Bixin and norbixin are two types of apocarotenoids that can be extracted from the seeds of cashew nuts (Bixa orellana). They are the main components of annatto, a natural food coloring agent, which is widely used in the food industry to impart yellow and orange-red hues to various food products. The objective of this study was to develop a method for the simultaneous determination of bixin and norbixin in different food samples using high performance liquid chromatography (HPLC-PDA). The samples were extracted with ethanol by ultrasonic vibration at room temperature for 20 minutes. The extracted samples were then analyzed by HPLC-PDA using XBridge C18 chromatography column (150 mm x 4.6 mm; 5 µm), and isocratic elution with a mobile phase consisting of 0.5 % formic acid in water: MeOH (15:85, v:v) at 450 nm detection wavelength. The method showed good specificity, high correlation coefficients (R2 > 0.9999) for the calibration curves, acceptable repeatability (1.9-4.2% for bixin and 4.1-5.9% for norbixin), satisfactory recovery (96.0-107% for bixin and 95.0-109% for norbixin), low limits of detection (LOD) (0.17-0.5 mg/kg for both bixin and norbixin), and low limits of quantification (LOQ) (0.56-1.31 mg/kg for both bixin and norbixin). The method was successfully applied to the simultaneous determination of bixin and norbixin in 35 food samples. The results showed: concentrations ranging from 736 mg/100g to 5911 mg/100g (bixin) and from 8.91 mg/100g to 51.9 mg/100g (norbixin) for raw material samples and from 5.85 mg/kg to 490 mg/kg (bixin) and 0.83 ng/kg to 8.02 mg/kg (norbixin) for food samples.
Bixin, norbixin, food, HPLC, PDA
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