Determination of Ergot Alkaloids: Comparison of extraction efficiencies in several cereal production chains by UPLC-MS/MS

doi:10.21203/rs.3.rs-4649298/v1

Download PDF

Research Article

Determination of Ergot Alkaloids: Comparison of extraction efficiencies in several cereal production chains by UPLC-MS/MS

https://doi.org/10.21203/rs.3.rs-4649298/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

To date, there are more than 80 ergot alkaloids identified, their distribution depends on different factors (e.g. geographic regions, host plants etc.) and, dependent on the frequency and concentration ingested and the frequency of ingestion, they can cause acute and chronic toxic effect on human health. These toxins commonly infect cereal crops such as triticale and rye, and also wheat, barley and oats. In this regard, considering the growing consumption of plant-based foods compared to food of animal origin, the European Food Safety Authority, has highlighted the need to develop risk assessment strategies.

For this reason, the emphasis has been placed on the optimization of extraction efficiency, to quantify the main ergot alkaloids and their epimers, that are now available on the market (ergosine, ergocristine, ergocriptine, ergocornine, ergosinine, ergocristinine, ergocriptinine and ergocorninine).

The effectiveness of three different extraction conditions (acidic, alkaline and neutral) followed by a rapid clean-up using dispersive solid-phase extraction with C₁₈ sorbent was evaluated by ultra performance liquid chromatography tandem quadrupole mass spectrometry (UPLC-MS/MS), resulting in a short chromatographic run (16 min). The method was developed and validated in five different cereal production chains (rye, oat, wheat, wheat gluten and baby food). The efficiency and efficacy of extraction methods were evaluated and compared as well as obtained recoveries. The verified linear range was 0.5–500 µg kg^− 1 for all the tested compounds, according to the legal limits. The limits of quantification were dependent on the analyte but almost independent from the matrices. Recovery values for the 8 ergot alkaloids spiked at levels of 2, 20 and 100 µg kg^− 1 were calculated for each matrix extracted with its best extraction protocol and were completely satisfactory. The applicability and the trueness of the method were examined by analysing a set of 54 samples, including also other cereals like spelt, tritordeum and triticale, and evaluating some reference materials.

Ergot alkaloids

Epimerization

Extraction procedures

liquid chromatography tandem quadrupole-mass spectrometry

cereals

baby food

One of the most notable fungal infections is Claviceps purpurea from the Claviceps genus. The ascospores from the fungus Claviceps purpurea contaminate the seeds heads of living plants at flowering time with the development of a sclerotia, also known as ergot body, which replaces the seed of the crops (Krska and Crews 2008). Ergot sclerotia contain ergot alkaloids (EAs), which are secondary metabolites since they are produced by the fungi but not involved in their normal physiological processes. They are a group of nitrogenous organic compounds (Komarova and Tolkachev 2001), concentrated on the outer edge of the sclerotia, with lower concentrations in the interior (Young et al. 1983).

The most susceptible crops to ergot are cross-pollinators such as triticale and rye, however, also wheat, barley, oats and millet can also become infected. Environmental conditions (e.g. temperature, humidity, fungal concentrations and nutrients) promote ergot infection in crops and grasses, especially seasons with heavy rainfall and wet soils (Agriopoulou 2021). The sclerotia can be harvested together with cereals or grass and thus, they can contaminate cereal-based food and feed products with EAs, whom ingestion can cause ergotism in humans and animals, in addition to acute and chronic effect (Krska and Crews 2008). There is also the possibility for post-harvest contamination (during storage, transportation and food processing), where these alkaloids are very stable and resistant to degradation (Liao et al. 2013), thus good agricultural and management processes, and home or industrial processing are not sufficient to eliminate them. Ergot alkaloid intoxication is still an issue in developing countries (Agriopoulou 2021) and remains a concern for livestock (Schummer et al. 2020).

EAs are characterized by the presence of tetracyclic ergoline rings methylated on the N-6 nitrogen atom, substituted on C-8 and with a double bond in the C8-C9 or C9-C10 positions. They can be classified into 3 main classes: short chain substituted amides of lysergic acid, clavine alkaloids, and ergopeptines, which are peptide alkaloids with an additional peptide half attached to the basic tetracyclic ring (Fig. 1) (Komarova and Tolkachev 2001; Krska and Crews 2008; EFSA 2012; Crews 2015).

There are six main ergot alkaloids produced by Claviceps species in the sclerotia, named ergometrine, ergotamine, ergosine, ergocristine, ergocryptine and ergocornine. These EAs are classified as ergopeptines, apart from ergometrine which is classified as a simple lysergic acid derivative. Each ergot sclerotia can contain a variable concentration of ergot alkaloids (Carbonell-Rozas et al. 2022). The total ergot alkaloid concentrations can vary from 0.01 to 1.04% in a single ergot body (Ruhland and Tischler 2008). The percentages of the six common ergot alkaloids within an ergot body depends on type of crop, fungi, individual ergot bodies, year, and geographical location (Grusie et al. 2017; Kodisch et al. 2020).

Ergot alkaloids have multiple chiral centers, the double bond at C9–C10 allows epimerisation at the carbon 8 chiral center forming C8-(R) isomers with left-hand rotation, which are known as ergopept-ines, and C8-(S) isomers with right-hand rotation, which are known as ergopept-inines (Komarova and Tolkachev 2001; Krska and Crews 2008). The

N6 gives the protonated EA pKa values of 5.0 to 7.4, they have a positive charge in acid solution and are neutral in alkali (Crews 2015). The R and S-epimers are interconvertible and pass through an intermediate configuration during their energetic conversion, that can occur under various conditions. Cherewyk et al. (2022a) underlined that the R-epimer can epimerize to the S-epimer under aprotic (e.g., acetonitrile, acetone, chloroform) and protic solvents (e.g., methanol or water:methanol mix), at room temperature over days, at 37°C in several matrices over hours, and during the extraction process of cereals (Krska et al. 2008a). The S-epimer can convert to the R-epimer in organic solvent, water, or acidic solutions (Komarova and Tolkachev 2001). The literature reported that R-epimers are believed to have biological activity, while S-epimers to be inactive, however, recent studies have demonstrated potential bio activity of the -inine epimers which are likely to be harmful similar to the -ine epimers (Crews 2015; Cherewyk 2022a). Thus, the CONTAM panel of the European Food Safety Authority EFSA (2012) recommended to conduct risk assessments on both forms (-ine and -inine), and to consider also the ergot alkaloids concentration, and not the presence of ergot sclerotia alone (EFSA 2012).The European Commission has established a maximum level for ergot sclerotia and alkaloids in food (Reg. EU No 2021/1399) considering the sum of the six common R and S-epimers (EU 2021). The lowest concentration is based on vulnerable populations such as infants and children at 20 µg/kg total ergot alkaloids. A maximum level of 500 µg/kg was established, but from the 1st July 2024, a reduction for some categories of foods should guarantee an higher level of safety. To safeguard humans’ health, EFSA reported a total daily intake (TDI) for ergot alkaloids of 0.6 µg/kg body weight per day, and an acute reference dose of 1 µg/kg body weight (EFSA 2012).

Determining EAs is of great importance and for this reason, a stable, efficient, and sensitive analytical method, able to determine and quantify them, should be developed (Chung 2021; Silva 2023; Cherewyk 2024). Considering the medium-high polarity of these compounds, according to the literature, the SLE (solid-liquid extraction) has mainly been used with non-polar organic solvents (like dichloromethane/ethylacetate/methanol/25% aqueous ammonia) under alkaline conditions (Muller 2009; Bryła 2015) or with polar solvents (like methanol and acetonitrile) under acidic conditions (Mulder 2015; Rubert 2012; Versilovskis 2020). Krska et al. (2008b) compared different extraction solvent protocols and highlight that acetonitrile mixtures with ammonium carbonate buffer (84:16, v/v) yielded the highest concentrations of EAs. Eight years later, Guo et al. (2016) exploited almost the same extraction solvent MeCN with ammonium carbonate buffer (85:15, v/v), comparing the matrix effect after three clean-up procedures: C₁₈ sorbent, PSA sorbent and MCX cartridge, discovering that C₁₈ sorbent cleaned the cereal matrix slightly better than PSA sorbent, and showed the best performance. Chung (2021) and Silva (2023), in their reviews, concluded that many methods have been developed for the determination and quantification of EAs, and those based on QuEChERS and dispersive-SLE (d-SPE) procedures, are the fastest, cheapest, and most efficient methods developed.

Although EU Commission established maximum levels for the sum of the –ine and –inine forms, the present study aims to develop a method able to quantify the main ergot alkaloids and their epimers, that are now available, without any legal restriction, on the market, to guarantee the applicability of the method to all entities involved in monitoring and quantification. The developed method determined, 4 ergot alkaloids (ergosine, ergocornine, α-ergocryptine and ergocristine) with their corresponding epimers (ergosinine, ergocorninine, α-ergocryptinine and ergocristinine).

