinks after dilution. Extracts were either passed manually though an immunoaffinity column (IAC) containing antibodies to both aflatoxins and OTA or were analyzed sequentially using an automated system with in-line reusable immunoaffinity cartridges for aflatoxins or OTA. In both cases, analysis was by LC with f...
There is an urgent need for natural water reference materials certified for nutrients. In 1996, N... more There is an urgent need for natural water reference materials certified for nutrients. In 1996, NRC collected seawater for a proposed CRM at a depth of 200 m in the North Atlantic; this was immediately filtered through 0.05-µm cartridge filters into 50-L carboys. The water was later homogenized in the NRC laboratories in Ottawa and stabilized via gamma irradiation. Over six
In the field of mycotoxin analysis, substantial problems shown by high between-laboratory standar... more In the field of mycotoxin analysis, substantial problems shown by high between-laboratory standard deviations and noncomparable and nontraceable results have been caused by the lack of proper calibrants for external calibration. During a large-scale Standard Measurement and Testing project of the European Commission (EC) dealing with preparation and certification of reference materials for determination of the mycotoxin zearalenone (ZON) in maize, a ZON calibrant in acetonitrile was prepared and checked for purity, homogeneity, and stability. Before certification, on the basis of preparation, the calibrant was checked in a mini-interlaboratory study by UV spectrophotometric determination. The molar absorptivities of ZON in acetonitrile at 236, 274, and 314 nm were established, and as a main result, a common reference wavelength of 274 nm with molar absorptivity of 12623 +/- 111 L/mol x cm can be recommended for ZON in acetonitrile. A concentration and expanded uncertainty of the ZON...
In the field of mycotoxin analysis, substantial problems shown by high between-laboratory standar... more In the field of mycotoxin analysis, substantial problems shown by high between-laboratory standard deviations and noncomparable and nontraceable results have been caused by the lack of proper calibrants for external calibration. During a large-scale Standard Measurement and Testing project of the European Commission (EC) dealing with preparation and certification of reference materials for determination of the mycotoxin zearalenone (ZON) in maize, a ZON calibrant in acetonitrile was prepared and checked for purity, homogeneity, and stability. Before certification, on the basis of preparation, the calibrant was checked in a mini-interlaboratory study by UV spectrophotometric determination. The molar absorptivities of ZON in acetonitrile at 236, 274, and 314 nm were established, and as a main result, a common reference wavelength of 274 nm with molar absorptivity of 12623 +/- 111 L/mol x cm can be recommended for ZON in acetonitrile. A concentration and expanded uncertainty of the ZON...
In this study, laboratory experiments have addressed the acute toxicity of two common mycotoxins,... more In this study, laboratory experiments have addressed the acute toxicity of two common mycotoxins, deoxynivalenol (DON) and zearalenone (ZON), in a range of freshwater organisms (including rotifers Brachionus calyciflorus, insects Chironomus riparius (larvae), crustaceans Daphnia pulex and Thamnocephalus platyurus, cnidarians Hydra vulgaris, molluscs Lymnaea stagnalis (embryos) and Protozoa Tetrahymena thermophila). Acute EC50 values highlight crustaceans as the most sensitive organisms to DON, with T. platyurus having a 24 h EC50 of 0.14 and D. magna having a 48 h EC50 of 0.13 mg DON/L. During exposures to ZON, H. vulgaris and L. stagnalis embryos showed the highest sensitivity; mortality EC50 values were 1.1 (96 h) and 0.42 mg ZON/L (7 d), respectively. Combining these novel invertebrate toxicity results, along with recent published data for freshwater plant and fish toxicity for analysis of Species Sensitivity Distributions, provides freshwater HC5 values of 5.2 μg DON/L and 43 μg ZON/L, respectively. Using highest reported environmental concentrations and following REACH guidelines, risk ratios calculated here show the risk of ZON to freshwater organisms is low. In contrast, DON may periodically because for concern in streams subject to high agricultural run-off, likely during certain times of year where cereal crops are susceptible to higher fungal infections rates and may pose increased risks due to climate change.
An interlaboratory study was performed on behalf of the UK Food Standards Agency to evaluate the ... more An interlaboratory study was performed on behalf of the UK Food Standards Agency to evaluate the effectiveness of an affinity column cleanup liquid chromatography (LC) method for the determination of zearalenone (ZON) in a variety of cereals and cereal products at proposed European regulatory limits. The test portion is extracted with acetonitrile:water. The sample extract is filtered, diluted, and applied to an affinity column. The column is washed, and ZON is eluted with acetonitrile. ZON is quantified by reversed-phase LC with fluorescence detection. Barley, wheat and maize flours, polenta, and a maize-based baby food naturally contaminated, spiked, and blank (very low level) were sent to 28 collaborators in 9 European countries and 1 collaborator in New Zealand. Participants were asked to spike test portions of all samples at a ZON concentration equivalent to 100 μg/kg. Average recoveries ranged from 91–111%. Based on results for 4 artificially contaminated samples (blind duplic...
Thirteen European laboratories experienced in the analysis of mycotoxins participated in an inter... more Thirteen European laboratories experienced in the analysis of mycotoxins participated in an intercomparison study within a European Commission-funded project. Goals of the study were to check the fitness for purpose of a small batch of gravimetrically prepared calibrants; to compare individually prepared calibrants with common calibrants; to check the feasibility of toxin mixtures as calibrant solutions; and to give recommendations on the production of future certified reference materials (CRMs) with regard to the nature of the calibrant and the means of certification. Each laboratory received ampules of each common calibrant containing single toxins [solution containing either deoxynivalenol (DON), 3-acetyl-DON (3-Ac-DON), nivalenol (NIV), or 15-acetyl-DON (15-Ac-DON)] and 3 ampules of toxinmixture (solutions of DON 3-Ac-DON NIV in acetonitrile) of known concentrations (about 20 g/mL). Ampules with single toxins (solution containing either DON, 3-Ac-DON, NIV, or 15-Ac-DON) and a to...
