Do You Want to Add Alternaria Toxins and Ergot Alkaloids in Your Multi-Mycotoxin Analysis? Part 2: Chromatographic Separation of Ergot Alkaloid Epimers

Previously we have discussed an established workflow providing a unique benefit of the simultaneous analysis of Alternaria toxins and ergot alkaloids with other major regulated mycotoxins. In this article, we would specifically discuss how the developed LC method could achieve the baseline separation of 6 critical ergot alkaloids (EAs) and their corresponding epimers for definitive quantification.

EAs are mycotoxins produced by the fungal species of the Claviceps genus. Contamination of EAs commonly occurs in cereal crops and wild grasses such as rye, triticale, wheat, oat, and barley. In fact, we have identified incurred EAs in rye flour, all purpose wheat flour, and baby wheat cereal obtained from local grocery stores. The ingestion of EAs could cause the so-called ergotism with symptoms including abdominal pain, vomiting, burning sensation of the skin, insomnia, and hallucinations. Of more than 40 known EAs, six compounds including ergocornine, ergocristine, ergocryptine, ergometrine, ergosine, and ergotamine, together with their isomeric -inine epimers are listed as regulated EAs by the European Union. The guidance is to quantify these 12 EAs separately and sum them up to a total EAs level. Therefore, it is necessary to chromatographically resolve all 12 epimeric EAs for unambiguous and accurate quantification. The most common practice is to utilize alkaline pH LC conditions to achieve the full separation of EA epimers on a C18 column. These restricted analytical conditions, however, have hindered the simultaneous analysis of EAs with other classes of mycotoxins.

Our goal was to develop an LC-MS friendly method under acidic conditions suitable for the simultaneous analysis of multi-class mycotoxin to include EAs. The chromatographic separation of all 12 epimeric EAs was the major factor for the decision making of the final LC method. For example, when the chromatographic performance was evaluated on multiple columns including Biphenyl, FluoroPhenyl, and C18 stationary phases bonded to superficially porous particles, it was realized that only the Biphenyl column could achieve the baseline separation of all 6 pairs of EA epimers under the acidic condition.