1. Introduction
Mycotoxins continue to be a concern for the safety and quality of food and feed [
1,
2]. Specifically, the fungus
Claviceps purpurea infects cereal crops forming visibly dark sclerotia that contain secondary metabolites known as ergot alkaloids [
3]. Ergot alkaloids have two configurations known as the
R-epimer and
S-epimer. Rotation of a functional group on the chemical structure at the carbon-8 defines the epimer. The
R-epimer exhibits a left-hand rotation, whereas the
S-epimer exhibits a right-hand rotation [
4,
5,
6]. Of the ergot alkaloids produced by
Claviceps purpurea, six
R and six corresponding
S epimers are quantified in food and feed samples [
7]. The six
R-epimers are, ergocornine, ergocristine, ergocryptine, ergometrine, ergosine and ergotamine [
8]. The corresponding
S-epimers are ergocorninine, ergocristinine, ergocryptinine, ergometrinine, ergosinine and ergotaminine. Ergot epimers have different concentrations within the sclerotia depending on the geographic location and crop type [
9].
In North America, diagnostic laboratories routinely include
R-epimers in their analytical methods for ergot alkaloid detection and quantification. However, the
S-epimers are often not included in diagnostic assays or analytical studies [
10,
11,
12]. The
S-epimers have been considered non [
13] or less [
14] bioactive compared to the corresponding
R-epimers. The
R-epimers are known to cause adverse effects when consumed by humans and animals [
15]. Similarly, the
S-epimers may also affect physiological systems [
16,
17]. It is, therefore, important to quantifying
S-epimers in ergot-contaminated samples.
Ergot alkaloids can epimerize between the
R and
S-epimer [
18]. Komarova and Tolkachev (2001) noted that the rate of conversion may be associated with the side group of a particular epimer. The conversion, also known as epimerization, between
R and
S-epimers, has also been associated with pH, light, matrix, and temperature [
19,
20,
21,
22]. If
S-epimers constitute a large portion of the total concentration of ergot epimers [
3], it is imperative to include them in an analysis for regulatory or diagnostic considerations.
Multiple instruments and techniques have been used to quantify ergot alkaloids. The use of an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method has been used to quantify mycotoxins [
23,
24]. The UHPLC-MS/MS technique can quantify both
R and
S-epimers of ergot alkaloids and have become the method of choice for detection [
25]. Additionally, methods such as high-performance thin-layer chromatography (HPTLC) [
26], and liquid chromatography with fluorescence detection [
27] have been recently utilized for the detection of the
R and
S-epimers of ergot alkaloids. Conversely, the use of an enzyme linked immunosorbent assay (ELISA) cannot distinguish differences among epimers [
28]. The European Union recommends that both
R and
S-epimers of ergot alkaloids should be quantified [
29]. In contrast, the Canadian Food Inspection Agency (CFIA) currently only considers the
R-epimers in their safety standards [
30].
Internal standards are regularly utilized in analytical quantification. Structural analogues, isotopic labeled, and deuterated compounds have been used as internal standards [
20]. Isotopically label internal standards are ideal since they behave in a similar manner to the analytes of interest. However, such labeled internal standards are limited for all ergot epimers. Usually, one or two internal standards are used to account for losses during extraction and analysis of ergot epimers [
3,
27,
31]. Internal standards that can successfully behave in a similar way to the analytes of interest and account for any losses of the analytes throughout extraction and analysis, are beneficial in analytical methods.
The simultaneous detection and quantification ergot R and S-epimers are beneficial in terms of time and ease of interpretation. Only low concentrations of ergot epimers in feed are accepted according to regulatory standards. Therefore, a sensitive and timely method for epimer quantification is required. Assessing similarities and differences between the R and S-epimers in terms of validation may provide useful information for future analytical methods. The objective of this study was to validate a new method for the simultaneous detection and quantification of R and S-epimers of ergot alkaloids utilizing deuterated lysergic acid diethylamide (LSD-D3) as a new internal standard. Deuterated lysergic acid diethylamide has not been utilized previously to quantify ergot epimers, to the authors knowledge. A second objective was to use the validated method to quantify R and S-epimers of ergot alkaloids in naturally contaminated hard red spring wheat samples.
3. Discussion
This study describes a new and validated sensitive method for the analysis of
R and
S-epimers of ergot alkaloids. The validation of this method followed the validation procedures outline in the Commission Decision 2002/657/EC [
33]. Various parameters were adapted from the ThermoFisher Scientific method [
34], however, improved sensitivity and matrix effects are observed in the present study. The improvements could be associated with differences in the methods and/or the use of an IS. During the method validation, a limited quantity of ergot epimer standards were available to use based on cost. A lesser amount of grain and solvent were utilized to minimize cost and excessive use of standards. This allows laboratories to save money and resources, especially when sample replication is necessary and spiking at the beginning of the extraction process is important.
