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

Ga-Yeong Lee1, Choong-In Yun2,3, Juhee Cho1, Young-Jun Kim1,2,*
Author Information & Copyright
1Department of Food Science and Technology, Seoul National University of Science and Technology, Seoul 01811, Korea.
2Research Institute of Food and Biotechnology, Seoul National University of Science and Technology, Seoul 01811, Korea.
3Department of Food Science and Biotechnology, Sungkyunkwan University, Suwon 16419, Korea.
*Corresponding Author: Young-Jun Kim. Phone: +82-2-970-6734. E-mail:

© Copyright 2023 Korean Society for Food Science of Animal Resources. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: Jul 06, 2023 ; Revised: Aug 14, 2023 ; Accepted: Aug 15, 2023

Published Online: Sep 08, 2023


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.

Keywords: bixin; norbixin; annatto; high-performance liquid chromatography; validation