Determination of pyrrolizidine alkaloids in Petasites hybridus leaf native CO2-extract by using UHPLC-HRMS

Due to increasing regulatory awareness, the content of hepatotoxic and genotoxic pyrrolizidine alkaloid (PA) content in herbal medicinal preparations has to be thoroughly monitored. Recently, new very low PA regulatory threshold concentrations have been proposed by the EMA/HMPC. We developed a highly sensitive and reproducible UHPLC-HRMS method for PA determination in a CO₂-extract of P. hybridus leaves. After sample preparation by liquid-liquid extraction and chromatographic separation by UHPLC, PAs senkrirkine, senecionine, seneciphylline, senecionine-N-oxide and seneciphylline-N-oxide were detected by HRMS (mass resolution 30000 FWHM at m/z 400) in positive ionisation mode. PAs were quantified using external standard series of all mentioned alkaloids. The standards were diluted in a matrix of PA-free P. hybridus CO₂-extract. Monocrotaline served as internal standard, and a correction factor for all alkaloids was calculated. Prior to quantitation by UHPLC-HRMS, PAs in the dried leaves of P. hybridus were concentrated by SPE on RP18 cartridges. Since no PA-free P. hybridus leaves were available for the standard solutions, recovery rates were determined with PA-free replacement matrix materials. Using different types of matrices, it became evident that quantification of PAs by UHPLC-HRMS was significantly affected by matrix effects. The UHPLC-HRMS method showed an LoQ of 2 ppb for all PAs. Recovery at the LoQ was between 88.9 and 141.9%, repeatability and precision were between 3.5 and 13.6%. Linearity of the five PAs showed correlation coefficients between 0.99948 and 0.99975, and coefficients of variation between 7.44 and 8.56. A working range between 2 ppb and 200 ppb could be fixed. The assay allows to determine low PA concentrations in P. hybridus leaf native CO₂-extract, and is suitable for analytical monitoring of PA concentration in accordance with regulatory requirements.

Keywords: pyrrolizidine alkaloids, PA, Petasites hybridus, UHPLC-HRMS, senkirkine, senecionine, seneciphylline