Article pubs.acs.org/JAFC
Occurrence of Ergot and Ergot Alkaloids in Western Canadian Wheat and Other Cereals Sheryl A. Tittlemier,* Dainna Drul, Mike Roscoe, and Twylla McKendry Grain Research Laboratory, Canadian Grain Commission, 1404-303 Main Street, Winnipeg, Manitoba, Canada S Supporting Information *
ABSTRACT: A new method was developed to analyze 10 ergot alkaloids in cereal grains. Analytes included both “ine” and “inine” type ergot alkaloids. Validation of the method showed it performed with good accuracy and precision and that minor enhancement due to matrix effects was present during LC-MS/MS analysis, but was mitigated by use of an internal standard. The method was used to survey durum and wheat harvested in 2011, a year in which ergot infection was particularly widespread in western Canada. A strong linear relationship between the concentration of ergot alkaloids and the presence of ergot sclerotia was observed. In addition, shipments of cereals from 2010−2012 were also monitored for ergot alkaloids. Concentrations of total ergot alkaloids in shipments were lower than observed in harvest samples, and averaged from 0.065 mg/kg in barley to 1.14 mg/ kg in rye. In shipments, the concentration of ergot alkaloids was significantly lower in wheat of higher grades. KEYWORDS: mycotoxins, monitoring, durum, grading, sclerotia, method validation
■
INTRODUCTION Ergot is a well-known fungal infection of cereal grains characterized by the presence of dark-colored sclerotia that replace healthy kernels. References to ergot and its health effects date back to 1100 BCE in China and occur through Ancient Egypt and Greece, through Europe in the Middle Ages, and up to the mid-1900s in France.1 A series of alkaloid secondary metabolites are produced in ergot sclerotia, and it is these ergot alkaloids that cause the hallucinations, convulsions, burning sensation, vasoconstriction, and gangrenous loss of limbs associated with consumption of ergot-contaminated grain. There are also risks to livestock from exposure to ergot-contaminated feed.2 Ergot alkaloids are types of indole alkaloids, and those most commonly associated with wheat, rye, and other food cereal grains are amide-like derivatives of lysergic acid.3 These ergot alkaloids that have an R configuration at C8 (indicated by an “ine” suffix in their names) contain a double bond at C9−C10 of the ergoline ring and undergo epimerization to C8(S). The names of the epimerized “ine” compounds end in “inine”. The R ergot alkaloids readily undergo transformation to the S enantiomers under alkaline conditions, as well as over lengthy periods of storage.4 The epimerization is reversible and can reach equilibrium.3 Ergot is associated mainly with rye, but is also relevant for wheat, barley, and oats. Infection by the causative organism Claviceps purpurea occurs during flowering of the host plant. Therefore, because rye is a cross-pollinator with large open florets, it is particularly susceptible to infection by C. purpurea.5 Ergot alkaloids are generally present at higher concentrations in rye and rye-based foods as compared to other cereal grains.6 Modern management of ergot is focused on limiting the presence of ergot sclerotia in cereal grain. There are currently no regulations for ergot alkaloids in grain as there are for other mycotoxins; however, many jurisdictions have set tolerances for ergot sclerotia. In Canada, the highest quality grades of durum Published 2015 by the American Chemical Society
and western red spring had a limit of 0.010% ergot sclerotia on a mass basis7 prior to August 1, 2014. Current tolerances are 0.02 and 0.04% ergot sclerotia on a mass basis,7 for top grades of durum and western red spring, respectively. The Codex Alimentarius, American, and European maximum for ergot sclerotia in wheat is 0.05%.8−10 Ergot sclerotia can be removed from healthy grain by sorting based on color or density. There were three main purposes of this work: (1) to develop and validate a new analytical method for the analysis of raw cereal grains to include both R and S ergot alkaloids; (2) to examine the relationship between the presence of ergot sclerotia and concentrations of ergot alkaloids in wheat; and (3) to survey the presence of ergot alkaloids in shipments of western Canadian wheat from 2010 through 2012.
