【作者】 张甜；

【导师】 杨华； 马俪珍；

【作者基本信息】 山西农业大学 ， 农产品加工及贮藏工程， 2017， 硕士

【摘要】 N-亚硝胺是一类公认的毒性化合物,广泛存在于各类食品尤其是肉制品中,一些挥发性N-亚硝胺可诱发细胞癌变,是人类日常饮食安全的重要隐患,因此检测N-亚硝胺的含量对于食品安全的监管和控制至关重要。肉制品基质十分复杂,且N-亚硝胺含量低,因此必须对肉样进行前处理以提取和富集N-亚硝胺。国标(GB/T5009.26-2003)中的样品前处理采用水蒸汽蒸馏法,需样量大,耗时长,消耗过多有机溶剂,检测时采用气相色谱-质谱仪,设备投入较高。如果能建立一种快速、简便的前处理方法,并结合常用气相色谱仪和液相色谱仪进行检测,将会显著提高N-亚硝胺的检测效率。本文以肉制品中常见的9种挥发性N-亚硝胺(NDMA,NMEA,NDEA,NPYR,NMOR,NDPA,NPIP,NDBA,NDPhA)为代表,首先重点研究了两种样品前处理方法:固相微萃取(SPME)法和超声波提取结合固相萃取(UE-SPE)法;进一步优化两种色谱检测方法:气相色谱-氮磷检测器(GC-NPD)法和高效液相色谱-紫外(HPLC-UN)法,最后以国标法为基准,分析了上述前处理与检测方法联用在肉糜制品中检测N-亚硝胺的精准度与效率。论文主要研究结果如下:1.利用固相微萃取(SPME)结合气相色谱-氮磷检测器(GC-NPD)测定肉制品中9种挥发性N-亚硝胺。通过优化萃取温度、萃取时间、NaCl浓度、转速、平衡时间和解析时间、来选取测定9种挥发性N-亚硝胺的最佳萃取条件。结果表明:以PDMS/DVB/CAR为萃取头,平衡时间10min、萃取温度40℃、萃取时间30min、搅拌速度400r/min、盐离子浓度0.36g/mL(饱和NaCl)、解析时间3min时能得到最佳萃取效果。采用SPME,结合GC-NPD测定9种N-亚硝胺的线性相关系数为0.9964～0.9996,检出限为0.01～10ng/mL,定量限为0.03～33 ng/mL,回收率为66.76%～93.20%,平行实验相对偏差(RSD)为3.27%～7.62%。2.采用高效液相色谱结合紫外检测器(HPLC-UV)对9种N-亚硝胺进行分离检测。通过优化样品溶剂、流动相种类、流动相配比和流动相pH来确定9种N-亚硝胺的最佳分离条件。结果表明,采用外标法定量,在0.1～4.0 μg/mL,线性相关系数达0.9995以上,9种N-亚硝胺的仪器检出限为0.010～0.057 μg/mL,定量限为0.033～0.190 μg/mL,平行试验相对标准偏差(RSD)为0.03%～0.29%,该方法不需要衍生化处理,可操作性强,灵敏度高,重现性好,适用于9种N-亚硝胺的分离检测。3.建立超声波提取-固相萃取-气相色谱-氮磷检测(UE-SPE-GC-NPD)和超声波提取-固相萃取-液相色谱-紫外检测(UE-SPE-HPLC-UV)测定肉制品中9种N-亚硝胺的方法。选取二氯甲烷作为提取试剂,探讨了萃取时间、有机溶剂用量和萃取功率对提取效果的影响,且比较C18、中性氧化铝和活性炭这3种固相萃取柱对超声波提取出的目标物的净化作用。结果表明:样品经过40mL二氯甲烷在400W功率下提取20min,N-亚硝胺的峰面积达到最大,即提取效果最好,且最终通过回收率试验选取了活性炭固相萃取小柱作为样品净化。UE-SPE-GC-NPD的回收率在66.80%～93.89%之间,且其精密度均在1.39%～3.07%之间,UE-SPE-HPLC-UV的回收率在65.80%～93.20%之间,且其精密度均在1.19%～3.42%之间,该方法简便、快速,具有良好的灵敏度、重复性,适用于同时测定肉及肉糜制品中9种N-亚硝胺类化合物的含量。4.比较 SPME-GC-NPD、UE-SPE-GC-NPD 和 UE-SPE-HPLC-UV 与国标方法测定肉制品中N-亚硝胺含量的差异,可知SPME-GC-NPD检测到的N-亚硝胺的种类较少,适合快速测定肉制品中的NDMA、NMEA和NDEA含量。UE-SPE-GC-NPD较UE-SPE-HPLC-UV方法检测的N-亚硝胺的检测限低,更适合N-亚硝胺含量少的肉制品的检测。对同种样品,本研究优选的三种方法检测的N-亚硝胺含量略高于国标法,但检测出的N-亚硝胺的种类少于国标法,根据实际情况酌情选择合适的方法。更多还原