Artificially contaminated samples of rye, oat, wheat, wheat gluten and baby food were extracted comparing three different solvents (acidic, neutral and alkaline) and extraction procedures with different amounts of d-SPE sorbent, to find the best combination of high extraction efficiency and effective clean-up results. To the best of our knowledge, this is the first paper addressing the determination of ergot alkaloids in all these matrices together. After the development and in-house validation, the performance of this approach was evaluated using certified reference materials analysing different naturally contaminated samples, over a range of cereal production chains.

2.1. Reagents, standards and materials

All solvents were HPLC – MS grade. Acetonitrile was purchased from VWR International, Ltd (Poole, United Kingdom). Formic acid and ammonium carbonate were obtained from Sigma Aldrich (Darmstadt, Germany). For the preparation of the mobile phase and all sample preparations experiments, water was purified using a Milli-Q system (Millipore, Bedford, MA).

Certified reference standards of four ergot alkaloids, ergosine, ergocristine, ergocriptine, ergocornine and the corresponding epimers, ergosinine, ergocristinine, ergocriptinine and ergocorninine were purchased as

dried down films from Romer Labs (Tulln, Austria) with a chemical purity > 98%.

Dispersive Solid Phase Extraction C₁₈ sorbent (Bondapak HC18 reference WAT035672), screw-capped amber glass vials (12 x 32 mm Screw Neck Qsert Vial, 300 uL, reference 186002803) and PTFE filters (wwPTFE Acrodisc Mini 0.2um 13mm, reference 186009314) were from Waters (Milford, MA. USA).

2.2. Extraction solvents

Three different extraction protocols were compared: (1) an alkaline mixture of acetonitrile – ammonium carbonate buffer 85:15 (v/v), (2) an acidic extraction with acetonitrile – water 84:16 (v/v) containing 0.5 % formic acid and (3) a neutral aqueous extraction with acetonitrile – water, 84:16 (v/v). The pH of the ammonium carbonate in the extraction solvent was investigated in the range pH 7.5 – 10.

2.3. Samples

Samples for method development and validation:

Wheat samples were obtained from the 2023 harvesting campaign. Rye samples came from the Sweden suppliers while oat and wheat gluten samples came from different Italian suppliers. Baby food sample (blended flour of rye, oat and wheat) was purchased at the local supermarket.

Certified reference materials were obtained from FAPAS (FERA Science Ltd. York, UK) and were two proficiency tests (PT) for ergot alkaloids in rye (PT 22190 and PT22203) and a quality control material for ergot alkaloids in baby food (T22196QC).

Survey samples for method applicability:

A set of 54 naturally contaminated samples (from 2020 to 2022) coming from different suppliers, covering rye, wheat (common and durum wheat) and other cereals like spelt, tritordeum and triticale.

2.4. Sample preparation procedure and clean-up

2.4.1. Wheat and Oat

Finely ground representative samples of wheat and oat (2.00g ± 0.01 g) were weighed into 50-mL polypropylene centrifuge tube with 20 mL of the alkaline mixture acetonitrile – ammonium carbonate buffer (200 mg/L, 3 mmol; pH 8.5), 85:15 (v/v), followed by short vortex mixing 30s, mild shaking for 20 min and a centrifugation step of 5 min (5000 x g) at 4 °C. During the extraction the falcon tubes were covered with an aluminium foil. A total of 5 mL of the supernatant for each sample was transferred to a 50-mL centrifuge tube containing 300 ± 1 mg of C₁₈sorbent for dSPE clean-up and vortex-mixed at high speed for 1 min.

2.4.2. Wheat Gluten, Rye and baby food

Finely ground representative samples of wheat gluten, rye and baby food (2.00g ± 0.01 g) were weighed into 50-mL polypropylene centrifuge tube with 20 mL of the alkaline mixture acetonitrile – ammonium carbonate buffer (200 mg/L, 3 mmol; pH 8.5), 85:15 (v/v), followed by short vortex mixing 30s, mild shaking for 20 min and a centrifugation step of 5 min (5000 x g) at 4 °C. During the extraction the falcon tubes were covered with an aluminium foil. A total of 5 mL of the supernatant for each sample was transferred to a 50-mL centrifuge tube containing 150 ± 1 mg of C₁₈sorbent for dSPE clean-up and vortex-mixed at high speed for 1 min.

For all the matrices, the mixture of extraction solvent and dSPE was centrifuged for 5 min (5000 x g) at 4 °C, and then the upper layer was filtered through the PTFE filter and transferred into a screw-capped amber glass vial prior to injection of 5 µL into the UPLC – MS/MS system. The samples were analysed immediately after extraction otherwise they were evaporated to dryness under a stream of N₂and reconstituted with the appropriate volumes of each extraction solutions.

2.5. Standard preparation for quantification

Following the indications of the manufacturer, the standards were reconstituted in 5 mL of MeCN, to achieve the concentrations of 100 µg mL^-1 for the -ine epimers and 25 µg mL^-1 for the inine-epimers. From these stock solutions, three working solutions containing all 8 ergot alkaloids were prepared in pure acetonitrile: (1) A first working solution at a concentration of 1 µg mL^-1, and this solution was used to prepare (2) a second working solution of 0.2 µg mL^-1, and this was used to prepare (3) a third working solution at 0.05 µg mL^-1.

During the analysis of the calibrants for quantification, we found that the best way of working was to prepare the standard freshly each time, or create aliquots in non-protic solvent and amber glass vials, evaporated to dryness under a gentle stream of N₂, stored at – 20°C and reconstituted just before use to avoid epimerization or possible degradation during storage.

2.5.1. Calibration

A nine-points matrix matched calibration curve was used to quantify the analytes (0.5, 2, 5, 10, 25, 50, 100, 200 and 500 ng/mL). To reach the desired concentrations, the calibration standards were freshly prepared by serial dilutions using blank sample extract (a multi-matrix blended flour composed of 1/3 rye, 1/3 oat and 1/3 wheat gluten) as diluent. A blank food material was chosen, in which, according to previous analysis, no ergot alkaloids were detected. The blank material was extracted with the three extraction solutions indicated above (see paragraph 2.2) in order to create three different matrix-matched curves and calculate the matrix effect for each of them. Matrix-matched curves were obtained by serial dilution of the calibration standard with different volumes of acetonitrile extract (for each extraction solutions) of a multi-matrix sample.

2.6. Epimerization of EAs during preparation

The stability of ergot alkaloids and their epimers was tested in rye after 30 min or overnight. Waiting 30 min before starting the extraction procedure allowed good recoveries to be obtained without any significant epimerization, while when the sample was left overnight, severe epimerization occurred for all the 3 extraction procedures tested. The decrease of -ines led to a corresponding increase of the -inines form and sometimes the S-epimer may convert to the R-epimer in organic solvent, water, or acidic solutions (Komarova and Tolkachev 2001). According to these results, spiking samples should be prepared and analysed on the same day.

Furthermore, the analytical run time should be minimized to avoid epimerization during the analysis. In addition, it was better to cover the falcon tube with aluminium foil during the extraction step, avoiding prolonged exposure to light during the other preparation steps and, also centrifuging at 4 °C in order to minimize epimerization during the sample preparation procedure. In general, the stability and epimerization of the R and S-epimers may depend on the specific ergot alkaloid assessed (Schummer et al. 2020).

2.7. UPLC – MS/MS analysis

The analyses were performed on an ACQUITY I-Class UPLC system coupled to a Xevo-TQS micro tandem quadrupole mass spectrometer (both from Waters Corp., Manchester, UK). The separation column used was an ACQUITY BEH C₁₈ column 2.1 x 150 mm, 1.7 μm (Waters Corp., Manchester, UK), heated at 40 °C. Mobile phase was 3 mM ammonium carbonate buffer (200 mg L^-1 = 3 mmol; pH 8.5±0.3) in water (eluent A), and pure MS grade acetonitrile (eluent B) at a flow rate of 0.4 mL/min. The autosampler was set to 8°C (which was beneficial to control any epimerisation during a sample batch) and the injection volume was 5 µl. The eluent gradient profile was set as follows: 0 – 1.0 min (5% B); 1.0 – 2.5 min (45% B); 2.5 – 9.0 min (95% B); 9.0 – 12.0 min (95% B); and from 12.0 to 12.2 min return to the initial condition. The system was re-equilibrated with the initial composition for 4 minutes prior to the next injection, yielding a total run time of 16 min.

For mass spectrometric acquisition, multiple-reaction monitoring (MRM) was conducted in positive ESI mode. The source temperature was set to 150 °C and the desolvation gas temperature was 500 °C. The desolvation gas flow was held at 700 L/h, and the collision pressure (argon) was 4.3 × 10⁻³ mbar. The capillary and cone voltages were 2.5 kV and 30 V, respectively.

