Two-Dimensional HPLC Combined with On-Line SPE for Determination of Sudan Dyes I–IV in Chili Oil

The determination of Sudan dyes in complex matrices (e.g., chili oil) often requires extensive sample preparation prior to HPLC analysis. Due to the fat-solubility of Sudan dyes, extraction with organic solvents such as acetonitrile, methanol, n-hexane, cyclohexane, and petroleum ether is typically used. Some supplementary means (e.g., ultrasound-assisted and microwaveassisted extractions) are applied to improve the extraction efficiency. The procedure following extraction is cleanup, which is the bottleneck for the sensitive determination of Sudan dyes in chili oil. Solid-phase extraction (SPE), gel permeation chromatography, thin-layer chromatography and dispersive solid-phase extraction have been reported for cleanup.

Here, a two-dimensional HPLC with on-line SPE intercolumn trapping method was developed for fast, effective determination of Sudan dyes I–IV in chili oil. Following extraction with methylene dichloride and acetonitrile, the analytes are separated in the first dimension and removed selectively to be absorbed on an SPE column. This eliminates numerous matrix interferences. When analyte trapping is complete on the SPE column, it is switched into the flow path of the second dimension, where the absorbed dyes are eluted from the SPE column, separated, and detected. The 2-D HPLC with on-line SPE intercolumn trapping system runs automatically on the Thermo Scientific Dionex UltiMate™ 3000 HPLC dual-pump system controlled by Thermo Scientific Dionex Chromeleon™ software, and provides the advantages of full automation, absence of operator influence, and strict process control compared to a typical off-line SPE method.