【Abstract】 N-nitrosamines are recogn ized as a class of compounds that have strong toxic and harmful,widely exist in cured meat products and heat processed foods.Some volatile N-nitrosamines can induce cell canceration,which is an important latent danger to human diet.The matrix of meat products is very complex,and the content of N-nitrosamines is extremely low,so it is very important for the extraction and enrichment of N-nitrosamines in the sample before the detection of N-nitrosamines.The pretreatment method of national standard method adopt steam distillation,that needs lots of samples,long time and more organic reagents,so it is necessary to establish a pretreatment and detection method can detect quickly and accurately for N-nitrosamines.The GB method adopt the GC-MS as the detection method,it is expensive,if it can use the common GC or HPLC for detection,there will make the detect method for N-nitrosamines much more convenient.In this paper,we make the 9 kinds of common N-nitrosamines(NDMA,NMEA,NDEA,NPYR,NMOR,NDPA,NPIP,NDBA,NDPhA)in meat products as the research object,Focus on studying two pretreatment method(Solid-phase microextraction,SPME,ultrasonic extraction with SPE)and two chromatographic method(GC-NPD,HPLC),finally,comparing the result of samples,we choose to use the established method with the GB method.1.Determination of Nine Volatile N-Nitrosamines in Meat Products by Solid-Phase microextraction(SPME)with GC-NPD.The best extraction conditions of the nine N-nitrosamines(N-nitrosodimethylamine,N-nitrosodiethylamine,N-nitrosopyrrolidine,N-nitrosodipropylamine,N-nitrosodibutylamine,N-nitrosopiperidine,N-nitrosomethy lethylamine,N-nitrosomorpholine,and N-nitrosodiphenylamine)were determined by optimization of equilibration time,extraction time,extraction temperature,NaCl concentration,stirring speed and analysis time.The results showed that the best extraction effect can be obtained when the extraction fiber used PDMS/DVB/CAR,the equilibrium time was 10 min,the extraction temperature was 40 ℃,the extraction time was 30 min,the stirring speed was 400 r/min,the salt concentration was 0.36 g/mL and the analytical time was 3 min.The linear correlation coefficients of the 9 kinds of N-nitrosamines were determined by SPME with GC-NPD in the range of 0.9964 to 0.9996,the detection limit was 0.01～10 ng/mL and the limit of quantification was 0.03～33 ng/mL,the recoverie was in the range of 66.76～90.4%,the RSD was 3.27～7.62%.2.Adopting high performance liquid chromatography(HPLC)with UV detector to separat and detect the 9 N-nitrosamines.Through optimizing the mobile phase type,ratio of mobile phase,the mobile phase pH and sample solvent to determine the optimum separation conditions of 9 kinds of N-nitrosoamines.The results show that adopting the HPLC-UV,external standard method to quantitate,between 0.1～4 μg/mL,the linear correlation coefficient is above 0.9995,the HPLC-UV of the detection limit is 0.01～0.085 μg/mL instrument 9 N-nitrosamines,the limit of quantification was 0.033～0.283μg/mL,the parallel test of relative standard deviation(RSD)was 0.03%～0.29%,and the method does not require derivatization,strong operability,high sensitivity,good reproducibility,it is suitable for separation and detection of 9 kinds of N-nitrosoamines.3.A solid phase extraction and liquid chromatography with ultraviolet detection(SPE-HPLC-UV)for simultaneous determination in meat products of NDMA,NMEA,NDEA,NDPA,NDBA,NPIP,NPYR,NMOR and NDpheA methods 9 N-nitrosoamines.Using dichloromethane as extraction reagent to investigate the effect of extraction consisting of extraction time,solvent amount and extraction power,compared the purification effect of C18,neutral alumina and activated carbon for ultrasonic extraction of sample.The results showed that using 40 mL dichloromethane,400 W,extract 20 min,the peak area of N-nitrosamines reached the maximum.And finally through the recovery experiment selected activated carbon solid-phase extraction as sample purification.The recovery of the method was 86%一99%,and the precision was 1.19～3.42%,the method is simple and rapid,with good sensitivity,repeatability,and can be applied to simultaneous determination of content in meat products of 9 kinds of N-nitrosamines compounds.4.Through the comparison of SPME-GC-NPD,SPE-GC-NPD and SPE-HPLC-UV and the national standard method in the determination of N-nitrosamines in samples shows that SPME-GC-NPD can detect N-nitrosoamines type less,more suitable for rapid determination of NDMA,NMEA and NDEA in meat products.The LOD of UE-SPE-GC-NPD was lower compared with UE-SPE-HPLC-UV methods,so it was more suitable to detect the lower content of N-nitrosamines in meat.For the same sample,the N-nitrosamines content of three methods optimised was higher lightly compared with GB method,but the species of N-nitrosamines was less to the GB method,so we choice the method to detect the N-nitrosamines should base the specific circumstance.更多还原