2.8. Data Management

Data were acquired using MassLynx (v4.2) and processed using TargetLynx XS Application Manager. The selection of MRM transitions and optimization of critical parameters was performed by infusion of individual solutions of each analyte. Table 1 summarizes conditions for all the MRM transitions including the retention times. The dwell time was set to 20 ms (12 points per peak) for each MRM transition.

Table 1 Retention times and mass spectrometric conditions for determination of 8 ergot alkaloids.

Analyte	R_t (min)	Precursor ion [M+H]⁺ *(m/z)*	Quantifier ion *(m/z)*	Collision energy 1 (eV)	Qualifier ion (m/z)	Collision energy 2 (eV)
Ergosine	4.57	548.2	223.1	31	268.1	25
Ergocornine	5.11	562.3	223.1	37	268.1	25
α-Ergocryptine	5.40	576.3	223.1	38	268.1	25
Ergocristine	5.48	610.3	223.1	35	268.1	26
Ergosininine	5.73	548.2	223.1	31	268.1	25
Ergocorninine	6.27	562.3	223.1	37	268.1	25
α-Ergocryptinine	6.61	576.3	223.1	38	305.2	30
Ergocristinine	6.74	610.3	223.1	35	268.1	26

2.9 Method Validation

The method was validated in-house determining the matrix effect (ME), linearity of the calibration, recovery rates, repeatability and both limits of detection (LOD) and quantification (LOQ).

Performance criteria from document Nº SANTE/11312/2021 (SANTE 2021) and document CEN/TR 16059:2010 on single laboratory validation of mycotoxin methods (CEN 20210), were used as references.

To evaluate the matrix effect, a comparison between the slope of the matrix-matched and the solvent calibration curves for the three-extraction solvents tested, was performed. Slope differences were calculated for each analyte applying the following equation (Eq. 1) (SANTE 2021):

Negative values represented signal suppression while positive values indicated signal enhancement caused by the matrix interferences.

Peak area was used as analytical response and linearity, expressed as determination coefficients (R²), was evaluated at the concentration range reported in paragraph 2.4.1.

Recovery data were performed using blank rye, oat, wheat, wheat gluten and baby food matrices artificially contaminated at three concentration levels: 2, 20 and 100 µg kg^-1, with three replicates each. These concentrations refer to each individual analyte in the sample. Blanks and spiked samples were then extracted and analysed as described in the previous section. The percentage of recovery was given by the calculated amount of the spiking minus the calculated amount of the blank sample divided by the theoretical spiked concentration.

The same dataset was used to calculate the method’s repeatability, which was expressed as the relative standard deviation (RSD) of six replicates.

The Limits of Detection (LOD) and Quantification (LOQ) were determined as the analyte concentration corresponding to the mean of 3 and 10 standard deviations (SDs), respectively, of the response in a blank sample matrix.

The method’s trueness was evaluated by analysing a quality control material for ergot alkaloids in baby food (T22196QC) and two proficiency tests (PT) for ergot alkaloids in rye (PT22190 and PT22203) provided by FAPAS (FERA Science Ltd. York, UK) in 2022-2023. Trueness was expressed as z-scores for the individual EAs compounds considered in the proficiency testing report. Participants’ z-scores were calculated as (Eq.2):

Where:

x participant’s reported results

x_aassigned value

σ_p standard deviation for proficiency

The number of z-scores for all the analytes in the range -2 ≤ ≤ 2 were calculated.

2.10 Method applicability

The method’s applicability was tested with a set of 54 naturally contaminated samples (harvesting season from 2020 to 2022) covering some validated matrices and other cereals like spelt, tritordeum and triticale. They were analysed in triplicate for the presence of the 8 EAs compounds covered in the current method.

3.1 Method Development

3.1.1 MS optimization

For the determination MRM transitions, single substance solutions of the 8 Ergot alkaloids were directly infused into the MS via a syringe pump and parameters (polarity, collision energy and cone voltage) were optimized for each toxin. Two MRM transitions with the best intensity and signal-to-noise (S/N) ratios in positive electrospray ionization (ESI+) were chosen for each toxin. The optimized mass transitions are summarized in Table 1.

3.1.2 Optimization of the chromatographic separation

Different mobile phases and elution gradients were tested and optimised.

In literature, some studies underlined that for ergot alkaloids analysis on C₁₈ columns, high pH conditions are required to achieve full separation. At the same time, there is considerable concern that using high pH could cause a decrease in sensitivity of mass-spectrometric detection under conditions that could suppress analyte ionization in solution. For that reason, acidic mobile phases are increasingly being used, such as the addition of volatile weak acids for enhancing the ionization of basic compounds in ESI+ (Rubert 2012; Carbonell- Rozas 2022; Liang 2022). Nevertheless, the methods exploiting acidic phases often are less suitable for the detection of both epimers (ines and inines), on the other hand several studies have reported the successful detection of EAs in electrospray positive mode when using high pH buffers in the mobile phase (Mulder 2015; Guo 2016; Veršilovskis 2020).

To optimize the chromatographic conditions and find the best solution for this study, we compared the analyte signal intensity of ergot alkaloids and their epimers, within a range of pKa values (4.8 ~ 6.2) (Krska et al. 2008b), in different pH mobile phases. For separation under acidic conditions, 0.1 % formic acid was added to water (Solvent A) and methanol (Solvent B). In the case of alkaline conditions, 3 mM ammonium carbonate buffers at two different pH (7.8; 8.5) (Solvent A) with acetonitrile (Solvent B) were tested. The pH was adjusted to 8.5 by adding 25% ammonia.

As shown in Figure 2, high pH mobile phases (blue) did not suppress the ionization of the compounds in ESI+; positive ions are formed abundantly, and analyte responses are often enhanced in high pH compared to acidic mobile phases (red).

Waters BEH C₁₈ column showed good stability for the alkaline conditions, 3 mM ammonium carbonate buffer, at the chosen pH 8.5, delivering the best compromise for more sensitive signals and better peak shapes (i.e., less tailing and higher S/N).

As the EAs are medium to rather polar compounds, the run started with higher aqueous proportions in the mobile phase (5% B). An isocratic step of 1 min was included at the beginning, increasing the solvent B to 45% after 2.5 min and to 95% after 9 min. Another isocratic step was included for 3 min (9.0 min – 12.0 min) and then, a steeper increase of the organic concentration was applied resulting in a baseline separation of all 8 EAs in a total run time of 12 min and an equilibration time of 4 min.

During the analysis of naturally contaminated samples (FAPAS) with the transition m/z 576 à 223 for α-ergocryptine (Rt = 5.40 min) an additional peak was detected at Rt = 5.48 min. We ascribed this peak to the presence of β-ergocryptine. On the other hand, α- and β-ergocryptinine isomers (m/z 576 à 305) cannot be separated by traditional liquid chromatography columns, they are isobaric by nature and cannot be distinguished by mass spectrometry unless ion mobility mass spectrometry is employed (Krska and Crews 2008; Chang 2021).

Fig. 3 shows a chromatogram of this transition obtained from naturally contaminated rye flour, where, despite the close elution, there is a clear separation between α- and β-ergocryptine compounds, while the related isomers cannot be separated. Thus, the sum of α- and β-ergocryptinine is commonly monitored.

3.1.3 Optimization of the sample extraction and clean-up

The selection of the right extraction procedure is critical to obtain satisfactory recovery of EAs. The main problem is that solvent, pH, temperature but also light exposure could induce epimerization at the C-8 position of these alkaloids (Komarova and Tolkachev 2001; Hafner et al. 2008; Krska et al. 2008a; Schummer et al. 2020), leading to different relative intensities of the -ine and -inine compounds and thus an incorrect relative quantification and risk assessment of them. The parameters influencing the extraction efficiency, were optimized.

Here, three different extraction protocols were compared: (1) an alkaline mixture of acetonitrile – ammonium carbonate buffer 85:15 (v/v), (2) an acidic extraction with acetonitrile – water 84:16 (v/v) containing 0.5 % formic acid and (3) a neutral aqueous extraction with acetonitrile – water, 84:16 (v/v). The pH of the ammonium carbonate in the extraction solvent was investigated in the range pH 7.5 – 10, finding that an increase in the extraction solvent buffer pH increased the overall extraction efficiency. However, increasing the pH also increased the epimerisation rate, for this reason, a pH of 8.5 was established as the best candidate.

Although fast extraction is commonly the goal for any separation procedure, we decided to compare two different extraction times (5 and 20 min) for each matrix. 5 minutes extraction time didn’t allow proper recoveries for all the matrices tested, recoveries were very high, while, a combination of short vortex mixing 30s, mild shaking for 20 min and 5 min centrifugation at -4°C, was found to increase the efficiency of separation in our study.

