*2.3. Occurrence of EAs in Swine and Dairy Feeds*

The method derived from this study was applied to explore the 11 ergot alkaloids in 200 feed samples consisting of swine (*n* = 100) and dairy feeds (*n* = 100). In the swine feed samples, more than 49% were contaminated with ergot alkaloids, especially ergocryptine (-ine) (40%), ergosine (-ine), and ergotamine (-ine) (37%). However, more than 50% of total samples were not detectable in 11 ergot alkaloids in the swine feed sample. The dairy feed samples had the same prevalent contaminants as the swine feed samples but with higher positive samples, especially for ergocryptine (-ine) (50%), ergosine (-ine) (48%), ergotamine (-ine), and ergocristine (-ine) (49%), as shown in Tables 4 and 5. The mycotoxin levels in all feed samples almost complied with the EU regulation (≤1000 mg/kg of 11 ergot alkaloids) [28]. There are several reports on the presence of EAs in the feed from different countries, with 86–100% of listed EAs detected in feed samples from Germany [1] and 83% of compound feeds containing EAs with an average concentration of 89 μg/kg and a maximum concentration of 1231 μg/kg in the Netherlands [19]. The major detected EAs were ergosine, ergotamine, ergocristine, and ergocryptine. Interestingly, Malysheva et al. [13] reported the occurrence of EAs over three years in 1065 cereal samples originating from 13 European countries, with 52% of rye, 27% of wheat, and 44% of total samples containing EAs (ergosine, ergocristine, and ergocryptine) ranging from less than 1 to 12,340 μg/kg. In Spain, the concentrations for individual ergot alkaloids ranged between 5.9 μg/kg for ergosinine to 145.3 μg/kg for ergometrine, while the total ergot alkaloid content ranged from 5.9 to 158.7 μg/kg in swine samples. About 12.7% revealed contamination by at least one ergot alkaloid, and among contaminated swine samples, 65% were contaminated by more than one [22].

The ergot contaminations and patterns were differences due to the geographical region and environmental conditions [10].


**Table 4.** Occurrence of 11 ergot alkaloids in swine feed samples.

**Table 5.** Occurrence of 11 ergot alkaloids in dairy feed samples.


#### **3. Conclusions**

EAs are hazardous mycotoxins in food and feed samples. Our results showed that the LC-ESI-MS/MS technique was an excellent tool for untargeted determination of 11 EAs in swine and dairy feed samples. The validated LC-MS/MS method using a multifunctional column was successfully performed according to the SANTE/11813/2017 standard. LODs and LOQs were recorded as 0.25 and 0.5 ng/g for EAs. Recoveries were 90.6–120%. When this technique was applied to real feed samples, it showed that 11 EAs were quantifiable in animal feeds. The mycotoxin levels in the swine and dairy samples almost complied with the EU regulations. The presence of ergot sclerotia is regulated to a maximum of 500 mg/kg in unprocessed cereal for humans [29] and 1000 mg/kg in feed materials and compound feed containing unground cereals [30]. However, further studies with a larger sample size are needed to confirm these as acceptable levels. The knowledge of toxigenic *Claviceps* species for better understanding of the production of EAs and to progress appropriate solutions for disease management should be investigated.

## **4. Materials and Methods**

#### *4.1. Reagents and Materials*

The LC-MS/MS grade reagents, consisting of ammonium carbonate and acetonitrile (MeCN), were purchased from Fluka (St. Louis, MO, USA). The Mycosep 150 multifunctional column for extraction clean-up was purchased from Romer Labs (Tulln, Austria). Deionized water was produced using a Milli-Q system (Millipore; Bedford, MA, USA).

#### *4.2. Analytical Standards*

The analytical standards of the ergot alkaloids (ergometrine, ergosine, ergotamine, ergocornine, ergocryptine, ergocristine, ergosinine, ergotaminine, ergocorninine, ergocryptinine, and ergocristinine) were purchased from Chiron (Trondheim, Norway).