■
MATERIALS AND METHODS
Samples. Samples of Canada western amber durum (CWAD; n = 23) and Canada western red spring (CWRS; n = 42) harvested in 2011 were obtained from the annual Canadian Grain Commission (CGC) Harvest Sample Program. These samples consisted of grain that was voluntarily sampled at harvest by producers and submitted to the CGC. Sampling instructions were provided to producers prior to harvest.11 Once received, samples were examined by CGC inspectors, and the percentage of ergot sclerotia by mass in each sample was determined as described in the Canadian Grain Grading Guide.7 This set of 65 samples collected for ergot alkaloid analysis was specifically selected to cover a range of ergot sclerotia percentages. After grading, the selected samples were stored at room temperature in paper envelopes until ergot alkaloid analyses were performed. Test portions of the harvest samples were prepared for ergot alkaloid analysis by grinding the entire whole grain sample (approximately 200−400 g) using a model KR 804 commercial coffee grinder (Ditting Maschinen AG, Switzerland). The sample was ground Received: June 16, 2015 Accepted: July 2, 2015 Published: July 2, 2015 6644
DOI: 10.1021/acs.jafc.5b02977 J. Agric. Food Chem. 2015, 63, 6644−6650
Journal of Agricultural and Food Chemistry
Article
Figure 1. Structures of ergot alkaloid analytes and internal standard. fine enough that ≥85% of mass passed through a U.S. 50 sieve (nominal sieve openings of 300 μm). In addition, samples from shipments of western Canadian cereals were provided by CGC Industry Services inspectors. Grains sampled included wheat (n = 117), durum (n = 46), barley (n = 25), and rye (n = 1). These samples represented randomly selected export shipments of cereal grains that occurred between August 1, 2010, and July 31, 2012. It should be noted that the year of transport does not necessarily correspond to the year in which grain was grown and harvested. During the loading of the shipments, moving grain was sampled according to standardized procedures at a constant interval using CGC-approved automated cross-stream sample diverters and dividers.12 The increments obtained during loading of a lot were combined into a composite sample that was considered to be representative of the entire lot. A Boerner divider (Seedburo Equipment Co.) was used to divide the composite sample into 10 kg laboratory samples. The 10 kg laboratory samples were stored at
room temperature in canvas bags for up to 5 weeks until further preparation. Test portions of the shipment samples were prepared from the 10 kg laboratory sample of whole grain according to the process described previously.13 The 10 kg laboratory sample of whole grain was comminuted using a Retsch SR 300 rotor beater mill fitted with a 750 μm screen and coupled with a Retsch DR 100 vibratory feeder. After comminution, the entire mass of ground grain was homogenized and divided into 10 × 1 kg portions on a rotary sample divider (Materials Sampling Solutions, Southport, Australia). After the first division into 10 × 1 kg, all 10 portions were recombined in the RSD hopper and divided into 10 × 1 kg again. One 1 kg portion of ground grain was randomly selected and further divided on another RSD into 10 subsamples of 100 g of ground grain. Subsamples of 100 g were stored at room temperature in closed high-density polyethylene or polypropylene containers. The containers were not airtight, but were sealed to prevent the possibility of insects entering the samples. 6645
DOI: 10.1021/acs.jafc.5b02977 J. Agric. Food Chem. 2015, 63, 6644−6650
Journal of Agricultural and Food Chemistry
Article
Analysis of Ergot Alkaloids. Ten ergot alkaloids were included in the analytical method (Figure 1). Standards of individual analytes were obtained as crystals (Sigma-Aldrich, Oakville, Canada; Novartis, Dorval, Canada; Biopure, Tulln, Austria); purities ranged from 97 to 99%. Four of the alkaloids were S epimers of R enantiomers. The two epimers that were not included in the method were not easily obtained at the time of analysis. Dihydroergotamine (11, Fluka, analytical standard grade) was used as an internal standard for the analytical method. A method based on that of Krska et al.4 was developed for the extraction and ultrahigh-pressure liquid chromatographic−tandem mass spectrometric (UPLC-MS/MS) analysis of the 10 ergot alkaloids. Ground sample (10 g) was extracted with 50 mL of a 84:16 (v/v) acetonitrile/3.03 mM aqueous ammonium carbonate in 250 mL polyethylene centrifuge bottles. This mixture was shaken on a flatbed shaker for 30 min and then centrifuged at 2450g for 10 min at room temperature. A 464 μL aliquot of the supernatant was transferred to a glass test tube and mixed with 20 μL of internal standard solution (4 ng/μL dihydroergotamine in acetonitrile) and 1536 μL of 3.03 mM aqueous ammonium carbonate. Samples were vortexed to mix well and filtered using a disposable plastic syringe and PTFE syringe filter prior to injection. Final extracts were autoinjected, with the autosampler tray held at 15 °C, and chromatographed on a 2.1 × 100 mm i.d., 1.7 μm, BEH C18 column (Waters) held at 30 °C on a Waters Acquity I-class UPLC. A gradient of two solutions (A = 3.03 mM ammonium carbonate and B = acetonitrile) was used to resolve the analytes. The mobile phase began at 80% A, changed to 20% A over 5 min, and returned to 80% A after a further 1 min. Analysis was performed using a Waters Xevo MS/MS in the positive electrospray ionization mode using multiple reaction monitoring. The source and desolvation temperatures were held at 150 and 500 °C, respectively, with the capillary at 0.50 kV and the extractor at 3.0 V. The cone, desolvation, and collision gas flows were 100 L/h, 700 L/h, and 0.15 mL/min, respectively. To avoid epimerization of analytes, diluted standard mixes were dried down and kept in a freezer until needed. The dried mixes were reconstituted in acetonitrile just prior to instrumental analysis. Exposure to light was minimized, and samples and extracts were kept covered during processing and were placed in the UPLC sample manager with the illumination option turned off once diluted. Analytes were considered to be positively identified and quantitated if their retention times were within 0.1 min of the average retention time of the corresponding analyte in the external calibration standards; the peak had a signal-to-noise ratio >9:1, and the ratio of qualification to quantitation ions was within acceptable tolerances.14 The analyte responses were normalized to the dihydroergotamine responses during calculation of concentrations. Blank samples fortified with a solution of standards and an in-house reference material were analyzed with each batch of samples and were used to monitor the performance of the analytical method.