An interlaboratory study was performed on behalf of the UK Food Standards Agency to evaluate the ... more An interlaboratory study was performed on behalf of the UK Food Standards Agency to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatographic (LC) method for the determination of deoxynivalenol in a variety of cereals and cereal products at proposed European regulatory limits. The test portion was extracted with water. The sample extract was filtered and applied to an immunoaffinity column. After being washed with water, the deoxynivalenol was eluted with acetonitrile or methanol. Deoxynivalenol was quantitated by reversed-phase LC with UV determination. Samples of artificially contaminated wheat-flour, rice flour, oat flour, polenta, and a wheat based breakfast cereal, naturally contaminated wheat flour, and blank (very low level) samples of each matrix were sent to 13 collaborators in 7 European countries. Participants were asked to spike test portions of all samples at a range of deoxynivalenol concentrations equivalent to 200–2000 ng/g deoxynivalenol....
Cereal grains are commonly contaminated with Fusarium mycotoxins and their plant-derived masked m... more Cereal grains are commonly contaminated with Fusarium mycotoxins and their plant-derived masked metabolites. The fate of masked mycotoxins in the human gut is poorly understood. Here we assess the metabolism and transport of glucoside metabolites of common trichothecenes (deoxynivalenol, nivalenol, T-2 toxin) and zearalenone compounds (zearalenone, α- and β-zearalenol) in the human gut in vitro. Masked mycotoxins were incubated with artificial digestive juices and absorption was assessed in differentiated Caco-2/TC7 cells. Colonic metabolism was studied using fecal batch cultures from 5 donors and mycotoxins were detected using LC-MS/MS. All masked mycotoxins were stable under upper GI tract conditions and no absorption was observed. Free trichothecenes were absorbed intact whereas free zearalenone compounds were absorbed and metabolized to undetected compounds by Caco-2/TC7 cells. Human gut microbiota efficiently hydrolyzed all masked mycotoxins. Trichothecenes were fully recovered...
A method is reported for the analysis of sterigmatocystin in various food and feed matrices using... more A method is reported for the analysis of sterigmatocystin in various food and feed matrices using a commercial sterigmatocystin immunoaffinity column (IAC) for sample clean-up prior to HPLC analysis by UV with mass spectrometric detection (LC-MS/MS). Cereals (wheat, oats, rye, maize and rice), sunflower seeds and animal feed were spiked with sterigmatocystin at levels from 0.75 to 50 µg kg(-1) to establish method performance. Using acetonitrile/water extraction followed by IAC clean-up, and analysis by HPLC with detection at 325 nm, recoveries ranged from 68-106%, with repeatability from 4.2 to 17.5%. The limit of quantification with UV detection in these matrices was 1.5 µg kg(-1). For the analysis of beer and cheese the sample preparation prior to IAC clean-up was changed to accommodate the different properties of the matrix, prior to analysis by LC-MS/MS. For beer and cheese spiked at 5.0 µg kg(-1) the recoveries were 94% and 104%, and precision (RSDs) was 1.9% and 2.9% respectively. The limits of quantification by LC-MS/MS in beer and cheese were 0.02 µg kg-1 and 0.6 µg kg(-1) respectively. The sterigmatocystin IAC was demonstrated to provide an efficient clean-up of various matrices to enable this mycotoxin to be determined by either HPLC with UV detection or LC-MS/MS.
This review highlights developments in mycotoxin analysis and sampling over a period between mid-... more This review highlights developments in mycotoxin analysis and sampling over a period between mid-2011 and mid- 2012. It covers the major mycotoxins aflatoxins, Alternaria toxins, ergot alkaloids, fumonisins, ochratoxin, patulin, trichothecenes, and zearalenone. A section on mycotoxins in botanicals and spices is also included. Methods for mycotoxin determination continue to be developed using a wide range of analytical systems ranging from rapid immunochemical-based methods to the latest advances in mass spectrometry. This review follows the format of previous reviews in this series (i.e. sections on individual mycotoxins), but due to the rapid spread and developments in the field of multimycotoxin methods by liquid chromatography-tandem mass spectrometry, a separate section has been devoted to advances in this area of research.
An automated HPLC method for the simultaneous detection of aflatoxins (AF) and ochratoxin A (OA) ... more An automated HPLC method for the simultaneous detection of aflatoxins (AF) and ochratoxin A (OA) has been developed. The method uses an immunoaffinity column containing antibodies specific to both AF and OA. The samples were extracted with an acetonitrile/water mixture and diluted with phosphate buffer saline (PBS). The aqueous extracts were then transferred to an ASPEC HPLC system for automated
A study has been carried out to assess appropriate statistical models for use in evaluating retai... more A study has been carried out to assess appropriate statistical models for use in evaluating retail sampling plans for the determination of mycotoxins in food. A compound gamma model was found to be a suitable fit. A simulation model based on the compound gamma model was used to produce operating characteristic curves for a range of parameters relevant to retail sampling. The model was also used to estimate the minimum number of increments necessary to minimize the overall measurement uncertainty. Simulation results showed that measurements based on retail samples (for which the maximum number of increments is constrained by cost) may produce fit-for-purpose results for the measurement of ochratoxin A in dried fruit, but are unlikely to do so for the measurement of aflatoxin B1 in pistachio nuts. In order to produce a more accurate simulation, further work is required to determine the degree of heterogeneity associated with batches of food products. With appropriate parameterization in terms of physical and biological characteristics, the systems developed in this study could be applied to other analyte/matrix combinations.
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