Profiling of pyrrolizidine alkaloids and quantitation of the retronecine-core in crude plant extracts: A Qual-Quan approach by UHPLC-TOF/MSe and UHPLC-TOF/MRM

Abstract

Pyrrolizidine alkaloids (PA) are secondary metabolites of high toxicological relevance. Their occurrence has triggered the development of several PA quantitative methodologies based on a limited number of certified standards, including time-consuming purification steps. Herein, we shed light on the variability of PAs by screening PA-containing extracts from Boraginaceae, Fabaceae and Compositae families. Additionally, we proposed a quantification methodology based on UHPLC-HRMS for the evaluation of the total PA content as retronecine-equivalents (RE) directly from crude matrices. In total, 105 PAs were identified using HRMSe experiments, specific MS/MS fragmentation patterns, a customized in-house library and literature data. Among them, 43 retronecine/heliotridine-type PAs, 13 platynecine-type derivatives, as well as their 45 corresponding N-oxides, were reported. Furthermore, three otonecine and one trachelanthamidine necine base were observed. Interestingly, 18 PAs were annotated as glycosylated derivatives, reported for the first time in the literature. The quantitative study recorded PA concentrations ranking from 8.64 ± 0.08 to 3096.28 ± 273.72 µg RE/g extract dry weight in shoots of Alkanna graeca and in leaves of Crotalaria retusa, respectively. The methodology showed LLOD and LLOQ values of 3.46 and 11.52 pg · mL−1, respectively, offering a good precision (2.81 – 7.60% RSD) and accuracy (96.96 – 105.19%). Besides, a correction factor coupled to the detailed dereplication process was developed for each extract (ranging from 1.96 to 2.48) in relation to their unique PA content. The present methodology will facilitate PA quantification directly from crude extracts and avoid the underestimation of PA real content in botanical extracts.