alkaloid analytes were fortified only at the medium concentration. During validation, separate sets of blanks were fortified with R and S ergot alkaloid analytes because a low amount of S enantiomers was found in the R enantiomer standard solutions. Ergosinine, ergocorninine, ergocryptinine, and ergocristinine were present at approximately 6% of the concentration of their analogues in the R enantiomer standard mix. Ergosine, ergocornine, ergocryptine, and ergocristine were also present in the S enantiomer standard mix, although at only about 2% of the concentration of their analogues. It was not clear if this was due to impurities in the analytical standards or due to epimerization during handling and use of the standard solutions over time. Both of these possibilities have been noted in the literature.15 Because of this apparent “contamination”, separate calibration standards were made for R and S enantiomers, and these were used in the validation and in all subsequent samples analyses. The method produced good results with recoveries from individual ergot alkaloids in individual fortified samples ranging from 60 to 132%. The overall mean recovery ± standard deviation for all ergot alkaloids over all fortification concentrations and days was 83 ± 12% for wheat and 88 ± 15% for rye. A comparison of individual ergot alkaloid peak areas from standard solutions prepared in solvent to postextraction fortified wheat and rye matrix matched standards indicated some enhancement of ergocorninine, ergocryptinine, and ergocristinine occurred due to matrix effects. The enhancement was somewhat higher for the rye matrix as compared to the wheat matrix (Table 1). The average ratio of ergot alkaloid Table 1. Ratios of Responses for Ergot Alkaloids and Internal Standard in Wheat and Rye Matrix Extract As Compared to Solvent ergonovine ergosine ergotamine ergocornine ergocryptine ergocristine dihydroergotamine ergosinine ergocorninine ergocryptinine ergocristinine
■
RESULTS AND DISCUSSION Validation and Method Performance. Validation of the analytical method was performed using fortified wheat and rye. Blank wheat and rye were created by hand-cleaning grain samples to remove ergot sclerotia. Even with this extra cleaning, some ergot alkaloids were still present in the blanks; therefore, during method validation concentrations of ergot alkaloids in the fortified samples were corrected by subtracting the concentration of analyte in the blanks prior to calculation of recoveries. Blank samples were fortified at three different concentrations and extracted and analyzed on multiple days. Seven replicates were fortified and extracted for each day/ concentration combination: low (2 μg/kg ergonovine and 10 μg/kg other analytes), medium (41 μg/kg ergonovine and 200 μg/kg other analytes), and high (407 μg/kg ergonovine and 2000 μg/kg other analytes). To conserve standards, the S ergot
wheat
rye
1.05 0.94 1.09 0.99 1.05 1.07 1.12 1.05 1.28 1.34 1.43
1.05 0.82 1.10 1.07 1.12 1.19 1.23 1.08 1.40 1.55 1.83
peak area in matrix to solvent was 1.13 for wheat and 1.22 for rye. These averages are very close to the ratios observed for the internal standard dihydroergotamine in wheat (1.12) and rye (1.23). This similarity indicates that normalization of individual ergot alkaloid responses to the dihydroergotamine internal standard would mitigate matrix effects on the measurement of the sum total concentration of all 10 ergot alkaloids analyzed. Limits of quantitation (LOQs) were 2 μg/kg for ergonovine and 10 μg/kg for the nine other ergot alkaloids. The LOQs were estimated as the lowest concentration to produce a chromatographic peak with a signal-to-noise ratio of at least 9:1 having an area that could be measured with good precision (i.e., a relative standard deviation of ≤20% for seven replicate injections). 6646
DOI: 10.1021/acs.jafc.5b02977 J. Agric. Food Chem. 2015, 63, 6644−6650
Journal of Agricultural and Food Chemistry
Article
