Abstract:

A novel and effective method was established for the qualitative analysis of impurities in auramine O samples of illegal food additives using high performance liquid chromatography-ion trap-time of flight mass spectrometry (HPLC-IT-TOF-MS). An impurity was identified in the auramine O sample using the optimized HPLC-IT-TOF-MS method. According to the exact mass of each fragment ion measured by multistage MS, the structure of the impurity was determined to be that of 4-(imino (4-(methylamino) phenyl) methyl)-N,N-dimethylaniline hydrochloride. The synthetic route of the auramine O and the source of the identified impurity were proposed. Simultaneously, a preparative high performance liquid chromatography (prep-HPLC) technique was successfully applied for the purification of the auramine O from complex samples. Prep-HPLC columns with particle sizes of 10 μm and 5 μm were used for the separation and purification with injection volumes of 1 mL and 500 μL, respectively. Finally an auramine O reference standard with 99.52% purity was obtained by secondarily purification and determined by the analytical HPLC area normalization method. Deducting the 0.34% moisture content and 0.13% ash content, the final purity of the sample was 99.05%, as determined by mass balance method. The chemical structure was examined using UV, IR, LC-MS, and NMR. The developed method is simple and efficient, and can be applied for the preparation of reference standard materials for other illegal food additives.

Key words: auramine O, high performance liquid chromatography-ion trap-time of flight mass spectrometry (HPLC-IT-TOF-MS), impurity identification, preparative high performance liquid chromatography (prep-HPLC), reference standard