In literature some of the methods exploited only SLE procedures leading to successful results, but co-extracted substances could induce matrix enhancement or suppression effects and shorten the lifetime of the column. For that reason, once EAs have been extracted by SLE, the aqueous supernatants of each different matrix, were purified through the dispersive SPE using C₁₈ sorbent as a rapid one-step clean-up.

Guo et al. (2016) compared the spiked recovery of EAs in rye using purification by dispersive SPE with C₁₈sorbent, PSA sorbent and MycoSep 150 cartridge ergot column supplied by Romer Laboratories. Amongst these purification procedures, C₁₈ cleaned the cereal matrix slightly better than the PSA sorbent and had also the best performance in terms of matrix effect.

Fig. (4 – 8) display the average extraction efficiency for each ergot alkaloid in every matrix tested, analysing the spiked samples at a medium-low level (20 µg/kg) in triplicates, using three different extraction solvents (green = neutral, red = acidic, blue = alkaline) and two different amounts of C₁₈ sorbents (150 mg and 300 mg).

For wheat samples (Fig. 4), the acidic and neutral extraction with the SLE and the dSPE with 150mg of C₁₈ sorbent, present acceptable recoveries (except for ergocornine), with an increasing trend for the epimers. Both extractions with the dSPE adding 300mg of C₁₈, showed worse recoveries.

With the alkaline extraction, the SLE procedure leads too higher recoveries for the epimers; the dSPE, with 150mg of C₁₈sorbent, cleans the extract and improves the trend but it still has high recovery values for the analytes ergosinine and a-ergocriptine (> 120%). The best performance in terms of reproducibility and stability was obtained adding 300mg of C₁₈sorbent, yielding recoveries between 74 to 102%.

For oat samples (Fig. 5), good results (70 – 120 %) were obtained for all three-extraction procedures (except for the neutral SLE for some analytes) and this represents the versatility of the matrix. However, the extraction procedure with the overall best performance in terms of signal abundance and highest recoveries is the alkaline extraction, without SLE, which still shows too high recoveries, while cleaning the sample with 300 mg of C₁₈sorbent yields recoveries between 80 to 120%.

Wheat gluten (Fig. 6) is a more complex matrix. The neutral procedure showed acceptable recoveries mainly with the SLE extraction, while cleaning the extract, recoveries decreased especially for the epimers. The acidic extraction with 300 mg of C₁₈sorbent yields good values and shows how the analytes suffer from ionic suppression caused by the matrix effect when the extract (SLE) is not cleaned.

Comparing the acidic with the alkaline extraction, recoveries are even higher with basic extraction (150 mg) of C₁₈sorbent, while when using 300 mg of C₁₈, the values dropped slightly, especially for ergocristine. In general, the alkaline extraction adding 150 mg of C₁₈led to greater overall recoveries, however, it is fair to point out that even an acid extraction with 300 mg C18 yielded good values, especially for ergosine and ergocristine.

For rye samples (Fig. 7), the neutral extraction with the SLE and the dSPE with 150 mg of C₁₈sorbent, presented higher recoveries compared to 300 mg, that cleaned the samples more by lowering the recoveries, especially for ergocornine. The acidic extraction is critical for the main EAs, while for ergocristine and α-ergocryptinine it yielded the highest recoveries (> 120%). With the alkaline extraction, the cleaning of the extract again showed better recoveries as can be observed from the differences between the SLE and the dSPE procedure. Comparing the latter’s performanceat 150 or 300 mg, the trend is quite similar, and both led to good results, which is why it was decided to continue with the 150mg approach (85 – 106 %) without increasing to 300mg.

For the baby food sample (Fig. 8), acid extraction did not perform very well, and of the three procedures tested, the one with 150 mg of C₁₈sorbent deliveredacceptable recoveries, but slightly above 70%. Similar but slightly higher results, especially for ergosine and its epimer, and for ergocristinine, were obtained with the neutral extraction with 150 mg of C₁₈sorbent. With alkaline extraction, the SLE procedure showed good recoveries, but the standard deviation trend was rather high, which is probably due to the multi-matrix composition (mixed rye, oat and wheat flour) of the baby food, which didn’t allow reproducibility in the measurement without cleaning the sample from the matrix. Comparing the performance of the C₁₈sorbent, in this case 150 mg, compared to 300 mg, seems to be sufficient for a good cleaning of the extract (as also observed in wheat gluten and rye) and produced recoveries between 73 and 96%.

In general, acceptable results were obtained with all three extraction solvents, also in terms of signal intensity, however, overall better recoveries for all the matrices tested, were achieved with the alkaline solution. This was also confirmed by Huybrechts (2021) when he compared a modified QuEChERS approach using an alkaline acetonitrile/water mixture with acidic aqueous methanol (MeOH/water/formic acid, 60/39/1, v/v/v). The alkaloids are deprotonated under alkaline conditions, and they tend to migrate more easily to the upper organic layer, leading the analytes to greater separation form the matrix and higher recoveries.

In addition, a cleaner extract with 300 mg of C₁₈ sorbent seemed to be the best solution for wheat and oat, while for more complex matrices, such as gluten, rye and baby food, a lower amount of C₁₈sorbent, 150 mg, showed excellent recoveries and good clean extracts.

3.2 Method Validation

3.2.1 Linearity and Matrix effect

Linearity was studied through matrix-matched calibration curves, spiked at nine concentration levels (see Paragraph 2.5.1), processed in duplicate and injected in triplicate. The linear range of the method was 0.5 – 500 µg/kg for all the different sample types. Good linear regression for all analytes was achieved, obtaining coefficient of determination (R²) values ≥ 0.997.

The precise quantitation of all ergot alkaloids in these complex matrices can be affected by matrix effects in ESI, responsible for enhancement or suppression of the ion abundance. Differences in the degree of matrix effects can be expected between different sample types as well as between the different extraction protocols. In cases where more than 20% signal suppression or enhancement is observed, ME need to be addressed in calibration (SANTE 2021).

The matrix effect here has been evaluated in a multi-matrix blended flour composed of rye, oat and wheat, extracted with the three different extraction protocols proposed (Table 2). As can be observed, with the acidic extraction (red) the majority of the analytes are affected by signal suppression except for three epimers ergosinine, ercocorninine and ergocristinine, for which slight ion enhancement is present. While with the neutral (green) and alkaline (blue) extraction, the analytes are affected by signal enhancement, and therefore matrix-matched calibration was used for quantitation purposes.

With the selected alkaline extraction, for all the matrices, the ME ranged between + 9.2 to +31.3.

Table 2 Matrix effect of 8 ergot alkaloids in a multi-matrix blended flour with three different extraction procedures and their relative coefficient of correlation, R² (linearity) values.

Analytes	ME % Neutral extraction	R²	ME % Acidic extraction	R²	ME % Alkaline extrcation	R²
ergosine	25.50	0.999	-6.8	0.997	23.3	0.997
ergocornine	9.89	0.999	-7.5	0.997	10.8	0.999
α-ergocriptine	15.38	0.999	-3.7	0.998	16.3	0.999
ergocristine	8.27	0.999	-5.5	0.997	9.2	0.999
ergosinine	21.47	0.999	17.2	0.999	28.9	0.999
ergocorninine	22.12	0.999	19.6	0.998	31.3	0.999
α-ergocriptinine	0.01	0.999	-0.7	0.998	9.3	0.999
ergocristinine	14.84	0.999	11.0	0.999	26.8	0.999

3.2.2 Recovery and precision

The recoveries of the analytes were measured by spiking blank samples (wheat, oat, wheat gluten, rye and baby food), previously checked for the presence of the target analytes, with EA standards at three different concentrations, 2, 20 and 100 µg/kg. Baby food samples was spiked at two concentrations (2 and 20 µg/kg) in line with the European Commission's MRL for ergot alkaloids of 20 µg/kg in processed cereal-based baby food (EU 2021). Samples were extracted, according to the procedure described above, with an alkaline extraction solvent combined with 300 mg of C₁₈sorbent for wheat and oat, and 150 mg of C₁₈sorbent for wheat gluten, rye and baby food. Raw data were processed using TargetLynx 4.2 software.

Results (Tables 3 - 7), expressed as mean percentage recovery of the three concentrations, showed good recovery values for all the analytes ranging from 70 to 105 % for wheat, 79 – 120% for oat, 70 – 118% for wheat gluten, 73 – 106% for rye and 71 – 96% for baby food. Overall, recovery rates in all validated matrices are in line with the recommendation (SANTE 2021) and in the range 70 – 120%.

