Pyrrolizidine alkaloids (PAs) are toxic natural products that present a major health concern. 1-2 unsaturated PAs can be bioactivated by CYP450 enzymes to yield pyrrole derivatives, which are involved in adverse effects, notably hepatic veno-occlusions, but also proved to be genotoxic. Even though these toxicities have been described since the 70’s, regulatory agencies only recently acknowledged the scope of the problem, encouraging efforts to collect more data on pyrrolizidine alkaloids in traditional herbal medicines, homeopathic medicines, and food and dietary supplements. However, there are still no universal methods able to detect all toxic PAs with high sensitivity.
Current PAs detection methods are mostly based on either HPLC-MS/MS or GC-MS. Nonetheless, even with state-of-the-art technological refinements, it is still difficult to detect and quantify every 1-2 unsaturated PAs in herb or food samples. As those approaches seem to reach stalemate, our work describes a new qPCR-based method of detection, combining the potential adduct-mediated genotoxicity of 1-2 unsaturated PA’s with the adduct-sensing capability of qPCR.
This approach consists in incubating specifically-designed synthetic double-strand oligonucleotides with chemically activated PAs, in order to yield adducted-DNA. The whole process follows a simple 4-steps plan: (i) SPE-SCX extraction of PAs from samples, (ii) chemical-metabolization to adducting moieties, (iii) incubation of oligonucleotides and alkaloid extracts and (iv) the qPCR itself.
Using retrorsine as standard, we are able to distinguish a 5 µM solution from a suitable blank, using a 48H, 67°C incubation of ds oligonucleotides. Optimization is in progress, aiming to decrease incubation time to a practical overnight and to lower the limit of detection to 0.5 µM in order to match HMPC/EMA skip testing requirements.