Determination of 34 pyrrolizidine alkaloids (PA) as contaminants in various plant extracts using UHPLC-ToF-HRMS

The very low PA regulatory threshold of a daily intake of 0.35 µg proposed by the EMA/HMPC requires sensitive and reproducible methods for determination. After establishing an UHPLC-ToF-HRMS method to determine five natural occurring PAs in in a CO₂ extract of P. hybridus leaves in 2014 (2), we now developed a contaminant assay for 34 PAs applicable for various extracts. To avoid matrix effects and to concentrate the analytes by the means of a second UHPLC pump and a trapping column, a 2D-chromatography system is integrated in the present UHPLC system. This replaces time-consuming sample preparation by means of SPE. Only the quantity of the sample to be injected is automatically purified and concentrated using autosampler and 2D-installation. The chromatographic separation avoids with two exceptions contemporaneous elution of PA isomers. PAs are detected by ToF-HRMS (mass resolution 30000 FWHM at m/z 400) in positive ionization mode. They are quantified using external standard series of all analyzed alkaloids. As a further important measure to avoid matrix effects, the standards are dissolved in a matrix solution of PA-free extracts. A detailed basic validation was executed with hops extract. For other matrices just linearity, precision, recovery and LoQ are validated. Due to fact that all external standards are dissolved in matrix solution the LoQs are slightly higher than with standards in pure solvents. Depending on the validated matrix they range from 10 to 20 ppb. Compared to methods without compensation of matrix effects, the measured values should be more close to the exact value.

[1] BfR (2013), Bestimmung von Pyrrolizidinalkaloiden (PA) in Pflanzenmaterial mittels SPE-LC-MS/MS. Methodenbeschreibung BfR-PA-Tee-1.0, http://www.bfr.bund.de/cm/343/bestimmung-von-pyrrolizidinalkaloiden.pdf, Last access: March 2015

[2] Schenk A, Siewert B, Toff S, Drewe, J (2015) UPLC TOF MS for sensitive quantification of naturally occurring pyrrolizidine alkaloids in Petasites hybridus extract (Ze 339)