The linearity of r
2 > 0.99 for the calibration curves are defined as good [
29]. The LOD and LOQ were calculated using the ‘Guidance Document on the Estimation of LOD and LOQ for Measurements in the Field of Contaminants in Feed and Food’ [
32]. Similarly, Arroyo-Manzanares et al., 2018 [
23] and Schummer et al., 2020 [
35] used the same approach. The calculated instrumental LOD and LOQ (µg/kg) from this study are low compared to Arroyo-Manzanares et al., 2018 [
23], using the same matrix. The LOD for all 12 epimers are below the lowest concentration on the linear calibration curve. Food for children and infants may contain a very low concentration of ergot alkaloids according to regulatory limits [
36], therefore, a new sensitive method with low LOD’s and LOQ’s are beneficial. This sensitive analytical method can be utilized in research or diagnostic research to obtain low and actuate concentrations.
For each R/S-epimer pair for each ergot alkaloids, similar and different LOD and LOQ values are observed. That could be associated with differing functional groups for certain R/S-epimer pairs. Potentially, the lower LOD and LOQ for most S-epimers could be associated with better ionization which may be related to the greater peak area (counts × minute) observed.
A common approach to determine the LOD and LOQ of a method includes the ‘mean of 3 and 10 SD’ in samples that are uninfected [
29] (p. 7037) or a signal to noise ratio of 3 and 10 [
31] (p. 295). The Guidance Document mentioned above suggests that common methods to determine the LOD and LOQ should not be utilized. Pascale et al., 2019 [
37] mentioned that the LOD and LOQ may vary between laboratories. Therefore, the guidance document describes an approach to measure those parameters in a way to incapsulate the whole procedure to help decrease those discrepancies. Sulyok et al., 2020 [
38] noted that the LOD and LOQ do not need to be reassessed for different matrices unless there is noise at a high concentration spike level. The rationale for utilizing a representative wheat matrix in the current study is supported.
Matrix effects are commonly observed when using UHPLC-MS/MS. Matrix effects of greater than 100% can infer signal enhancement, while matrix effect of less than 100% can infer signal suppression [
29,
31]. Variable matrix effects are observed in multiple matrices when analyzing ergot epimers [
31]. Similarly, the variability in matrix effects between studies can be associated with the differences in analytical methods and instruments. In the current study, the matrix effects appear to be minimal. This is associated with ME values for each epimer occurring around 100%. There are no guidelines on the acceptable amount of matrix effects [
38]. Matrix effects can be defined as soft, moderate or strong depending on the plus minus from 100%. According to that classification, the current study observed soft enhancement (100 + 20%). Associated with reasonable matrix effects and the use of an internal standard, matrix match calibration curves were not deemed necessary. Solvent calibration curves have also been utilized in recent analytical studies assessing mycotoxins [
38,
39]. Likewise, injecting small quantities of a matrix can minimize the need for matrix matched calibration curves [
37], which was utilized in the current method.
The S-epimers of all ergot alkaloids had lower matrix effects, closer to 100%, compared to the R-epimers. This could be associated with the differences in ionization between the two epimer configurations. A lower matrix effect for the S-epimers may allow for a more accurate concentration analyzed.
Recovery results in the present study are similar to other analytical methods analyzing similar analytes. The low (0.75 µg/kg) spike concentration had similar recoveries for all ergot epimers (79–115%) compared to the mid (5 µg/kg) spike concentration (68–119%). Arroyo-Manzanares et al., 2018 [
23] observed similar values for percent recovery with a range of 60–89% for all ergot epimers using higher spike concentrations (10 and 150 µg/kg). Tittlemier et al., 2015 [
3] also observed percent recoveries ranging from 60–132% for 10 ergot epimers. Interestingly, Tkachenko et al., 2021 [
39] saw greater accuracy/recovery for the
S-epimers compared to the
R-epimers. Similarly, this study observed the same trend except for ergometrine/-inine. However, the recovery for ergometrinine and ergometrinine are very similar. Factors such as greater ionization or greater stability may be associated with the greater recovery of the
S-epimers compared to the
R-epimers.
The percent recovery observed in the current study aligns with other mycotoxin standards stated in the European Commission Regulation (2006) [
40], although it is stated that recovery in terms of mycotoxins is under review [
38]. Krska et al., 2008 [
6] related the criteria for mycotoxins as having satisfactory values of 60–120% for recovery and a RSD < 30%. Ergot alkaloids are not explicitly defined in terms of recovery in the European Commission (EC) Regulation (2006) [
40].