The precision of the proposed method (expressed as percentage relative standard deviation, RSD %) was evaluated in terms of repeatability (intraday precision), assessed by performing six consecutive injections on the same day of the samples spiked at each concentration level tested. According to the CEN (2010) document and SANTE (2021) guideline, repeatability RSD_r should be ≤ 20% for each spike level tested. Satisfactory results were obtained at the concentration level evaluated, as the RSD_r were between 1 – 12% (Tables 3 - 7).

Table 3 Validation data for ergot alkaloids in wheat samples measured at three spiking levels (2, 20 and 100 µg/kg). Average recovery rates and repeatability measure (expressed as relative standard deviation, RSD_r) with n= 6 replicate each, LOD and LOQ.

Analyte	Level 2 µg/Kg		Level 20 µg/Kg		Level 100 µg/Kg		LOD µg/kg	LOQ µg/kg
Analyte	% Recovery	RSDr %	% Recovery	RSDr %	% Recovery	RSDr %	LOD µg/kg	LOQ µg/kg
ergosine	80	3.7	86	4.0	71	3.1	0.04	0.1
ergocornine	90	3.5	94	3.9	80	2.7	0.03	0.1
α - ergocriptine	85	3.7	92	3.2	83	2.4	0.03	0.1
ergocristine	70	4.9	74	4.7	86	4.9	0.07	0.2
ergosinine	82	3.8	99	3.8	91	2.8	0.03	0.1
ergocorninine	82	3.5	95	2.6	81	3.4	0.03	0.1
α-ergocryptinine	105	3.0	102	2.8	79	2.9	0.03	0.1
ergocristinine	82	3.9	93	4.0	87	4.5	0.04	0.1

Table 4 Validation data for ergot alkaloids in oat sample measured at three spiking levels (2, 20 and 100 µg/kg). Average recovery rates and repeatability measure (expressed as relative standard deviation, RSD_r) with n= 6 replicate each, LOD and LOQ.

Analyte	Level 2 µg/Kg		Level 20 µg/Kg		Level 100 µg/Kg		LOD µg/kg	LOQ µg/kg
Analyte	% Recovery	RSDr %	% Recovery	RSDr %	% Recovery	RSDr %	LOD µg/kg	LOQ µg/kg
ergosine	96	3.4	101	3.5	102	3.9	0.04	0.1
ergocornine	88	6.1	119	4.7	109	3.8	0.03	0.1
α - ergocriptine	87	5.4	108	4.2	106	4.1	0.03	0.1
ergocristine	88	7.2	97	6.1	82	6.4	0.07	0.2
ergosinine	93	4.3	114	4.8	97	4.3	0.03	0.1
ergocorninine	79	4.0	117	5.2	115	4.6	0.03	0.1
α-ergocryptinine	93	5.7	120	4.7	118	5.2	0.03	0.1
ergocristinine	85	4.6	80	5.1	93	5.3	0.04	0.1

Table 5 Validation data for ergot alkaloids in wheat gluten sample measured at three spiking levels (2, 20 and 100 µg/kg). Average recovery rates and repeatability measure (expressed as relative standard deviation, RSD_r) with n= 6 replicate each, LOD and LOQ.

Analyte	Level 2 µg/Kg		Level 20 µg/Kg		Level 100 µg/Kg		LOD µg/kg	LOQ µg/kg
Analyte	% Recovery	RSDr %	% Recovery	RSDr %	% Recovery	RSDr %	LOD µg/kg	LOQ µg/kg
ergosine	115	4.7	83	3.0	118	3.1	0.04	0.1
ergocornine	84	4.5	92	3.9	90	2.7	0.03	0.1
α-ergocryptine	81	4.7	90	3.2	98	2.4	0.03	0.1
ergocristine	70	4.9	73	3.7	87	2.9	0.07	0.2
ergosinine	90	4.8	97	4.8	108	2.8	0.03	0.1
ergocorninine	67	4.5	93	3.6	78	3.4	0.03	0.1
α-ergocryptinine	93	4.0	99	3.8	89	2.9	0.03	0.1
ergocristinine	79	4.9	91	4.0	83	4.5	0.04	0.1

Table 6 Validation data for ergot alkaloids in rye sample measured at three spiking levels (2, 20 and 100 µg/kg). Average recovery rates and repeatability measure (expressed as relative standard deviation, RSD_r) with n= 6 replicate each, LOD and LOQ.

Analyte	Level 2 µg/Kg		Level 20 µg/Kg		Level 100 µg/Kg		LOD µg/kg	LOQ µg/kg
Analyte	% Recovery	RSDr %	% Recovery	RSDr %	% Recovery	RSDr %	LOD µg/kg	LOQ µg/kg
ergosine	74	5.1	91	2.1	84	11.3	0.04	0.1
ergocornine	73	3.6	88	9.9	82	5.8	0.03	0.1
α-ergocryptine	76	8.7	105	4.4	99	12.4	0.03	0.1
ergocristine	74	11.9	85	10.2	87	10.7	0.07	0.2
ergosinine	73	8.9	102	4.8	89	11.5	0.03	0.1
ergocorninine	79	12.0	106	6.0	97	9.8	0.03	0.1
α-ergocryptinine	73	11.9	87	10.2	91	10.7	0.03	0.1
ergocristinine	80	13	88	6.3	94	10.9	0.04	0.1

Table 7 Validation data (mean recoveries and relative standard deviation) for ergot alkaloids in baby food sample measured at two spiking levels (2 and 20 µg/kg). Average recovery rates and repeatability measure (expressed as relative standard deviation, RSD_r) with n= 6 replicate each, LOD and LOQ.

Analyte	Level 2 µg/Kg		Level 20 µg/Kg		LOD µg/kg	LOQ µg/kg
Analyte	% Recovery	RSDr %	% Recovery	RSDr %	LOD µg/kg	LOQ µg/kg
ergosine	80	5.6	91	3.2	0.04	0.1
ergocornine	78	4.0	85	3.6	0.03	0.1
α-ergocryptine	87	3.4	96	4.2	0.03	0.1
ergocristine	71	6.3	73	5.4	0.07	0.2
ergosinine	77	4.4	88	3.6	0.03	0.1
ergocorninine	79	4.1	94	3.8	0.03	0.1
α-ergocryptinine	83	3.4	96	3.6	0.03	0.1
ergocristinine	86	5.5	79	5.7	0.04	0.1

3.2.3 LOD and LOQ

Assessed limits of detection (LOD) ranged from 0.03 to 0.07 μg kg^-1and estimated limits of quantification (LOQ) were 0.1 μg kg^-1, except for ergocristine, at 0.2 μg kg^-1(Tables 3 – 7). The low LOQ values were additionally confirmed with the recovery and repeatability values obtained from the lowest tested level of the calibration curve, 0.5 ng/mL.

The results presented here show that the method is very sensitive and yields favourable performance characteristics. It is fast and simple, without needing the time consuming SPE approach exploiting only dispersive sorbent and it also enables results in compliance with the new values proposed by the European Commission that will come into force 1.07.2024 (EU 2021). Table 8 represents a comparison with other method presented in the literature.

Table 8 Comparison of the performance characteristics of the proposed method with other published methods in literature.

Sample	Extraction + clean-up	Analytical technique	LOQ (μg/kg)	Reference
Rye	ACN:2mM (NH₄)₂CO₃(84:16, v/v) + neutral alumina based	LC-IT-MS/MS	1.0 – 3.0	Bryla et al. 2015
Rye flour, wheat flour, bread and noodles	ACN:(NH₄)₂CO₃(85:15, v/v) + C₁₈ sorbent	UPLC-MS/MS	0.2 – 0.5	Guo et al. 2016
Bread samples (wheat, multi-grain, rye and wheat-rye)	MeOH:H₂O:CH₂O₂ (60:40:0.4, v/v/v)	LC-MS/MS	0.3 – 1.2	Versilovskiks et al. 2020
Wheat Barley	ACN:(NH₄)₂CO₃(85:15, v/v) + C₁₈/Z-SEP+	UHPLC-MS/MS	0.49 – 3.33 0.50 – 3.92	Carbonell-Rozas 2021
Oat	ACN:(NH₄)₂CO₃(85:15, v/v) + C₁₈/Z-SEP+	UHPLC-MS/MS	0.2 – 3.2	Carbonell-Rozas 2022
Baby food (breakfast cereals and cookies)	MeOH:H₂O:CH₂O₂ (60:40:0.4, v/v/v)	LC-MS/MS	4	Mulder et al. 2015
Dry cereal-based baby food	ACN:3mM (NH₄)₂CO₃(84:16, v/v) + 6g MgSO4/1.5 g NaCl	UHPLC-MS/MS	0.1 – 0.3	Huybrechts et al. 2021
Oat, Barley, Wheat	H2O:ACN + 5% FA + 4 g MgSO4 and 1 g NaCl	HPLC-MS/MS	0.16 – 0.36 0.10 – 0.39 0.19 – 0.36	Kim et al. 2022
Wheat, oat, rye, wheat gluten and baby food	ACN:3mM (NH₄)₂CO₃(84:16, v/v) + C₁₈ sorbent	UPLC-MS/MS	0.1-0.2	Present study

3.2.4 Method’s trueness

To evaluate the trueness of the method, two proficiency test samples FAPAS (ergot alkaloids in rye flour, PT 22190 and PT22203) were purchased, extracted with the 3 different extraction procedures, and the z-scores were calculated for the EA compounds (Tables 9 – 10). Here, we sum the results of α-ergocryptine and β-ergocryptine to meet the request of the FAPAS test. Z scores between +2 and −2 are considered a satisfactory performance, Z scores between +2 and +3 or between −2 and −3 are considered questionable performance, and anything outside this range (> +3 or < −3) are considered unsatisfactory. In our analysis, the diverse extraction procedures delivered satisfactory results, except for ergocristine (Table 10), which had a questionable performance with the dispersive SLE (300 mg) approach. In general, better performances were obtained with the alkaline extraction with 150 mg of C₁₈sorbent, as confirmed also in the Paragraph 3.1.3, optimization of sample extraction and clean-up.

