Quantification by LC-MS/MS

The LC-MS/MS analysis was performed using an HPLC Agilent 1200 series coupled with Agilent QQQ 6460 (Agilent Technologies) according to the ISO protocol (ISO 18465, 2017). A column Supelco Discovery C18, 10 cm × 2.1 mm × 5 μm (Merck, 569220-U) was used with an isocratic elution of 10% of the mobile phase A (10 mM ammonium formate in water with 0.1% formic acid) and 90% of phase B (pure acetonitrile with 0.1% formic acid) and a flow rate of 0.4 ml/min at 40°C.

The mass spectrometer was operated in a positive ESI mode (ESI Low Tuning Mix G1969-85000, Agilent Technology) with a capillary voltage of 3,500 V, a nozzle voltage of 1,000 V, a gas temperature of 325°C, a gas flow of 10 L/min and a nebulizer at 35 psi. The sheath gas temperature and gas flow were at 250°C and at 8 L/min, respectively. MS/MS analysis was performed in multiple reaction monitoring (MRM) mode.

Cereulide peaks were confirmed when the relative deviation of q/Q area ratio of cereulide in the samples compared to the average ratio measured in standards did not exceed ±20%.

Agilent Masshunter Workstation Data Acquisition and Agilent QQQ quantitative Analysis (Quant-my-way) software were used for the acquisition and quantification, respectively.

The amounts of cereulide were obtained in ng/g of food sample. The limit of quantification (LoQ) was defined with spiked matrices based on a S/N ratio ≥ 6 for the cereulide qualifier. On the Agilent 6460, the LoQ was determined at 0.2 ng/g in the tested food matrices. The recovery rate was checked for all the runs to have at least 95% recovery. In addition, samples expected to have high or low cereulide levels, falling outside the calibration curve range, were diluted or concentrated, respectively.