Stability of Ergot Alkaloids during Sample Storage. An additional Canadian western rye sample was provided by CGC grain inspectors for use as an in-house reference material. This sample was ground and homogenized following the procedure used for the shipment samples described above. The ground and homogenized sample was stored, and test portions were weighed prior to each run and analyzed to monitor for changes in ergot alkaloid concentrations during storage. Portions of the in-house reference material were analyzed 27 times over a 10 month period. There was variation in the sum total ergot alkaloid concentrations over this period, but no overall decreasing or increasing tendency in sum total ergot alkaloid concentration was apparent. There were also no visible trends in individual ergot alkaloid concentrations or as fractions of the sum total ergot alkaloid concentrations over the storage period. The absence of trends in ergot alkaloid concentrations supports the appropriateness of storage at room temperature for ergot alkaloids in ground cereals. Ergot Alkaloids in Harvest Samples and Grain Shipments. The amounts of ergot sclerotia determined in all 2011 CWAD and CWRS submitted to the CGC Harvest Sample Program are shown in Figure 2, along with results from other harvests between 2002 and 2013 for context. The number of samples inspected for the presence of ergot sclerotia each year ranged from approximately 3200 to 5000 for CWRS and
from 820 to 2000 for CWAD. Figure 2 shows that the occurrence and severity of ergot infection (as shown by the percentage of samples downgraded due to ergot and the mean percentage of ergot in submitted samples, respectively) vary from year to year in both CWRS and CWAD. The mean percentages of ergot in 2011 were 0.030 and 0.026 for CWAD and CWRS, respectively. These means were both lower than in 2010; however, for CWRS the percentage of samples submitted to the CGC Harvest Sample Program that were downgraded due to the presence of ergot in 2011 was the highest between 2002 and 2013; for CWAD the second highest percentage of samples was downgraded in 2011. These data indicate that ergot was more widespread in 2011, even though the levels of infection in CGC Harvest Sample Program submitted grain were not the most serious that had been observed between 2002 and 2013. Overall, the data illustrated in Figure 2 suggest that the occurrence and severity of ergot infection in CWRS and CWAD have increased over the time period from 2002 to 2013. The incidence of ergot in the United Kingdom also increased during the mid-2000s.16 Changes in agronomical practices, such as the use of grass field margins and zero-tillage treatment of fields, have been implicated as factors contributing to the increase of ergot. Because burying ergot sclerotia prevents the release of ascopores and subsequent infection of flowering cereal plants,17 zero-tillage practices result in more inoculum on the field. The adoption of zero tillage in the Canadian Prairie provinces grew during the 2000s. From 2002 to 2008, the percentage of area in zero tillage increased from about 35% to 55%.18 Field margins act not only as a reservoir for C. purpurea inoculum but as a local source of secondary inoculum as well. Bayles et al.16 reported a higher risk of ergot infection to wheat mainly at the edges of wheat fields due to secondary inoculum from infected grasses in field margins. These practices would affect the presence of ergot in addition to environmental factors such as precipitation, temperature, and soil conditions,19 which have also been implicated as affecting ergot in cereals. Total concentrations of ergot alkaloids in CWAD and CWRS harvest samples from 2011 are shown in Figure 3. Concentrations of sum total ergot alkaloids in the harvest
Figure 2. Occurrence of ergot in wheat (CWRS) and durum (CWAD) samples submitted to the Canadian Grain Commission Harvest Sample Program. The percentage of samples downgraded due to ergot is provided by the vertical bars, and the mean percent ergot observed in the samples is indicated by the line graph. Samples with no ergot sclerotia were set to 0 for the calculation of the annual mean percent ergot.
