*2.1. General Conditions for LC-ID-MS*/*MS Separation*

The fine-tuning of ion transitions in the MS/MS instrument was carried out with individual standard solutions (2 μg/mL) in methanol and employing electrospray (ESI) source in negative ion mode according to our previous paper [13]. The isotopically labeled ISTDs were also tuned using the ISTD mixture (Section 4.1). The two most intense ion transitions of target compounds used for the MS/MS detection are detailed in Table 1. The ALT, AOH, and AME toxins are weak acidic molecules (Figure 1) and show an appropriate retention on C-18 HPLC columns and high sensitivity during MS or MS/MS detection [9–36]. TEN can be considered as a neutral molecule but can be also measured by LC-MS/MS method with negative ionization and fit-for-purpose sensitivity [8,10–13]. In the case of TEA, however, special HPLC conditions are necessary due to its different isomer forms appearing in aqueous phase at acidic pH [13,19,20]. Therefore, chemical derivatization with 2,4-dinitrophenylhidrazine was introduced for TEA that makes it a suitable compound for HPLC analysis [13,19,20]. The drawback of the derivatization approach is the longer sample preparation time, lower selectivity, and increased noise of analysis [13]. Recently, HPLC separations at alkaline pH conditions on C-18 column were reported for TEA separation in food matrices [6,21,22,30,31,33]. A pH above 8.0 results in reproducible retention time and peak shape for TEA but decreases its retention time due to the deprotonated hydrophilic form of TEA at alkaline pH (Figure 1).


**Table 1.** MS/MS detection parameters for *Alternaria* toxins detected in APCI and ESI ionization modes employing negative or positive ion mode. The quantifier ion transition is highlighted with bold.

This condition, however, does not require derivatization, hence, it was tested in the present study with a HPLC column suitable for separation at above pH 8.0. A Zorbax Extend C-18 column allows separation at pH up to 11.5. The alkaline condition (Section 4.7) resulted in baseline separation for the five toxins and appropriate retention for TEA on this column (Figure 2). The pH of the mobile phase was tested between 8.0 and 9.0. Retention time shift and difference in sensitivity were not observed. The apparent retention factor (k') calculated for TEA was higher than 2.0 under all conditions. However, the ESI source did not result in enough sensitivity for the analysis, and the instrumental limit of quantification (LOQ) was not lower than 50 ng/mL, but the aim was to detect AME and AOH below 10 ng/mL [6]. Consequently, the ESI positive ionization mode was also tested (Table 1) under acidic pH condition (Section 4.7), but a better sensitivity could not be achieved. Moreover, the peak shape of TEA was irreproducible under acidic condition using this HPLC column mentioned above. Therefore, the atmospheric pressure chemical ionization (APCI) source was also tested in negative ion mode and under alkaline HPLC condition. It should be pointed out that the same ion transitions were used for performing the detections in both ESI and APCI modes. Only different polarities (positive or negative) resulted in various ion traces. This instrument gave increased sensitivity for these toxins with APCI source using negative ionization. The instrumental LOQ could be lowered at least with one order of magnitude for all compounds in comparison to those values obtained with ESI probe. The best LC-MS/MS conditions were obtained using alkaline pH condition for HPLC separation at pH 8.8 and employing APCI source with negative ionization mode (Figure 2).

**Figure 2.** Total ion current chromatogram of five toxins at 10 μg/kg using LC-APCI(-)-MS/MS separation at pH 8.8. Compounds: TEA (5.1 min); ALT (9.8 min); AOH (10.2 min); TEN (10.9 min); and AME (12.0 min). The concentrations of ISTDs were: TEA-13C2 (83 μg/kg), ALT-d6 (33 μg/kg), AOH-d3 (17 μg/kg), TEN-d3 (17 μg/kg), and AME-d3 (17 μg/kg).
