CN108896694B - 一种动物性食品中农药残留的LC-QToF-MS筛查分析方法 - Google Patents
一种动物性食品中农药残留的LC-QToF-MS筛查分析方法 Download PDFInfo
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- CN108896694B CN108896694B CN201810729412.7A CN201810729412A CN108896694B CN 108896694 B CN108896694 B CN 108896694B CN 201810729412 A CN201810729412 A CN 201810729412A CN 108896694 B CN108896694 B CN 108896694B
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/89—Inverse chromatography
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
本发明公开了一种动物性食品中农药残留的LC‑QToF‑MS筛查分析方法。它包括如下步骤:(1)提取:将动物性食品试样均质后与提取溶剂混合,经振荡提取得到提取液,然后将所述提取液经除水、离心后,取上清液;(2)净化:将步骤(1)得到的上清液与净化剂混合进行净化,收集上清液,然后对得到的所述上清液依次进行加水稀释、过滤,得到待液样液a;(3)色谱质谱检测:对所述待测样液a采用反相液相色谱法对农药残留进行分离;然后采用带有ESI离子源的QToF‑MS筛查分析所述农药残留,外标法进行定量,即得到动物性食品中的农药残留含量。本发明操作简单、灵敏度高、重现性好,可广泛应用于动物性食品中多种农药残留的筛查分析。
Description
技术领域
本发明涉及一种动物性食品中农药残留的LC-QToF-MS筛查分析方法,属于食品卫生、农产品质量安全检测领域。
背景技术
动物性食品不仅面临公众熟知的兽药残留污染风险,也可能经由很多途径受到农药的污染。例如,在饲料类作物种植、贮存过程中使用的除草剂、杀虫剂、杀鼠剂等,可能会通过食物链转移至动物性食品中;动物的饲养过程中,也可能会使用杀螨剂喷洒笼舍以控制体外寄生虫,造成这些农药在动物性食品中的残留,并最终经由食物链的富集作用导致其在人体内的累积,威胁人类健康。因此,如何快速、高通量、高精度地筛查分析动物性食品中的农药残留,是食品检测与分析工作面临的新的挑战。
目前对动物性食品中农药残留筛查分析方法的研究较少,报道的分析方法主要是对蔬菜、水果中农药残留的检测分析。相比蔬菜、水果等,动物性食品产品基质成分较为复杂,且理化性质各不相同,传统的提取方法,如索氏提取法、固液萃取法、液液萃取法等,往往步骤繁琐、耗时较长且需要消耗大量有机溶剂。QuEChERS提取净化方法中,常用的传统吸附剂伯仲胺(PSA)、石墨化炭黑(GCB)等因为会对含有特殊结构的农药分子产生强烈的吸附效应,效果也很局限。
发明内容
本发明的目的是提供一种动物性食品中农药残留的LC-QToF-MS筛查分析方法,本发明操作简单、灵敏度高、重现性好,可广泛应用于动物性食品中多种农药残留的筛查分析。
本发明提供的一种动物性食品中农药残留的LC-QToF-MS筛查分析方法,包括如下步骤:
(1)提取:将动物性食品试样均质后与提取溶剂混合,经振荡提取得到提取液,然后将所述提取液经除水、离心后,取上清液;
(2)净化:将步骤(1)得到的上清液与净化剂混合进行净化,收集上清液,然后对得到的所述上清液依次进行加水稀释、过滤,得到待液样液a;
(3)色谱质谱检测:对所述待测样液a采用反相液相色谱法对农药残留进行分离;然后采用带有ESI离子源的QToF-MS筛查分析所述农药残留,外标法进行定量,即得到动物性食品中的农药残留含量。