Figure 3. Relationship between the amount of ergot sclerotia and ergot alkaloids in Canada Western Amber Durum (CWAD) and Canada Western Red Spring (CWRS) samples from the 2011 harvest. 6647
DOI: 10.1021/acs.jafc.5b02977 J. Agric. Food Chem. 2015, 63, 6644−6650
Journal of Agricultural and Food Chemistry
Article
Table 2. Total Concentration of 10 Ergot Alkaloids Quantitated in Cereals Shipped between August 1, 2010, and July 31, 2012
a
grain
n
n > LOQa
mean ΣEA (mg/kg)
median ΣEA (mg/kg)
range ΣEA (mg/kg)
90th percentile ΣEA (mg/kg)
wheat durum barley rye
117 46 25 1
117 46 21 1
0.229 0.225 0.065 1.138
0.195 0.193 0.047
0.012−0.666 0.040−0.584
Occurrence of Ergot and Ergot Alkaloids in Western Canadian Wheat and Other Cereals Sheryl A. Tittlemier,* Dainna Drul, Mike Roscoe, and Twylla McKendry Grain Research Laboratory, Canadian Grain Commission, 1404-303 Main Street, Winnipeg, Manitoba, Canada S Supporting Information *
ABSTRACT: A new method was developed to analyze 10 ergot alkaloids in cereal grains. Analytes included both “ine” and “inine” type ergot alkaloids. Validation of the method showed it performed with good accuracy and precision and that minor enhancement due to matrix effects was present during LC-MS/MS analysis, but was mitigated by use of an internal standard. The method was used to survey durum and wheat harvested in 2011, a year in which ergot infection was particularly widespread in western Canada. A strong linear relationship between the concentration of ergot alkaloids and the presence of ergot sclerotia was observed. In addition, shipments of cereals from 2010−2012 were also monitored for ergot alkaloids. Concentrations of total ergot alkaloids in shipments were lower than observed in harvest samples, and averaged from 0.065 mg/kg in barley to 1.14 mg/ kg in rye. In shipments, the concentration of ergot alkaloids was significantly lower in wheat of higher grades. KEYWORDS: mycotoxins, monitoring, durum, grading, sclerotia, method validation
■
INTRODUCTION Ergot is a well-known fungal infection of cereal grains characterized by the presence of dark-colored sclerotia that replace healthy kernels. References to ergot and its health effects date back to 1100 BCE in China and occur through Ancient Egypt and Greece, through Europe in the Middle Ages, and up to the mid-1900s in France.1 A series of alkaloid secondary metabolites are produced in ergot sclerotia, and it is these ergot alkaloids that cause the hallucinations, convulsions, burning sensation, vasoconstriction, and gangrenous loss of limbs associated with consumption of ergot-contaminated grain. There are also risks to livestock from exposure to ergot-contaminated feed.2 Ergot alkaloids are types of indole alkaloids, and those most commonly associated with wheat, rye, and other food cereal grains are amide-like derivatives of lysergic acid.3 These ergot alkaloids that have an R configuration at C8 (indicated by an “ine” suffix in their names) contain a double bond at C9−C10 of the ergoline ring and undergo epimerization to C8(S). The names of the epimerized “ine” compounds end in “inine”. The R ergot alkaloids readily undergo transformation to the S enantiomers under alkaline conditions, as well as over lengthy periods of storage.4 The epimerization is reversible and can reach equilibrium.3 Ergot is associated mainly with rye, but is also relevant for wheat, barley, and oats. Infection by the causative organism Claviceps purpurea occurs during flowering of the host plant. Therefore, because rye is a cross-pollinator with large open florets, it is particularly susceptible to infection by C. purpurea.5 Ergot alkaloids are generally present at higher concentrations in rye and rye-based foods as compared to other cereal grains.6 Modern management of ergot is focused on limiting the presence of ergot sclerotia in cereal grain. There are currently no regulations for ergot alkaloids in grain as there are for other mycotoxins; however, many jurisdictions have set tolerances for ergot sclerotia. In Canada, the highest quality grades of durum Published 2015 by the American Chemical Society
and western red spring had a limit of 0.010% ergot sclerotia on a mass basis7 prior to August 1, 2014. Current tolerances are 0.02 and 0.04% ergot sclerotia on a mass basis,7 for top grades of durum and western red spring, respectively. The Codex Alimentarius, American, and European maximum for ergot sclerotia in wheat is 0.05%.8−10 Ergot sclerotia can be removed from healthy grain by sorting based on color or density. There were three main purposes of this work: (1) to develop and validate a new analytical method for the analysis of raw cereal grains to include both R and S ergot alkaloids; (2) to examine the relationship between the presence of ergot sclerotia and concentrations of ergot alkaloids in wheat; and (3) to survey the presence of ergot alkaloids in shipments of western Canadian wheat from 2010 through 2012.