Precision is commonly calculated using percent relative standard deviation (% RSD). Arroyo-Manzanares et al., 2018 [
23] had a RSD of 13% or lower for all mycotoxins analyzed. Likewise, Guo et al., 2016 [
29] had RSD values of lower than 15%. In the current study, the intra-day precision ranged from 1.86–13.81% RSD and inter-day precision ranged from 5.88–23.9% RSD for both spike concentrations. Diana Di Mavungu et al., 2012 [
31] had similar results for repeatability (12–26% RSD) and within laboratory reproducibility (12–24% RSD) for the lowest spiked concentration used analyzing 12 ergot epimers. A RSD of less than 20% conforms with the European Commission Regulation (2006) [
40]. An acceptable RSD ≤ 20% for 97% of the analytes quantified in different food matrices has been reported [
41]. In the current study, only one analyte, ergometrine, had >20% RSD for inter-day precision. There appeared to be no trends in the similarity or differences between the
R and
S-epimers in terms of precision.
Internal standards are used in analytical methods to account for the loss of analytes throughout the extraction process and analytical procedure. Internal standards account for losses associated with the cleanup and detection of ergot alkaloids [
20]. Ideally, for each ergot epimer analyzed, there would be an isotopically labeled internal standard. However, such internal standards are not available [
36]. Therefore, in the current study, the internal standard, LSD-D
3, was used to account for any potential losses throughout the extraction procedure and analysis. Without the internal standard, a decrease in concentration was observed which was deemed inaccurate. The observed decrease in concentration observed was associated with the dry down of the extraction solvent. Through recovery assessment, it was deemed that the use of the internal standard accounted sufficiently for the loss of all the epimers. The concentration of epimers with the correction of the internal standard were close to the actual concentration spiked. Therefore, the internal standard can be added before the dry down step to account for any potential losses. Tittlemier et al., 2015 [
3] and Fabregat-Cabello et al., 2016 [
42] added IS to their samples in a similar manner. The LSD-D
3 has similar physiochemical properties to the ergot epimers, similar shape to the ergoline ring of the ergot epimers and elutes on the chromatogram similar to some epimers. A limitation of LSD-D
3 is the deuterated atoms are cleaved during fragmentation [
20]. However, the molecular ion with the deuterated atoms still attached is used for quantification. Additionally, deuterated compounds must not to have the same molecular weight as naturally occurring isotopes. Three or more deuterated atoms are recommended. The current internal standard utilized may not be ideal for all epimers analyzed compared to C13 isotopically labeled internal standard, however, it acts sufficiently for accounting for any losses. Holderied et al., 2019 used lysergic acid diethylamide (LSD) as their internal standard to detect and quantify ergot epimers associated with structural and chemical similarities. Conveniently, LSD-D
3 can be easily purchased and is readily available, whereas LSD is a control substance in Canada. The LSD-D
3 can now be included as an internal standard to quantify ergot alkaloids and may be adopted into current or future methods.
Epimerization of ergot epimers in analytical methods can be of concern. Cool temperature autosamplers are recommended to minimize epimerization [
31,
43], which was utilized in the present study. Epimerization was also minimized using amber vials and black plastic bags to limit light exposure throughout sample handling and extraction. The ultra-high performance liquid chromatography (UHPLC) column was maintain at a temperature of 40 °C. A previous method also utilized the same temperature for the quantification of ergot alkaloids [
34]. As noted in the chromatogram (see materials and methods), the epimers have sharp peaks and, with the exception of ergotamine/ergotaminine, do not have a ‘saddle’ between their peaks, indicating on column epimerization is unlikely [
44].
This current validated method quantified 12 ergot epimers in naturally contaminated wheat. These 12 epimers are the major ergot alkaloids produced by
Claviceps purpurea and constitute a large portion of the ergot alkaloid metabolome [
45]. Wheat samples from western Canada have had similar concentrations to the present study [
3]. The
R-epimers of each ergot alkaloid had greater concentrations than the
S-epimers. However, the concentrations of some
R/
S-epimer pairs were similar to each other. Ergocristine and ergocristinine had the greatest concentrations of all
R and
S-epimers analyzed. Similarity, they have been reported to be the most dominate
R and
S-epimer in terms of concentration [
19]. Interestingly, independent sample number 4 had low concentrations of ergocornine/ergocorninine compared to the other independent samples. The high proportion of
S-epimers in contaminated grain may be contributed to the epimerization of
R to
S-epimers over time. Consequently, the large concentration of
S-epimers supports the quantification in a diagnostic and analytical setting. Similarly, including the
S-epimers of ergot alkaloids in food and feed safety standards is important.