Development and In-House Validation of a Liquid Chromatography-Electrospray-Tandem Mass Spectrometry Method for the Simultaneous Determination of Sudan I, Sudan II, Sudan III and Sudan IV in Hot Chilli Products

An accurate method based on the use of reversed-phase (RP) liquid chromatography–tandem mass spectrometry interfaced with electrospray (LC–ESI–MS/MS) was devised for the determination of Sudan I, Sudan II, Sudan III and Sudan IV in hot chilli food samples. A simple sample treatment procedure entailing the use of an extraction step with acetone without clean-up was developed. A C18 column with an aqueous formic acid/methanol mixture as the mobile phase was used under isocratic conditions. Mass spectral acquisition was done in positive ion mode by applying selected reaction monitoring of three fragmentation transitions per compound to provide a high degree of selectivity. The method was in-house validated in terms of detection limits (LOD), quantitation limits (LOQ), linearity, sensitivity, accuracy, recovery, and selectivity on two kinds of hot chilli sauces. Good results in the low ng/g level were obtained for LOD and LOQ of all analytes in matrices. Under both intra-day repeatability (R.S.D. between 1 and 13%) and intermediate precision (about 5–15% R.S.D. for both chilli sauce matrices) conditions, precision proved to be typical of determinations based on electrospray LC–MS and acceptable for routine monitoring purposes. Extraction recoveries for all four azo-dyes in chilli tomato sauce ranged from 92 to 103% at a spiking level of 5g/kg, whereas values between 72 and 97% were calculated in chilli tomato and cheese sauce at the same concentration level. The applicability of the method to the determination of Sudan azo-dyes in hot chilli products was demonstrated.