Separation of pyrrolizidine alkaloids in different Senecio species using ultra-high performance supercritical fluid chromatography

Different Senecio species, especially S. inaequidens - a neophyte native to South Africa - have widely spread across Europe and now are found worldwide. The entire genus is known to contain toxic pyrrolizidine alkaloids (PAs), which renders them a possible health hazard to humans and livestock. As they can enter the food chain or occur as contaminants in herbal crops and phytopharmaceutical formulations (e.g. teas), efficient and straightforward assays for their qualitative and quantitative analysis are in high demand. Different techniques have been used for this purpose, most commonly HPLC or GC. As the analysis of PAs is a challenging task, alternative methodologies like ultra-high performance SFC (UHPSFC) may offer an additional benefit in terms of their separation efficiency and orthogonal selectivity. In this study an UHPSFC approach for the simultaneous determination of six PAs (free bases as well as N-oxides) is presented, which achieved the baseline separation of all standard compounds in seven min. Optimal separation was carried out in gradient mode on a TorusTM DEA column with 0.05% ammonia in methanol as modifier. The column temperature was 25 degrees C, ABPR 1900 psi and flow rate 1.1 mL/min, with a detection wavelength of 215 nm. The assay was validated and fulfilled all ICH criteria exhibiting good linearity (R2 >= 0.9994), precision (inter-day variance <= 3.67%, intra-day variance <= 3.92%) and recovery rates (96.3-104.1%), with detection limits typical for SFC-PDA (<= 4.24 mu g/mL). Further-more, it could conveniently be coupled to MS-detection, which increased the sensitivity significantly. To confirm practical suitability of the method, different Senecio samples were analyzed, indicating a high qualitative as well as quantitative difference in their PA profile (e.g. total amounts of PA between 0.09 and 4.63 mg/g).