|Cheng, Chi-fai Raymond
|Analytical studies on N-nitroso and related compounds
Nitroso compounds -- Analysis
Hong Kong Polytechnic University -- Dissertations
|Department of Applied Biology and Chemical Technology
|xx, 177 leaves : ill. ; 30 cm
|N-nitroso compounds (NOCs) are putative carcinogens the majority of which have been shown to be carcinogenic in laboratory animals. While the analytical methodology for volatile NOCs, namely N-nitrosamines, was well established, there is a lack of progress in the development of sensitive analytical methods for determination of non-volatile NOCs in foods. Furthermore, existing methodology relies mainly on the application of gas chromatography-thermal energy analyser (GC-TEA), which is susceptible to interference from other nitrogen-containing compounds. Development of alternative methods for their analysis is needed to confirm and validate qualitative and quantitative results derived from GC/TEA analysis. In this study, we have successfully developed an alternative analytical method based on combined use of reverse-phase ion-pair high performance liquid chromatography (RP-IP-HPLC) and electrospray ionization mass spectrometry (ESI-MS). Four non-volatile N-nitrosoamino acids, namely nitrososarcosine (NSAR), nitrosoproline (NPRO), N-nitorsothiazolidine-4-carboxylic acid (NTCA), N-nitroso-2-methyl-thiazolidine-4-carboxylic acid (NMTCA) were completely separated by isocratic elution using a 2.1 mm x 150 mm C18 (5u) HPLC column and 1.4mM C16-cetyltrimethylammonium chloride in 60: 35 :5 (v/v) MeOH: H2O: MeCN mobile phase. The HPLC conditions, such as alkyl chain length and concentration of tetraalkyl ammonium ion-pairing reagent and mobile phase composition were systematically studied, evaluated and optimized with regard to chromatographic resolution, economy of analysis time and sensitivity of detection by electrospray ionization mass spectrometry. Non-volatile N-nitrosoamino acids were sensitivity detected by negative electrospray ionization in the form of the deprotonated carboxylate molecular anion, [M-H]. The effect of co-eluting anion derived from the ion-pairing reagent was evaluated and optimized. Interfering cluster ion formation was avoided by careful manipulation of the mobile phase composition. A linear working range of 250pg to 2Ong/injection of N-nitrosoamino acids was established for quantitative analysis. The detection limit was 250 pg/injection for NSAR, NPRO, NTCA and NMTCA at a S/N ratio of ≥ 3. Our study is the first report on the development and application of RP-IP-HPLC/ESI-MS method to analysis of non-volatile NOCs in food. Relatively high levels of NSAR and NPRO at 2,200 ± 260 and 154 ± 15 ug/kg, respectively, was found in Chinese salted fish. This is also the first time that non-volatile N-nitroso compounds were analysed in an example of preserved Chinese food. As a supplementary study a RP-IP-HPLC method for simultaneous of nitrosating agents, i.e. nitrite and nitrate, and inhibitor of nitrosation, i.e. ascorbic acid in food was also developed. The method makes use of 0.010 M octylammonium orthophosphace as the ion interacting reagent and 20% (v/v) aqueous methanol as the mobile phase. The content of nitrite, nitrate (expressed as nitrite ion and nitrate ion, respectively) and ascorbic acid in commercial brands of canned tomato, carrot and mixed vegetable juices were surveyed. The quantitative results indicated that nitrite was very probably added as a preservative in the canned vegetable juices. The total amount of nitrite and nitrate ion per can in two commercial brands of vegetable juices was found to exceed the acceptable daily intake recommended by the 1996 Joint FAO/WHO Expert Committee on Food Additives for an average adult of 60 kg body weight.
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