■
MATERIALS AND METHODS
Samples. Samples of Canada western amber durum (CWAD; n = 23) and Canada western red spring (CWRS; n = 42) harvested in 2011 were obtained from the annual Canadian Grain Commission (CGC) Harvest Sample Program. These samples consisted of grain that was voluntarily sampled at harvest by producers and submitted to the CGC. Sampling instructions were provided to producers prior to harvest.11 Once received, samples were examined by CGC inspectors, and the percentage of ergot sclerotia by mass in each sample was determined as described in the Canadian Grain Grading Guide.7 This set of 65 samples collected for ergot alkaloid analysis was specifically selected to cover a range of ergot sclerotia percentages. After grading, the selected samples were stored at room temperature in paper envelopes until ergot alkaloid analyses were performed. Test portions of the harvest samples were prepared for ergot alkaloid analysis by grinding the entire whole grain sample (approximately 200−400 g) using a model KR 804 commercial coffee grinder (Ditting Maschinen AG, Switzerland). The sample was ground Received: June 16, 2015 Accepted: July 2, 2015 Published: July 2, 2015 6644
DOI: 10.1021/acs.jafc.5b02977 J. Agric. Food Chem. 2015, 63, 6644−6650
Journal of Agricultural and Food Chemistry
Article
Figure 1. Structures of ergot alkaloid analytes and internal standard. fine enough that ≥85% of mass passed through a U.S. 50 sieve (nominal sieve openings of 300 μm). In addition, samples from shipments of western Canadian cereals were provided by CGC Industry Services inspectors. Grains sampled included wheat (n = 117), durum (n = 46), barley (n = 25), and rye (n = 1). These samples represented randomly selected export shipments of cereal grains that occurred between August 1, 2010, and July 31, 2012. It should be noted that the year of transport does not necessarily correspond to the year in which grain was grown and harvested. During the loading of the shipments, moving grain was sampled according to standardized procedures at a constant interval using CGC-approved automated cross-stream sample diverters and dividers.12 The increments obtained during loading of a lot were combined into a composite sample that was considered to be representative of the entire lot. A Boerner divider (Seedburo Equipment Co.) was used to divide the composite sample into 10 kg laboratory samples. The 10 kg laboratory samples were stored at
room temperature in canvas bags for up to 5 weeks until further preparation. Test portions of the shipment samples were prepared from the 10 kg laboratory sample of whole grain according to the process described previously.13 The 10 kg laboratory sample of whole grain was comminuted using a Retsch SR 300 rotor beater mill fitted with a 750 μm screen and coupled with a Retsch DR 100 vibratory feeder. After comminution, the entire mass of ground grain was homogenized and divided into 10 × 1 kg portions on a rotary sample divider (Materials Sampling Solutions, Southport, Australia). After the first division into 10 × 1 kg, all 10 portions were recombined in the RSD hopper and divided into 10 × 1 kg again. One 1 kg portion of ground grain was randomly selected and further divided on another RSD into 10 subsamples of 100 g of ground grain. Subsamples of 100 g were stored at room temperature in closed high-density polyethylene or polypropylene containers. The containers were not airtight, but were sealed to prevent the possibility of insects entering the samples. 6645
DOI: 10.1021/acs.jafc.5b02977 J. Agric. Food Chem. 2015, 63, 6644−6650
Journal of Agricultural and Food Chemistry
Article
Analysis of Ergot Alkaloids. Ten ergot alkaloids were included in the analytical method (Figure 1). Standards of individual analytes were obtained as crystals (Sigma-Aldrich, Oakville, Canada; Novartis, Dorval, Canada; Biopure, Tulln, Austria); purities ranged from 97 to 99%. Four of the alkaloids were S epimers of R enantiomers. The two epimers that were not included in the method were not easily obtained at the time of analysis. Dihydroergotamine (11, Fluka, analytical standard grade) was used as an internal standard for the analytical method. A method based on that of Krska et al.4 was developed for the extraction and ultrahigh-pressure liquid chromatographic−tandem mass spectrometric (UPLC-MS/MS) analysis of the 10 ergot alkaloids. Ground sample (10 g) was extracted with 50 mL of a 84:16 (v/v) acetonitrile/3.03 mM aqueous ammonium carbonate in 250 mL polyethylene centrifuge bottles. This mixture was shaken on a flatbed shaker for 30 min and then centrifuged at 2450g for 10 min at room temperature. A 464 μL aliquot of the supernatant was transferred to a glass test tube and mixed with 20 μL of internal standard solution (4 ng/μL dihydroergotamine in acetonitrile) and 1536 μL of 3.03 mM aqueous ammonium carbonate. Samples were vortexed to mix well and filtered using a disposable plastic syringe and PTFE syringe filter prior to injection. Final extracts were autoinjected, with the autosampler tray held at 15 °C, and chromatographed on a 2.1 × 100 mm i.d., 1.7 μm, BEH C18 column (Waters) held at 30 °C on a Waters Acquity I-class UPLC. A gradient of two solutions (A = 3.03 mM ammonium carbonate and B = acetonitrile) was used to resolve the analytes. The mobile phase began at 80% A, changed to 20% A over 5 min, and returned to 80% A after a further 1 min. Analysis was performed using a Waters Xevo MS/MS in the positive electrospray ionization mode using multiple reaction monitoring. The source and desolvation temperatures were held at 150 and 500 °C, respectively, with the capillary at 0.50 kV and the extractor at 3.0 V. The cone, desolvation, and collision gas flows were 100 L/h, 700 L/h, and 0.15 mL/min, respectively. To avoid epimerization of analytes, diluted standard mixes were dried down and kept in a freezer until needed. The dried mixes were reconstituted in acetonitrile just prior to instrumental analysis. Exposure to light was minimized, and samples and extracts were kept covered during processing and were placed in the UPLC sample manager with the illumination option turned off once diluted. Analytes were considered to be positively identified and quantitated if their retention times were within 0.1 min of the average retention time of the corresponding analyte in the external calibration standards; the peak had a signal-to-noise ratio >9:1, and the ratio of qualification to quantitation ions was within acceptable tolerances.14 The analyte responses were normalized to the dihydroergotamine responses during calculation of concentrations. Blank samples fortified with a solution of standards and an in-house reference material were analyzed with each batch of samples and were used to monitor the performance of the analytical method.
