Improvement of analytical method for three azo dyes in processed milk and cheese using HPLC-PDA

This study aims to develop and validate a method for simultaneously measuring three azo dyes (azorubine, brilliant black BN, lithol rubine BK) not designated in Korea. The HPLC-PDA analysis method was validated based on the ICH guidelines, and the color stability was evaluated. The milk and cheese samples were spiked with azo dyes, the correlation coefficient of calibration curve ranged from 0.999 to 1.000 and the recovery rates of azo dyes were 98.81 ∼ 115.94%, with RSD of 0.08 ∼ 3.71%. The LOD and the LOQ in milk and cheese ranged from 1.14 to 1.73 μg/mL and 3.46 to 5.25 μg/mL, respectively. In addition, the expanded uncertainties of the measurements ranged from 3.3421 to 3.8146%. The azo dyes appeared to be color stable for more than 14 days. The results indicate that this analytical method is suitable for extracting and analyzing azo dyes in milk and cheese samples, which are not permitted in Korea.