#### *4.3. Preparation of Standards Solution*

The analytical standard ergot alkaloid stock solutions were prepared in acetonitrile to provide a working standard solution of 100 μg/mL concentration for ergometrine, ergosine, ergotamine ergocryptine, ergocristine, and ergocornine and 25 μg/mL for ergosinine, ergotaminine, ergocryptinine, ergocristinine, and ergocorninine. For method validation of the spiking experiments, working standard solutions were freshly prepared at 1.0 μg/mL and were stored in amber vials at −20 ◦C for one week.

#### *4.4. Sample Collection*

A total of 200 feed samples consisting of swine feed (*n* = 100) and dairy feed (*n* = 100) were randomly collected from animal farms in different regions of Thailand. All samples were ground in a rotor mill ZM200 (Retsh GmbH, Hann, Germany) into a fine powder (0.50 mm) and stored at −20 ◦C before analysis.

#### *4.5. Sample Preparation*

The sample preparation protocol applied was developed based on Krska et al. [10]. Briefly, 5 g of homogenized feed sample was weighed into a 50 mL polypropylene (PP) centrifugation tube, followed by the addition of 25 mL of acetonitrile–ammonium carbonate buffer (3.03 mM), 84:16 (*v/v*). The tube was closed and shaken using a laboratory shaker (IKA Labortechnik; Staufen, Germany) for 30 min at 240 rpm. The extract was passed through Whatman No. 4 filter paper, and 4 mL of the extract was transferred to the Mycosep 150 multifunctional column (Romers lab, Tulln, Austria). Then, 1 mL of the purified extract was evaporated to dryness at 40 ◦C. The residue was reconstituted in 500 μL 50% mobile phase, and the mixture was passed through a 0.22 μm nylon filter before being used in the LC-MS/MS analysis.

#### *4.6. UHPLC-MS/MS Analysis*

The 11 target ergot alkaloids were analyzed using the UHPLC-MS/MS method. Chromatographic separation was developed according to Krska et al. [10]. The analysis used a Shimadzu LC-MS 8060 system (Shimadzu, Tokyo, Japan) that was equipped with a Gemini analytical column (150 × 2.0 mm i.d., 5.0 μm particle size; Phenomenex; Torrance, CA, USA) maintained at 30 ◦C. The mobile phase for analyses used 3.03 mM ammonium carbonate in deionized water (A) and MeCN (B) in ESI (+). The gradient elution was identical initially. The proportion of B was immediately increased from 5% to 17% within 1 min and further linearly increased to 47%, 54%, and 80% after 2, 10, and 15 min, respectively. Subsequently, the proportion of B was decreased to the initial conditions (5%) over 1 min, followed by a holdtime of 5 min, resulting in a total run-time of 21 min. The flow rate was stable at 0.5 mL/min throughout the run; 10 μL of sample extract was injected into the LC-MS/MS system.

The Shimadzu LC-MS 8060 system (Shimadzu, Japan) was equipped with an electrospray (ESI) ion source operated in positive mode. The ion source parameters were a nebulizing gas flow of 3 L/min, a heating gas flow of 10 l/min with an interface temperature: 300 ◦C, a CDL temperature of 250 ◦C, a heating block temperature of 400 ◦C, and a drying gas flow of 10 L/min. The dwell time (ms), Q1 Pre Bias (V), CE (V), and Q3 Pre Bias (V) were optimized during infusion of individual analytes (100 ng/mL) using automatic infusion. The MRM transitions of 11 ergot alkaloid-dependent parameters are summarized in Table 6.


**Table 6.** MS/MS parameters for determination of 11 ergot alkaloids.

#### *4.7. Method Validation Procedure*

The method performance characteristic parameters was determined to assess the efficiency of analytical method from this study by evaluating the linearity, accuracy, precision, LOD, and LOQ for EA contamination in swine and dairy feed samples. The analytes were quantified using a matrix-matched calibration standard with a pre spiking calibration curve for the 11 EAs for levels in the range 0.5–100.0 ng/g. The accuracy and precision (%RSD) were determined within the day by analyzing five replicates at three levels. The inter-day precision was determined at the same level as the within-day precision on three different days (*n* = 15). LODs and LOQs were calculated by analyzing the spiked samples at low level concentrations. LODs were determined as the lowest concentration of the analyte for which a signal-to-noise (S/N) ratio was 3:1, whereas S/N ratio was 10:1 for LOQs.