Table 9 Z -scores of ergot alkaloids in rye flour with the three extraction protocols from the 2023 FAPAS proficiency test.

Analyte	SLE 84:16	84:16 + 150 mg C18	84:16 + 300 mg C18	SLE 84:16 + 05FA%	84:16 + 05FA% + 150 mg C18	84:16 + 05FA% + 300 mg C18	SLE 85:15	85:15 + 150 mg C18	85:15 + 300 mg C18
Ergosine	0.9	0.2	0.4	-0.9	-0.3	-0.9	-0.5	-0.3	-1.0
ergocornine	-0.3	-0.8	-1.1	-0.6	-0.5	-0.2	-0.3	0.0	-1.4
α+β ergocryptine (sum)	0.1	-0.5	-0.4	0.9	0.7	0.5	1.1	0.6	0.1
ergocristine	1.3	0.7	1.5	-1.2	+1.3	+1.4	1.2	0.1	-1.5
ergosinine	-1.0	-1.5	-1.4	-1.2	-0.5	-1.9	-1.3	-0.8	-1.6
ergocorninine	-1.6	-0.7	-1.7	-1.6	-1.5	-1.2	-1.4	-1.0	-1.5
α+β ergocryptinine	-1.1	-1.5	-1.8	-0.9	-0.9	-1.0	-0.6	-0.8	-0.5
ergocristinine	-0.5	-1.1	-1.5	-0.5	-0.3	-1.3	-0.3	-0.3	-0.2

Table 10 Z -scores of ergot alkaloids in rye flour with the three extraction protocols from the 2022 FAPAS proficiency test.

Analyte	SLE 84:16	84:16 + 150 mg C18	84:16 + 300 mg C18	SLE 84:16 + 05FA%	84:16 + 05FA% + 150 mg C18	84:16 + 05FA% + 300 mg C18	SLE 85:15	85:15 + 150 mg C18	85:15 + 300 mg C18
Ergosine	-0.6	-1.2	-1.7	-0.9	-0.2	0.2	-0.1	-0.4	-1.3
ergocornine	1.6	-0.2	-1.8	-1.0	-1.2	-1.6	0.1	0.8	-0.4
α+β ergocryptine (sum)	-0.5	-0.5	-1.0	-0.4	-0.2	-0.5	1.0	0.5	-0.9
ergocristine	1.2	-0.5	-2.7	1.0	1.1	+2.4	1.1	-0.1	-2.5
ergosinine	-0.2	-0.2	-0.3	-1.0	-0.8	-0.7	-0.2	-0.1	-0.7
ergocorninine	-0.4	-0.4	-0.5	-0.6	-0.5	-0.6	-0.4	-0.2	-0.6
α+β ergocryptinine	-1.5	-1.7	-2.1	-1.9	-1.2	-1.5	-0.7	-0.6	-1.6
ergocristinine	-0.9	-1.3	-1.2	-0.2	0.2	-0.4	-1.2	-0.3	-0.4

In addition to the PT samples, a baby food quality control (QC) material (FAPAS) was analyzed, with the three extraction protocols presented above. The results are summarized in the Table 11.

In totally, 5 -ine and -inine ergot alkaloids were present in the sample. Ergotamine and its epimer are present in the QC material but they are not quantified in our study, but to calculate the total EAs content we have considered their value, for all three extraction solutions, as the optimal value given by the test sample (21.8 µg/kg for ergotamine and 5 µg/kg for ergotaminine).

In this case, the acidic extraction yields poor results for ergosine and ergocornine and quantify a total number of ergot alkaloids below the range proposed by the QC. The neutral extraction presents better results for ergosine but lower results for ergocornine. In this case, the total amount of EAs obtained with SLE is centred in the proposed range but, as the sample is not being cleaned, it may have a higher value due to the ME. In fact, we see a lower result when the sample is cleaned by the dispersive C₁₈clean-up. With the alkaline extraction, the SLE procedure delivers good results, but as the sample is not being cleaned, the performance of 150 mg of C₁₈sorbent seems to be sufficient for cleaning the extract and yields suitable values within the proposed range.

In conclusion, the achievement of the required z-scores benchmark was confirmation of the trueness of the final method.

Table 11 Comparison between values for ergot alkaloids in FAPAS QC baby food cereals and values obtained with the three extraction protocols tested.

Analyte	Assigned value µg/kg	^aRange for z ≤ 2	SLE 85:15	85:15 + 150 mg C18	85:15 + 300 mg C18	SLE 84:16	84:16 + 150 mg C18	84:16 + 300 mg C18	SLE 84:16 + 05FA%	84:16 + 05 FA% + 150 mg C18	84:16 + 05 FA% + 300 mg C18
Ergosine	8.9	4.98 - 12.82	7.9	7.8	6.5	7.7	7.0	6.0	0.1	0.4	2.8
ergocornine	14.4	8.0 - 20.7	15.3	15.6	15.7	6.6	5.2	4.3	9.8	9.0	6.0
α+β ergocryptine (sum)	21.7	12.1 - 31.2	25.7	24.2	23.5	18.0	16.0	13.4	19.0	18.1	13.3
ergocristine	//		34.1	36.7	34.6	50.0	46.0	42.0	26.4	26.4	24.5
ergosinine	//		0.9	0.5	0.4	0.9	0.7	0.5	<	<	<
ergocorninine	//		5.2	4.5	3.2	0.7	0.3	0.2	1.8	1.7	0.9
α+β ergocryptinine	//		5.6	6.6	5.7	3.4	3.0	2.6	3.6	3.7	2.5
ergocristinine	//		5.8	6.8	4.9	8.0	6.6	6.0	6.0	5.9	4.6
ergotamine	21.8		21.8	21.8	21.8	21.8	21.8	21.8	21.8	21.8	21.8
ergotaminine	5		5.0	5.0	5.0	5.0	5.0	5.0	5.0	5.0	5.0
total	161	93 - 228	127	130	121	122	112	102	93	91	79

^aThe range for |z| ≤ 2 is the concentration range within the limits of ±2 z scores. The assigned value and its range has been established from the proficiency test data and are suitable for use by laboratories as a fit-for-purpose quality control measure.

3.3 Method’s applicability: occurrence of ergot alkaloids

To evaluate the applicability of the validated method, a set of 54 samples (from 2020 to 2022) covering some validated matrices and other cereals like spelt, tritordeum and triticale, were analysed to monitor the natural occurrence of EAs. Here, we report only the positive cereals tested and results showed that 27 of the 54 samples contain EA compounds, some of which are above the legal limit (Table 12).

For tritordeum and triticale, no regulatory limit for EAs content has been set, so we have decided 'by convention' to consider them to be like other cereals, which have a maximum legal limit of 100 μg/kg that it will become of 50 μg/kg from 1^st July 2024.

Rye samples from 2020 are highly contaminated, with one sample that largely exceed the maximum legal limit (1302 μg/kg) (EU 2021). In the same year, also common wheat, spelt and tritordeum (Fig. 9) present EAs above the MRLs, especially if we consider the limit that will come into force from 1 July 2024.

Considering this small survey, rye was confirmed to be the crop more susceptible to the fungal infection (EAs content up to 1302 μg/kg) but also tritordeum, in the following two years, presents high values.

Ergocryptine and ergocristine (sometimes also ergosine) compounds are the toxins with the highest occurrence in the cereals tested. These results are in line with other previous studies that also reported they predominance in the literature (Malysheva 2014; Agriopoulou 2021). Therefore, it is extremely important to monitor their presence in these matrices due to the potential health risk that they can cause and try to establish maximum levels for them in order to guarantee the safety of the consumers.

