2.9.1. Sample Preparation and HPLC-ESI-MS/MS Quantification of the Total PA-Content

All analyzed samples were in homogenous form, lyophilized and homogenized in 50 mL conical centrifuge tubes using a mixer mill (MM 400; Retsch, Haan, Germany) for 2 min at 30 Hz. Three-hundred mg from each sample was soaked in a 15 mL polypropylene, conical centrifuge tube using 11 mL H2SO4 0.05 M. Then, 40 μL of internal standard solution (2 μg/mL in methanol) was added to each tube and the tubes were thoroughly mixed and extracted overnight using a continuous tube rotator (Multi Bio RS-24, Biosan, Riga, Latvia) and the following settings: orbital: 21/01, reciprocal: 15/01, vibrio: 5/1, duration: 14 h. Sample preparation to analyze the total PA-content of the individual samples was carried out according to the methods of Cramer et al. [28] and Letsyo et al. [29]. During the course of sample preparation all 1,2-unsaturated retronecine- and heliotridine-type ester PAs/PANOs were converted into the corresponding core structures, i.e., retronecine and/or heliotridine. After derivatization, these analytes were analyzed by HPLC-ESI-MS/MS, generating a single signal which could be quantified by the use of the added isotopically labeled IST, allowing the calculation of the total content of all 1,2-unsaturated retronecine- and heliotridine-type ester PAs including all metabolites thereof, which still bear both necessary features of PA-toxicity (1,2-unsaturation and ester-PAs). Using an internal deuterated standard allowed the direct quantification across all matrices (matrix effects were covered and the IST was detected in all analyzed samples). A limit of detection (LoD) of 1 μg PA/kg and a limit of quantification (LoQ) of 5 μg PA/kg was obtained across all different matrices. A detailed description of this method and the sum parameter approach as well as the underlying calculations was added to the Supplementary Material or can be found at Cramer et al. [28]. Individual samples (positive and negative analytical results) were re-analyzed randomly, to confirm the integrity of the method. Data analysis and integration was achieved with Analyst 1.6.2 Software (Applied Biosystems MDS Sciex, Darmstadt, Germany). All analytical values are presented based on dry weight (d.w.).