alkaloid analytes were fortified only at the medium concentration. During validation, separate sets of blanks were fortified with R and S ergot alkaloid analytes because a low amount of S enantiomers was found in the R enantiomer standard solutions. Ergosinine, ergocorninine, ergocryptinine, and ergocristinine were present at approximately 6% of the concentration of their analogues in the R enantiomer standard mix. Ergosine, ergocornine, ergocryptine, and ergocristine were also present in the S enantiomer standard mix, although at only about 2% of the concentration of their analogues. It was not clear if this was due to impurities in the analytical standards or due to epimerization during handling and use of the standard solutions over time. Both of these possibilities have been noted in the literature.15 Because of this apparent “contamination”, separate calibration standards were made for R and S enantiomers, and these were used in the validation and in all subsequent samples analyses. The method produced good results with recoveries from individual ergot alkaloids in individual fortified samples ranging from 60 to 132%. The overall mean recovery ± standard deviation for all ergot alkaloids over all fortification concentrations and days was 83 ± 12% for wheat and 88 ± 15% for rye. A comparison of individual ergot alkaloid peak areas from standard solutions prepared in solvent to postextraction fortified wheat and rye matrix matched standards indicated some enhancement of ergocorninine, ergocryptinine, and ergocristinine occurred due to matrix effects. The enhancement was somewhat higher for the rye matrix as compared to the wheat matrix (Table 1). The average ratio of ergot alkaloid Table 1. Ratios of Responses for Ergot Alkaloids and Internal Standard in Wheat and Rye Matrix Extract As Compared to Solvent ergonovine ergosine ergotamine ergocornine ergocryptine ergocristine dihydroergotamine ergosinine ergocorninine ergocryptinine ergocristinine
■
RESULTS AND DISCUSSION Validation and Method Performance. Validation of the analytical method was performed using fortified wheat and rye. Blank wheat and rye were created by hand-cleaning grain samples to remove ergot sclerotia. Even with this extra cleaning, some ergot alkaloids were still present in the blanks; therefore, during method validation concentrations of ergot alkaloids in the fortified samples were corrected by subtracting the concentration of analyte in the blanks prior to calculation of recoveries. Blank samples were fortified at three different concentrations and extracted and analyzed on multiple days. Seven replicates were fortified and extracted for each day/ concentration combination: low (2 μg/kg ergonovine and 10 μg/kg other analytes), medium (41 μg/kg ergonovine and 200 μg/kg other analytes), and high (407 μg/kg ergonovine and 2000 μg/kg other analytes). To conserve standards, the S ergot
wheat
rye
1.05 0.94 1.09 0.99 1.05 1.07 1.12 1.05 1.28 1.34 1.43
1.05 0.82 1.10 1.07 1.12 1.19 1.23 1.08 1.40 1.55 1.83
peak area in matrix to solvent was 1.13 for wheat and 1.22 for rye. These averages are very close to the ratios observed for the internal standard dihydroergotamine in wheat (1.12) and rye (1.23). This similarity indicates that normalization of individual ergot alkaloid responses to the dihydroergotamine internal standard would mitigate matrix effects on the measurement of the sum total concentration of all 10 ergot alkaloids analyzed. Limits of quantitation (LOQs) were 2 μg/kg for ergonovine and 10 μg/kg for the nine other ergot alkaloids. The LOQs were estimated as the lowest concentration to produce a chromatographic peak with a signal-to-noise ratio of at least 9:1 having an area that could be measured with good precision (i.e., a relative standard deviation of ≤20% for seven replicate injections). 6646
DOI: 10.1021/acs.jafc.5b02977 J. Agric. Food Chem. 2015, 63, 6644−6650
Journal of Agricultural and Food Chemistry
Article