本发明中,LC-QToF-MS,中文名称为液相色谱-四级杆飞行时间质谱。
上述的方法中,所述动物性食品包括鸡肉、猪肉、牛肉、羊肉、鸭肉、鹅肉、鸡蛋、鸭蛋、鹅蛋、鹌鹑蛋、鸡肝、猪肝和羊肝中的至少一种;
所述提取溶剂包括乙腈、含有1%甲酸的乙腈或含有1%乙酸的乙腈;
所述净化剂包括EMR-Lipid。
本发明中,所述EMR-Lipid的英文全称为Enhanced Matrix Removal-Lipid,中文翻译为新型分散固相萃取材料。
本发明中,所述含有1%甲酸的乙腈、所述含有1%乙酸的乙腈中百分含量均以体积百分比计。
上述的方法中,所述动物性食品试样的质量与所述提取溶剂的体积比可为1g:2~4mL,具体可为1g:2mL;
所述振荡提取的时间可为10~60s,具体可为10s。
上述的方法中,步骤(1)中,对所述提取液进行除水,在提取液中加入过量的无水硫酸镁(MgSO4)、干燥的氯化钠(NaCl),以及质量比可为2:1的柠檬酸钠和柠檬酸二钠盐的混合物,所述除水的时间可为1~5min,具体可为1min;
对所述提取液进行离心的温度可为0~4℃,具体可为4℃,转速可为2500~5000×g,具体可为4000×g,时间可为5~10min,具体可为5min。
上述的方法中,步骤(2)中还包括对净化后收集的所述上清液加入过量的无水MgSO4和干燥的NaCl进行干燥的步骤;
步骤(2)中还包括将所述净化剂与水混合活化5~15s的步骤,具体可为活化10s;所述净化剂与所述水的体积比可0.4g:5~10mL,具体可为0.4g:5mL;
步骤(1)得到的所述上清液的体积与所述净化剂的质量比可5mL:0.4~0.8g,具体可为5mL:0.4g;
所述净化的离心的温度可为0~4℃,具体可为4℃,转速可为2500~5000×g,具体可为4000×g,时间可为5~10min,具体可为5min;
步骤(2)中收集所述上清液进行离心的温度可为0~4℃,具体可为4℃,转速可为2500~5000×g,具体可为4000×g,时间可为5~10min,具体可为5min。
本发明中,步骤(2)中所述净化的操作具体可为混合涡旋。
上述的方法中,稀释时,步骤(2)中收集的所述上清液与所述水的体积比为4:1~3;所述过滤采用0.22μm滤膜。
上述的方法中,所述反相液相色谱法采用的色谱柱为C18反相液相色谱柱。所述反相色谱柱具体可为规格为2.1mm×100mm、2.7μm的Poroshell 120 EC-C18反相色谱柱。
上述的方法中,所述反相液相色谱法的色谱条件液如下:流动相A为含5mmol/L甲酸铵和0.1%甲酸的水溶液;流动相B为含5mmol/L甲酸铵和0.1%甲酸的乙腈溶液;梯度洗脱程序为:0min:5%B,0.5min:5%B,3.5min:50%B,17min:100%B,20min:100%B,20.1min:5%B,后运行2min;流速:0.4mL/min;柱温:35℃;进样量:2μL。
本发明中,所述流动相A和所述流动相B的含量均按照本领域公知的体积百分比计。
上述的方法中,所述带有ESI离子源的QToF-MS方法的质谱条件如下:QToF-MS质谱仪的离子源喷雾电压:3500V;离子源温度:450℃;干燥气流量6L/min;鞘流气流速11L/min;鞘流气温度为350℃;喷雾气压35psig;全扫描质荷比为50~1700,并使用内标参比溶液对仪器质量精度进行实时校正,通过Agilent MassHunter Workstation Software对质谱检测进行数据采集与处理。
本发明中,所述LC-QTOF-MS质谱仪具体可为Agilent 6545和6530型号。
上述的方法中,所述农药残留包括除草剂、杀虫剂、杀菌剂、杀螨剂、杀鼠剂、杀线虫剂和植物生长调节剂类别中的至少一种;
