Validation and measurement uncertainty of HPLC method for bixin and norbixin in processed foods of animal resources distributed in Korea

This research aimed to validate a high-performance liquid chromatography (HPLC) method for the quantitative determination of bixin and norbixin in various foods. The Diode Array Detector (DAD) (495 nm) technique was used. Method was validated for specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, and accuracy, and the measurement uncertainty was assessed. The calibration curve showed excellent linearity (r²≥0.9999) over the tested concentration range of 0.2-25 mg/L. The LOD and LOQ were 0.03–0.11 and 0.02–0.05 mg/L for bixin and norbixin, respectively. The intra- and inter-day accuracies and precisions were 88.0±1.3–97.0±0.5% and 0.2–2.6% RSD for bixin and 88.2±0.8–105.8±0.8% and 0.3–2.7% RSD for norbixin, respectively. Inter-laboratory validation for accuracy and precision was conducted in three laboratories, and these results all met the AOAC guidelines. In addition, the relative expanded uncertainty (<22%) satisfied the CODEX recommendation. Furthermore, products distributed in Korea were monitored for annatto extracts using the proposed method to demonstrate its application. The developed analytical method is reliable for quantifying bixin and norbixin in various foods.