To conclude, the developed analytical method was found to be applicable to a wide variety of different cereals, including tritordeum, triticale and spelt, highlighting the possibility of its use in different cereal chains, in addition to those tested.

Table 12 Occurrence of ergot alkaloids in different cereal samples and the total amount per sample.

Cereals	Years	Analytes (μg/kg)								Total (μg/kg)
Cereals	Years	ergosine	ergocornine	ergocryptine	ergocristine	ergosinine	ergocorninine	ergocryptinine	ergocristinine	Total (μg/kg)
Rye	2020	53.8	35.8	63.8	33.5	18.3	6.5	15.5	8.6	236
Rye	2020	185.3	133.0	551.5	136.0	85.9	36.6	138.3	35.5	1302
Common Wheat	2020	3.3	3.6	5.3	55.8	3.7	4.3	3.2	14.7	94
Common Wheat	2020	72.1	5.6	6.3	125.3	36.4	4.9	6.8	21.2	279
Common Wheat	2020	8.5	3.8	3.8	32.0	4.8	3.8	3.2	4.3	64
Common Wheat	2020	5.3	4.5	3.5	4.6	2.8	3.2	3.8	2.5	30
Durum Wheat	2020	5.3	< LOQ	1.9	5.3	3.6	< LOQ	< LOQ	3.3	19
Durum Wheat	2020	3.6	< LOQ	1.5	3.3	1.9	< LOQ	< LOQ	1.8	12
Spelt	2020	5.1	< LOQ	3.9	131.9	3.6	< LOQ	< LOQ	36.8	181
Tritordeum	2020	33.3	13.6	15.3	66.8	15.9	6.3	8.8	33.5	194
Tritordeum	2020	8.5	13.3	30.5	< LOQ	6.6	6.9	16.8	< LOQ	83
Tritordeum	2020	11.9	3.8	5.8	56.3	8.3	3.1	3.8	18.5	112
Tritordeum	2020	58.5	58.5	83.5	50.6	35.3	15.3	31.8	8.5	342
Rye	2021	36.1	3.8	3.8	139.3	10.9	3.5	5.3	15.6	218
Rye	2021	3.1	5.0	3.0	8.3	3.5	3.6	5.3	3.1	35
Rye	2021	8.0	55.9	33.0	3.0	3.9	8.8	9.6	3.3	126
Common wheat	2021	3.8	3.0	3.3	3.1	< LOQ	< LOQ	< LOQ	0.1	13
Tritordeum	2021	31.6	3.0	3.5	86.9	15.0	3.3	3.3	33.3	180
Tritordeum	2021	3.6	3.6	3.5	6.8	3.3	3.1	3.3	3.5	31
Triticale	2021	3.9	3.8	3.6	3.3	3.5	3.3	3.3	0.8	26
Triticale	2021	5.1	< LOQ	1.8	5.1	3.8	< LOQ	0.1	0.9	17
Rye	2022	5.5	18.8	15.3	10.6	6.5	8.6	8.5	5.3	79
Rye	2022	3.5	5.6	5.9	5.3	5.9	3.1	3.3	3.0	36
Rye	2022	15.6	3.3	3.0	55.1	16.8	3.6	3.6	18.5	120
Spelt	2022	3.8	3.0	1.6	< LOQ	1.5	3.3	1.5	< LOQ	15
Tritordeum	2022	15.1	3.1	3.9	109.8	13.0	3.6	3.6	38.5	191
Tritordeum	2022	3.1	6.5	6.8	3.3	5.8	5.3	6.3	1.8	39

3.2 Conclusion

The present study developed and validated an accurate and detailed UPLC-MS/MS method for the quantification of ergot alkaloids (-ine and -inine isomers) in five different matrices, wheat, oat, durum wheat, rye and baby food.

The MRLs in the European Union are based on the sum of the –ine and –inine form, implying that epimerisation is not so impactful. However, in our method, these compounds were analysed individually, and it was chosen to quantify only those ergot alkaloids that are now available on the market without any legislative permission, to guarantee the applicability of the method to all entities involved in monitoring and quantification. The critical parameters that could induce epimerization (solvent, pH, temperature and light) were evaluated and the conditions chosen for both the preparation of standards and the extraction, i.e., alkaline conditions combined with acetonitrile, minimizing epimerization as much as possible. Data presented on extraction performance of various protocols gave valuable information for future applications and method developments. Dispersive SPE, using C₁₈ sorbent as clean-up step, alleviated the problem of matrix effect. It allowed proper recoveries addressing the needs of the industry in each category of matrix tested, especially for cereal-based food for infants in which the legal limit is set to 20 µg/kg. Moreover, the method enabled results in compliance with the new values proposed by the European Commission that will come into force from 1^st July 2024.

Satisfactory results from participation in the proficiency tests and analysis of a quality control material, confirmed that this method was fit for this purpose. Applicability was confirmed by analysing a set of several samples covering some validated matrices and other cereals like spelt, tritordeum and triticale. Here, results showed that 27 of the 54 samples contain EA compounds, some of which are above the legal limits.

To conclude, in the future, more data focusing on the behaviour of ergot alkaloids during processing and/or storage with the possible formation of modified or transformation products, will be needed for a complete and reliable risk assessment.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict of interest.

Funding

The present research was supported by the European Union funded Project: NextGenerationEU - National Resistance and Resilience Plan (PNRR) - Mission 4 Component 2 Investment 1.3 - Notice No. 341 of 15 March 2022 of the Ministry of Universities and Research, with the following detailed coordinates:

- Project Code PE00000003, MUR Directive Decree No. 1550 of 11 October 2022 granting of funding, CUP D93C22000890001;

- Project Title "ON Foods - Research and innovation network on nutrition and nutrition Sustainability, Safety - Working ON Foods".

Author Contribution

E. Rollo: Conceptualization, Investigation, Methodology, Formal Analysis, Writing; D. Catellani: Methodology; C. Dall’Asta: Supervision, Funding acquisition, Project administration; N. Dreolin: Methodology, Conceptualization, Review & editing; M. Suman: Supervision, Conceptualization, Review & editing, Project administration.

Acknowledgement

The authors would like to thanks Dr. Peter Hancock (Waters Corporation, UK) for his valuable suggestions within the reviewing phase.

