Study on the simultaneous determination of bixin and norbixin in foods by high-performance liquid chromatography

Abstract

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.