Yared Getachew Lijalem (Korea Research Institute of Standards and Science)

Kihwan Choi (Korea Research Institute of Standards and Science)

Byungjoo Kim (Korea Research Institute of Standards and Science)

Yared Getachew Lijalem (Korea Research Institute of Standards and Science)

Hyeonwoo Yu (Korea Research Institute of Standards and Science)

Kihwan Choi (Korea Research Institute of Standards and Science)

Byungjoo Kim (Korea Research Institute of Standards and Science)

This study presents the development of an isotope dilution ultrahigh-performance liquid chromatography–tandem mass spectrometry (ID-UHPLC–MS/MS) method for accurately determining aflatoxins (AFs) B₁, B₂, G₁, and G₂ in red peppers. Uniformly labeled ¹³C₁₇-AF analogs were used as internal standards. Unlike grains and coffee, red peppers showed distinct matrix interferences during immunoaffinity column (IAC) cleanup, leading to target analyte breakthrough, potentially introducing measurement biases. Using a large extraction solvent volume (10 mL) exacerbated this issue, resulting in reduced recovery. The optimized sample preparation method, involving 5 mL of an acetonitrile/water (80/20; v/v) mixture followed by cleanup with an AflaStar™ IAC, effectively minimized matrix interferences and prevented analyte breakthrough during IAC cleanup, ensuring consistent target AF recovery. The proposed method demonstrated high accuracy (97 %–103 %), low limits of detection (0.003–0.01 µg/kg), and remarkable precisions (intraday: 0.5 %–3.5 %; interday: 1.0 %–4.3 %). Moreover, the measurement uncertainty remained below 6 % (at a 95 % confidence level). Therefore, the method developed exhibits superior metrological quality in comparison with existing methodologies and can serve as a reference measurement procedure for AF analysis in red peppers.