Convenient and Sensitive HPLC Method for Determination of Nitrosamines in Foodstuffs Based on Pre-column Fluorescence Labeling

The determination of trace nitrosamines (NAs) has received more and more attention due to NAs' strong carcinogenicity and wide existence in foodstuffs. But the convenient and sensitive method for simultaneously analyzing multiple NAs remains poorly investigated. In the present study, a new method was developed for the determination of four NAs in foodstuffs based on fluorescence labeling followed by high-performance liquid chromatography with fluorescence detection (HPLC-FLD), which did not require complex pretreatments and expensive equipment. NAs were firstly denitrosated using hydrobromic acid-acetic acid to obtain the secondary amines. Subsequently, a fluorescence labelling procedure was immediately carried out to label the secondary amines using 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC). A response surface methodology (RSM) based on a Box-Behnken design (BBD) was used to optimize the main parameters affecting the denitrosation. Under the optimal HPLC conditions, the quantitative linear range of analytes was 0.05-200 nmol L-1, and the correlation coefficients were more than 0.9998. Detection limits for all NA derivatives were achieved within the range of 1.3-2.5 ng L-1. The proposed method was successfully applied for the determination of trace NAs in several foodstuffs. This method was proven to be simple, inexpensive, sensitive, accurate and reliable for trace NA determination in foodstuffs.