Study And Application Of Fluorescence Spectrophotometric Determination Of Nitrite In Food

Nitrite is one of the common food additives and is ubiquitous within milk, meat and preserved foods. However, excessive amount of nitrite in food will do great harm to people’s health. Therefore, a sensitive and specific method for the determination of nitrite in food is of great importance. Great efforts have been made to establish new method to determine nitrite in food. In comparison to other methods, spectrofluorimetry possesses more merits due to its convenience, simplicity, high sensitivity and selectivity, low limits of detection and low-cost. More and more attention has been paid to the spectrofluorimetric determination of nitrite in food. This thesis is divided into three parts. The first part of the main work is to develop a simple, sensitive, selective and convenient spectrofluorimetric method for detection of nitrite based on the highly efficient and specific reaction of nitric oxide with a dihydropyridine derivative to form a highly fluorescent pyridine derivative, in HCl solution. Under optimum conditions, excellent detection linear range was achieved from 2.0 × 10-7 to 4.5 × 10-6 mol·L-1 with a detection limit of 1.9 × 10-8 mol·L-1(S/N=3). The detection limit was lower than or comparable to most of reported methods. It is demonstrated that this spectrofluorimetric method could be used to quantitatively determine nitrite levels in food samples such as ham and sausage, with R.S.D.(n=6) less than 3.2% and recoveries of 92.3% ~ 106.7%. The second part of the main work is to study the reaction of nitrite with DAF-FM DA in HCl medium to form a highly fluorescent triazole compound. The fluorescence enhancement at 517 nm was proportional to nitrite concentration in the range of 5.0 × 10-8 to 1.5 × 10-6 mol·L-1 with a detection limit of 3.3 × 10-8 mol·L-1(S/N = 3), lower than those of other spectrofluorimetric methods. The proposed method has been applied to the determination of nitrite in ham and pickled mustard tuber, with R.S.D.(n = 6) less than 5.1% and recoveries of 84.8% ~ 99.0%. Nitrite containts in six food samples were determined simultaneously using DAF-FM DA and DHP-b. The results indicated that the newly synthesized dihydropyridine probe was more precise and accurate than the vicinal phenyldiamine probe. In the third part of the main work, the interference of ascorbic acid on the detecting results acquired by spectrophotometric method and DHP-b were studied. The results showed that ascorbic acid did not affect the determination of nitrite with the newly synthesized dihydropyridine probe DHP-b. Howerver, spectrophotometric method seriously suffered interference from ascorbic acid. More significantly, the newly synthesized dihydropyridine probe has the advantages of desirable photophysical properties including a large stokes shift, high fluorescence quantum efficiency, excellent photo-stability, free from interference of oxidants, facile synthesis, low-toxicity, high sensitivity and specificity for nitrite. The dihydropyridine-based fluorogenic probes we have synthesized, with the modular design, should serve as a very useful, readily expandable prototype for the subsequent development of a series of analogous fluorogenic probes for determination of nitrite and NO. Properties such as further enhanced sensitivity, expanded absorption and emission ranges, improved water solubility, as well as compatibility to a variety of conditions are expected for such probes.