Investigation On Analytical Methods For Spontaneous Detection Of Nine N-nitrosamines In Pickled Food

N-nitrosamine is a strong carcinogenic contaminant, which has been widelydetected in the pickled and heat-treated food. This substance can lead to cancerousorgans and some other daily health problems. Therefore, the key measures to cutdown its hazard are to establish rapidly as well as accurately detecting method andthe effective inhibitors. The fast detection methods for9kinds of nitrosamines(NDMA, NMEA, NDEA, NPYR, NMOR, NDPA, NPIP, NDBA and NDPhA) by theHigh Performance Liquid Chromatography (HPLC) are systematically introduced inthis thesis. Combining with the statistics analysis method, my studying results haverevealed the key factors of NDMA formation as well as the inhibitor effect ofgardenia yellow pigment on NDMA, and established the experiment, which willoffer the basis of experimental technique and theory foundation for further researchof inhibition kinetics.Taken the HPLC-DAD as the main qualitative and quantitative method andHPLC-MS as the auxiliary qualitative authentication method, nine N-nitrosamineswere identified with two detectors (Photodiode Array Detector, Mass Spectrometry)combined together. The result shows that the limit of detection (LOD) and the limitof quantitation (LOQ) of HPLC-DAD are0.2~0.49μg／mL and0.77~1.62μg／mL,respectively. The scope of relative standard deviation (RSD) of parallel experimentis0.03~0.19%. The LOD of HPLC-MS is0.007~0.133μg／mL. The two methodsare very suitable for9nitrosamines analysis for the derivatization are not needed,and the processes are operational as well as reproducible.Based on the solid phase extraction (SPE) technology, we had compared therecoveries of nine nitrosamines with four kinds of single column material (Extrelut,Alumina, Silica Gel and C18) and two kinds of dual blended material (Alumina-Silica, Alumina-C18). Moreover, a new SPE method with dual packing columnmaterial (Alumina-C18) was developed for enriching and purifying the nine nitrosamines simultaneously. Spiked recoveries of blank samples show that, theaverage recovery rates of six parallel tests on three levels are85.17%~101.5%, theRSD is0.8%~3.12%. The statistics significance analysis results of linear regressionequations which based on the recovery rates and spiked levels shows that, therecoveries of NPYR、NMOR、NDPA and NDBA are higher than the theoreticalvalue`nitrosamines (NDMA、NDEA、NMEA、NPIP and NDPhA) are lower than thetheoretical value in the rang of5~15μg／mL.Combined with flame ionization detector (FID) and the dual packing columnmaterials SPE method, a SPE-GC-FID method was established by using the gaschromatography (GC). The accuracy and precision of the method was comparedwith SPE-HPLC-DAD, and the results shows that, the LOD and LOQ of theGC-FID are1.27~1.83μg／mL and4.23~6.10μg／mL, the spiked recoveries ofblank samples with GC method are37%~65%and RSD ranges in0.93%~5.61%.The statistics significance analysis results shows that the all the nine nitrosaminesrecoveries by SPE-GC-FID are lower than the theoretical values. Compare with theSPE-HPLC-DAD, the precisions of the two methods have no significant difference,and the GC has low recovery.Both of the two methods SPE-GC-FID and SPE-HPLC-DAD were used todetermine the nitrosamines content levels in Chinese traditional pickled food. Theresult shows that, the spiked recoveries of actual samples are83.66%~97.28%byHPLC-DAD and35.17~78.29%by GC-FID, and RSD is0.53~2.36%and0.16%~1.38%, respectively. All the nine nitrosamines in different degree are detected inthe samples by HPLC-DAD as the total nitrosamines content is0.49~92.08μg／mL.The detection rate of all samples is50%. Five nitrosamines were detected bySPE-GC-FID, which the total nitrosamines content is6.45~96.89μg／mL, anddetection rate in all the samples is30%. The result of MS qualitative identificationshows that, the false positive rate of NDEA and NDPA are25%and33.34%by themethod HPLC-DAD. Meanwhile, the false positive rate of NDPA、 NDBA and NDPhA by GC-FID are75%、33.34%and66.67%. Therefore, the SPE-GC-FIDshows a poor anti-interference ability and incredible result, and theSPE-HPLC-DAD is more suitable for the detection of nitrosamines.Through researching the forming process of NDMA in vitro simulated gastricenvironment, we have found that pH is the critical factor which affects the NDMAformation. The highest yield of nitrosamines was achieved when the pH is2; whenadding the inhibitor gardenia yellow pigment with the volume ratio10%andconcentration0.7mg／L into the reaction system which contains10mmol／Lprecursor, the inhibition ratio to nitrosamines is up to46.4%. The inhibition actionsites and inhibition kinetics need the further deep study.