Determination of Pyrrolizidine Alkaloids in Tea and Herbal Infusions by LC-MS/MS

Pyrrolizidine alkaloids (PAs) and N-oxides (PANOs) are food-safety hazards subject to regulatory limits, and their occurrence in teas and herbal infusions (THI) represents a dietary route. This study aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determining five PAs and their PANOs in THI. Samples were extracted using hot acidified water to enhance PAs and PANOs extraction and subjected to solid-phase extraction (SPE) for clean-up and analyte preconcentration. Chromatographic separation was performed on a Luna^® PFP (2) 3 µm 100 Å column using a gradient of 10 mM ammonium formate with 0.1% formic acid in water, ethanol, and methanol. The method was validated according to international guidelines, achieving detection and quantification limits of 0.41-6.60 ng/g and 1.25-20.0 ng/g. It was applied to 53 THI samples from five European countries, primarily from Portugal as a pilot monitoring study. N-oxides were the predominant form, with senecionine-N-oxide, heliotrine-N-oxide, and retrorsine-N-oxide most frequently detected in Portuguese-marketed products. The total target PA/PANO content was approximately twofold higher in herbal infusions than in teas, with chamomile showing the highest levels, followed by lemon verbena. The validated method provides a sensitive and suitable tool for preliminary assessment of PAs and PANOs in THI.