| Summary: | Ptaquiloside, a naturally occurring cancer-causing substance in bracken fern, has been detected in the meat and milk of cows fed a diet containing bracken fern. A rapid and sensitive method for the quantitative analysis of ptaquiloside in bracken fern, meat, and dairy products was developed using the QuEChERS method and liquid chromatography–tandem mass spectrometry. The method was validated according to the Association of Official Analytical Chemists guidelines and met the criteria. A single matrix-matched calibration method with bracken fern has been proposed, which is a novel strategy that uses one calibration for multiple matrices. The calibration curve ranged from 0.1 to 50 µg/kg and showed good linearity (<i>r</i><sup>2</sup> > 0.99). The limits of detection and quantification were 0.03 and 0.09 µg/kg, respectively. The intraday and interday accuracies were 83.5–98.5%, and the precision was <9.0%. This method was used for the monitoring and exposure assessment of ptaquiloside in all routes of exposure. A total of 0.1 µg/kg of ptaquiloside was detected in free-range beef, and the daily dietary exposure of South Koreans to ptaquiloside was estimated at up to 3.0 × 10<sup>−5</sup> µg/kg b.w./day. The significance of this study is to evaluate commercially available products in which ptaquiloside may be present, to monitor consumer safety.
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