Agriopoulou S (2021) Ergot alkaloids mycotoxins in cereals and cerealderived food products: Characteristics, toxicity, prevalence, and control strategies. Agronomy 11:931. https://doi.org/10.3390/agron omy11050931
Bryła, M.; Szymczyk, K.; J˛edrzejczak, R.; Roszko, M. (2015) Application of Liquid Chromatography/Ion Trap Mass Spectrometry Technique to Determine Ergot Alkaloids in Grain Products. Food Technol. Biotechnol. 53, 18–28. https://doi.org/10.17113/ftb.53.01.15.3770
Carbonell-Rozas L, Mahdjoubi CK, Arroyo-Manzanares N et al (2021) Occurrence of ergot alkaloids in barley and wheat from Algeria. Toxins 13:316. https://doi.org/10.3390/TOXINS13050316
Carbonell-Rozas L, Hernandez-Mesa M, Righetti L et al (2022) Ion mobility-mass spectrometry to extend analytical performance in the determination of ergot alkaloids in cereal samples. J Chromatogr A 1682:463502. https://doi.org/10.1016/j.chroma.2022.463502
Cherewyk JE, Grusie-Ogilvie TJ, Parker SE, Blakley BR, Al-Dissi AN (2023b) The impact of storage temperature and time on ergot alkaloid concentrations. Toxins 15:497. https://doi.org/10.3390/toxins15080497
Cherewyk J, Grusie-Ogilvie T, Blakley B, Al-Dissi A (2022a) Validation of a new sensitive method for the detection and quantification of R and S-Epimers of ergot alkaloids in Canadian spring wheat utilizing deuterated lysergic acid diethylamide as an internal standard. Toxins 14:22. https://doi.org/10.3390/TOXINS14010022
Cherewyk J, Grusie-Ogilvie T, Blakley B, Al-Dissi A (2024) The C‑8‑S‑isomers of ergot alkaloids — a review of biological and analytical aspects. Mycotoxin Research. 40:1–17. https://doi.org/10.1007/s12550-023-00507-0
Chung SWC (2021) A critical review of analytical methods for ergot alkaloids in cereals and feed and in particular suitability of method performance for regulatory monitoring and epimerspecific quantification. Food Addit Contamim A 38:997–1012. https://doi.org/10.1080/19440 049.2021.1898679
Commission E (2021) Commission Regulation 2021/1399 of 24 August 2021 amending Regulation (EC) No 1881/2006 as regards maximum levels of ergot sclerotia and ergot alkaloids in certain foodstuffs. Off J Eur Communities 301:1–5
Crews C (2015) Analysis of ergot alkaloids. Toxins 7:2024–2050. https://doi.org/10.3390/toxins7062024
European Food Safety Authority (EFSA). TDI. Glossary. https://www.efsa.europa. eu/ en/ gloss ary/ tdi#: ~: text= The% 20tol erable% 20dai ly% 20int ake% 20(TDI,an% 20app recia ble% 20risk% 20to% 20hea lth. Accessed 1 Jun 2023
European Food Safety Authority (EFSA) (2012) Scientific opinion on ergot alkaloids in food and feed. EFSA J 10:1–158. http://doi.wiley.com/10.2903/j.efsa.2012.2798 Accessed 12 Jun 2023
European Food Safety Authority (EFSA) (2005) Opinion on ergot as undesirable substance in animal feed. EFSA J 225:1–27. http://www.efsa.eu.int/science/contam/contam_ opinions/941/contamop_ ej225_ergot_en1.pdf . Accessed 12 Jun 2023
Grusie T, Cowan V, Singh J et al (2017) Correlation and variability between weighing, counting and analytical methods to determine ergot (Claviceps purpurea) contamination of grain. World Mycotoxin J 10:209–218. https://doi.org/10.3920/WMJ2016.2174
Guo Q, Shao B, Du Z, Zhang J (2016) Simultaneous determination of 25 ergot alkaloids in cereal samples by ultraperformance liquid chromatography−tandem mass spectrometry. J Agric Food Chem 64:7033–7039. https://doi.org/10.1021/acs.jafc.6b02484
Hafner M, Sulyok M, Schuhmacher R et al (2008) Stability and epimerisation behaviour of ergot alkaloids in various solvents. World Mycotoxin J 1:67–78. https://doi.org/10.3920/
Huybrechts, B.; Malysheva, S.V.; Masquelier, J. A Targeted (2021) UHPLC-MS/MS Method Validated for the Quantification of Ergot Alkaloids in Cereal-Based Baby Food from the Belgian Market. Toxins, 13, 531. https://doi.org/10.3390/toxins13080531
Komarova EL, Tolkachev ON (2001) The chemistry of peptide ergot alkaloids. Part 1. Classification and chemistry of ergot peptides. Pharm Chem J 35:504–513. https://doi.org/10.1023/A:1014050926916
Kodisch A, Oberforster M, Raditschnig A et al (2020) Covariation of ergot severity and alkaloid content measured by HPLC and One ELISA method in inoculated winter rye across three isolates and three European countries. Toxins 12:676. https://doi.org/10.3390/toxins12110676
Krska R, Berthiller F, Schuhmacher R et al (2008a) Determination of ergot alkaloids: purity and stability assessment of standards and optimization of extraction conditions for cereal samples. AOAC Int 91:1363–1371 https://doi.org/10.1093/jaoac/91.6.1363
Krska R, Crews C (2008) Significance, chemistry and determination of ergot alkaloids: a review. Food Addit Contam 25:722–731. https://doi.org/10.1080/02652030701765756
Krska R, Stubbings G, MacArthur R, Crews C (2008b) Simultaneous determination of six major ergot alkaloids and their epimers in cereals and foodstuffs by LC-MS-MS. Anal Bioanal Chem 391:563–576. https://doi.org/10.1007/s00216-008-2036-6
Liang H, York J, Konschnik J, Majer H, Steimling J (2022) Comprehensive mycotoxin analysis: simultaneous determination of Alternaria toxins, ergot alkaloid epimers, and other major mycotoxins in various food matrices by LC-MS/MS, Restek Corporation https://www.restek.com/global/it/articles/multi-mycotoxin-method-for-alternaria-toxins-ergot-alkaloid-epimers-and-other-major-mycotoxins-in-food
Liao, C.D.;Wong, J.W.; Zhang, K.; Hayward, D.G.; Lee, N.S.; Trucksess, M.W. (2013) Multi-Mycotoxin Analysis of Finished Grain and Nut Products Using High-Performance Liquid Chromatography-Triple-Quadrupole Mass Spectrometry. J. Agric. Food Chem. 61, 4771–4782 https://doi.org/10.1021/jf4000677
Malysheva SV, Diana Di Mavungu J, Goryacheva IY, De Saeger S (2013) A systematic assessment of the variability of matrix effects in LC-MS/MS analysis of ergot alkaloids in cereals and evaluation of method robustness. Anal Bioanal Chem 405:5595– 5604. https://doi.org/10.1007/s00216-013-6948-4
Mulder PPJ, Pereboom-de Fauw DPKH, Hoogenboom RLAP et al (2015) Tropane and ergot alkaloids in grain-based products for infants and young children in the Netherlands in 2011–2014. Food Addit Contam Part B 8:284–290. https://doi.org/10.1080/19393210.2015.1089947
Muller C, Kemmlein S, Klaffke H, Krauthause W, Preiss-Weigert A, Wittkowski R. (2009). A basic tool for risk assessment: a new method for the analysis of ergot alkaloids in rye and selected rye products. Mol Nutr Food Res. 53:500–507. https://doi.org/10.1002/mnfr.200800091
Rubert, J.; Dzuman, Z.; Vaclavikova, M.; Zachariasova, M.; Soler, C.; Hajslova, J. (2021) Analysis of Mycotoxins in Barley Using Ultra High Liquid Chromatography High Resolution Mass Spectrometry: Comparison of Efficiency and Efficacy of Different Extraction Procedures. Talanta, 99, 712–719. https://doi.org/10.1016/j.talanta.2012.07.010
Ruhland M, Tischler J (2008) Determination of ergot alkaloids in feed by HPLC. Mycotoxin Res 24:73–79. https://doi.org/10.1007/BF02985284
[SANTE] Directorate General for Health and Food Safety. SANTE/11312/2021. Guidance Document on Analytical Quality Control and Method Validation Procedures for Pesticide Residues and Analysis in Food and Feed. Brussels (Belgium). Accessed 2022 Jan 1.
Silva, Â.; Mateus, A.R.S.; Barros, S.C.; Silva, A.S (2023) Ergot Alkaloids on Cereals and Seeds: Analytical Methods, Occurrence, and Future Perspectives. Molecules, 28, 7233. https://doi.org/10.3390/molecules28207233
Schummer C, Zandonella I, van Nieuwenhuyse A, Moris G (2020) Epimerization of ergot alkaloids in feed. Heliyon 6:e04336. https://doi.org/10.1016/j.heliyon.2020.e04336
Standard Recommendation S.R. CEN/TR 16059:2010 (2010) Food Analysis - Performance Criteria for Single Laboratory Validated Methods of Analysis for the Determination of Mycotoxins. ICS 07.100.30. European Committee for Standardization.
Veršilovskis A, Mulder PPJ, Pereboom-de Fauw DPKH et al (2020) Simultaneous quantification of ergot and tropane alkaloids in bread in the Netherlands by LC-MS/MS. Food Addit Contam Part B 13:215–223. https://doi.org/10.1080/19393210.2020.1771777
Young JC, Chen ZJ, Marquardt RR (1983) Reduction in alkaloid content of ergot sclerotia by chemical and physical treatment. J Agric Food Chem 31:413–415. https://doi.org/10.1021/jf00116a057

No competing interests reported.

Download PDF

Editorial decision: Revision requested
30 Jul, 2024
Reviews received at journal
30 Jul, 2024
Reviews received at journal
25 Jul, 2024
Reviewers agreed at journal
11 Jul, 2024
Reviewers agreed at journal
10 Jul, 2024
Reviewers invited by journal
10 Jul, 2024
Editor assigned by journal
08 Jul, 2024
Submission checks completed at journal
08 Jul, 2024
First submitted to journal
27 Jun, 2024

You are reading this latest preprint version

Determination of Ergot Alkaloids: Comparison of extraction efficiencies in several cereal production chains by UPLC-MS/MS

Status:

Version 1

Abstract

Figures

1. introduction

2. Materials and methods

2.1. Reagents, standards and materials

2.2. Extraction solvents

2.3. Samples

2.4. Sample preparation procedure and clean-up

2.4.2. Wheat Gluten, Rye and baby food

2.5. Standard preparation for quantification

2.6. Epimerization of EAs during preparation

2.7. UPLC – MS/MS analysis

2.8. Data Management

2.9 Method Validation

2.10 Method applicability

3 Results and discussion

3.1 Method Development

3.1.1 MS optimization

3.1.2 Optimization of the chromatographic separation

3.1.3 Optimization of the sample extraction and clean-up

3.2 Method Validation

3.2.1 Linearity and Matrix effect

3.2.2 Recovery and precision

3.2.3 LOD and LOQ

3.2.4 Method’s trueness

3.3 Method’s applicability: occurrence of ergot alkaloids

3.2 Conclusion

Declarations

Compliance with ethical standards

Funding

Author Contribution

Acknowledgement

References

Additional Declarations

Status:

Version 1