Stability of Ergot Alkaloids during Sample Storage. An additional Canadian western rye sample was provided by CGC grain inspectors for use as an in-house reference material. This sample was ground and homogenized following the procedure used for the shipment samples described above. The ground and homogenized sample was stored, and test portions were weighed prior to each run and analyzed to monitor for changes in ergot alkaloid concentrations during storage. Portions of the in-house reference material were analyzed 27 times over a 10 month period. There was variation in the sum total ergot alkaloid concentrations over this period, but no overall decreasing or increasing tendency in sum total ergot alkaloid concentration was apparent. There were also no visible trends in individual ergot alkaloid concentrations or as fractions of the sum total ergot alkaloid concentrations over the storage period. The absence of trends in ergot alkaloid concentrations supports the appropriateness of storage at room temperature for ergot alkaloids in ground cereals. Ergot Alkaloids in Harvest Samples and Grain Shipments. The amounts of ergot sclerotia determined in all 2011 CWAD and CWRS submitted to the CGC Harvest Sample Program are shown in Figure 2, along with results from other harvests between 2002 and 2013 for context. The number of samples inspected for the presence of ergot sclerotia each year ranged from approximately 3200 to 5000 for CWRS and
from 820 to 2000 for CWAD. Figure 2 shows that the occurrence and severity of ergot infection (as shown by the percentage of samples downgraded due to ergot and the mean percentage of ergot in submitted samples, respectively) vary from year to year in both CWRS and CWAD. The mean percentages of ergot in 2011 were 0.030 and 0.026 for CWAD and CWRS, respectively. These means were both lower than in 2010; however, for CWRS the percentage of samples submitted to the CGC Harvest Sample Program that were downgraded due to the presence of ergot in 2011 was the highest between 2002 and 2013; for CWAD the second highest percentage of samples was downgraded in 2011. These data indicate that ergot was more widespread in 2011, even though the levels of infection in CGC Harvest Sample Program submitted grain were not the most serious that had been observed between 2002 and 2013. Overall, the data illustrated in Figure 2 suggest that the occurrence and severity of ergot infection in CWRS and CWAD have increased over the time period from 2002 to 2013. The incidence of ergot in the United Kingdom also increased during the mid-2000s.16 Changes in agronomical practices, such as the use of grass field margins and zero-tillage treatment of fields, have been implicated as factors contributing to the increase of ergot. Because burying ergot sclerotia prevents the release of ascopores and subsequent infection of flowering cereal plants,17 zero-tillage practices result in more inoculum on the field. The adoption of zero tillage in the Canadian Prairie provinces grew during the 2000s. From 2002 to 2008, the percentage of area in zero tillage increased from about 35% to 55%.18 Field margins act not only as a reservoir for C. purpurea inoculum but as a local source of secondary inoculum as well. Bayles et al.16 reported a higher risk of ergot infection to wheat mainly at the edges of wheat fields due to secondary inoculum from infected grasses in field margins. These practices would affect the presence of ergot in addition to environmental factors such as precipitation, temperature, and soil conditions,19 which have also been implicated as affecting ergot in cereals. Total concentrations of ergot alkaloids in CWAD and CWRS harvest samples from 2011 are shown in Figure 3. Concentrations of sum total ergot alkaloids in the harvest
Figure 2. Occurrence of ergot in wheat (CWRS) and durum (CWAD) samples submitted to the Canadian Grain Commission Harvest Sample Program. The percentage of samples downgraded due to ergot is provided by the vertical bars, and the mean percent ergot observed in the samples is indicated by the line graph. Samples with no ergot sclerotia were set to 0 for the calculation of the annual mean percent ergot.
Figure 3. Relationship between the amount of ergot sclerotia and ergot alkaloids in Canada Western Amber Durum (CWAD) and Canada Western Red Spring (CWRS) samples from the 2011 harvest. 6647
DOI: 10.1021/acs.jafc.5b02977 J. Agric. Food Chem. 2015, 63, 6644−6650
Journal of Agricultural and Food Chemistry
Article
Table 2. Total Concentration of 10 Ergot Alkaloids Quantitated in Cereals Shipped between August 1, 2010, and July 31, 2012
a
grain
n
n > LOQa
mean ΣEA (mg/kg)
median ΣEA (mg/kg)
range ΣEA (mg/kg)
90th percentile ΣEA (mg/kg)
wheat durum barley rye
117 46 25 1
117 46 21 1
0.229 0.225 0.065 1.138
0.195 0.193 0.047
0.012−0.666 0.040−0.584