所述除草剂包括2,4-D、(Z)-嘧草醚、阿特拉通、胺苯磺隆、胺唑草酮、稗草胺、稗草畏、苯草醚、苯草酮、苯达松、苯磺隆、苯嗪草酮、苯噻草胺、苯酮唑、苯氧乙酸、吡草胺、吡草酮、吡氟草胺、吡氟甲禾灵、吡氟氯禾灵、吡嘧磺隆、吡唑草胺、吡唑解草酯、吡唑特、苄草丹、苄草隆、苄草唑、苄磺隆、丙苯磺隆、丙草胺、草不隆、草达津、草净津、除草丹、除草定、除草醚、哒草特、达草止代谢产物、得杀草、敌稗、敌草胺、敌草净、敌草隆、敌乐胺、地乐胺、地散磷、叠氮净、丁草胺、丁草特、丁嗪草酮、啶嘧磺隆、毒草安、毒莠定、恶草灵、恶唑隆、二丙烯草胺、二甲吩草胺、二甲戊灵、二氯苯甲酰胺、二氯吡啶酸、二氯喹啉酸、非草隆、砜嘧磺隆、呋草酮、呋嘧醇、伏草隆、氟胺磺隆、氟吡甲禾灵、氟丙嘧草酯、氟草敏、氟草烟、氟草烟异辛酯、氟哒嗪草酯、氟丁酰草胺、氟啶酮、氟咯草酮、氟禾灵、氟磺胺草醚、氟乐灵、氟硫草定、氟氯氢菊脂、氟噻草胺、氟烯草酸、禾草敌、禾草灵、禾草畏、环丙津、环丙嘧磺隆、环草隆、环草特、环庚草醚、环磺酮、环嗪酮、环莠隆、磺草胺唑、磺草灵、磺草酮、磺乐灵、磺噻隆、磺酰草吡唑、甲磺胺磺隆、甲磺隆、甲基苯噻隆、甲基碘磺隆钠盐、甲基氟嘧磺隆、甲基咪草烟、甲羧除草醚、甲酰胺磺隆、甲氧丙净、甲氧隆、甲氧咪草烟、甲氧醚隆、解草酮、解毒喹、精吡氟禾草灵、精恶唑禾草灵、精喹禾灵、卡草胺、克草猛、枯草隆、喹草酸、喹禾糖酯、利谷隆、亮氨酸、另丁津、隆草特、绿谷隆、绿麦隆、氯苯胺灵、氯吡嘧磺隆、氯草敏、氯氟草醚乙酯、氯磺隆、氯嘧磺隆、氯酯磺草胺、麦草伏甲酯、麦草伏异丙酯、麦草畏、咪草酸、咪草烟、咪唑磺隆、咪唑烟酸、咪唑乙烟酸、醚苯磺隆、醚黄隆、嘧苯胺磺隆、灭草喹、灭草隆、灭藻醌、牧草胺、萘丙胺、脲嘧啶、哌草磷、扑草净、扑灭津、扑灭通、嗪草酸甲酯、氰氟草酯、炔草酯、乳氟禾草灵、噻草定、噻吩草胺、噻氟隆、噻磺隆、噻酮磺隆、赛克津、三氟羧草醚、杀草胺、杀草丹、双苯恶唑酸、双苯酰草胺、双氟磺草胺、特草定、特草克、特丁净、特丁噻草隆、特丁通、甜菜安、甜菜宁、肟草酮、戊炔草胺、戊酰苯草胺、西草净、西玛津、西玛通、烯草酮、烯禾啶、酰嘧磺隆、辛酰溴苯腈、新燕灵、溴苯腈、溴丁酰草胺、溴谷隆、溴杀草敏、烟嘧磺隆、野草枯、野麦畏、乙草胺、乙羧氟草醚、乙氧苯草胺、乙氧氟草醚、乙氧嘧磺隆、异丙草胺、异丙甲草胺、异丙净、异丙乐灵、异丙隆、异恶草胺、异恶唑草酮、异戊乙净、抑草磷、抑草生、莠灭净、莠去津、仲丁通、唑草酮和唑嘧磺草胺中的至少一种;
所述杀虫剂包括3-酮呋喃丹、3-羟基克百威、S-氰戊菊酯、Z-甲基毒虫畏、Z-氯氰菊酯、胺丙畏、胺菊酯、巴丹、倍硫磷、倍硫磷砜、倍硫磷亚砜、苯胺磷砜、苯线磷、苯线磷亚砜、苯氧菊酯、苯氧威、吡丙醚、吡虫啉、吡螨胺、吡蚜酮、吡唑硫磷、避蚊胺、苄呋菊酯、丙虫磷、丙硫克百威、丙硫特普、丙烯菊酯、丙烯酰胺、丙溴磷、残杀威、虫酰肼、除虫脲、哒嗪硫磷、氘代克百威、敌百虫、敌敌畏、敌蝇威、地安磷、地虫硫磷、丁二酸二丁酯、丁基嘧啶磷、丁硫克百威、丁酮砜威、丁酮威、丁烯磷、啶虫丙醚、毒虫畏、毒杀芬、毒死蜱、对硫磷、对氧磷、多杀菌素、恶虫酮、恶虫威、恶唑磷、二甲基二硫醚、二嗪农、发硫磷、伐灭磷、酚线磷、丰索磷、砜吸磷、砜吸磷亚砜、呋虫胺、呋喃虫酰肼、呋线威、伏杀磷、氟胺氰菊酯、氟苯嘧啶醇、氟虫腈、氟虫脲、氟环脲、氟铃脲、氟氯苯菊酯、氟乙酰胺、氟蚁腙、高效氯氰菊酯、庚烯磷、谷硫磷、环虫酰肼、磺吸磷、混灭威、加普胺、甲胺磷、甲拌磷、甲丙硫磷、甲基吡啶磷、甲基毒死蜱、甲基对硫磷、甲基对氧磷、甲基嘧啶磷、甲硫威、甲硫威砜、甲氯磷、甲萘威、甲氰菊酯、甲氧虫酰肼、久效磷、久效威、抗蚜威、克百威、克敌菊酯、克来范、喹硫磷、乐果、联苯菊酯、莨菪碱、邻苯二甲酸丁苄酯、邻苯二甲二丁酯、邻苯二甲酸二环己酯、磷酸三苯酯、硫环磷、硫磷嗪、硫双威、氯氟氰菊酯、氯甲亚胺硫磷、氯菊酯、氯氰菊酯、氯烯炔菊酯、氯辛硫磷、氯唑磷、马拉代氧气磷、马拉松、猛杀威、醚菊酯、嘧啶磷、灭多威、灭害威、灭克磷、灭蚜磷、灭蝇胺、内吸磷、内吸硫磷、氰戊菊酯、炔丙菊酯、噻虫啉、噻虫嗪、噻嗪酮、噻唑磷、赛硫磷、三氟氯氰菊酯、三硫磷、三溴磷、三唑磷、杀虫丹砜、杀虫磺、杀虫脒、杀虫畏、杀扑磷、杀线威、莎稗磷、生物苄呋菊酯、生物丙烯菊酯、双硫磷、水胺硫磷、顺式氯氰菊酯、四氟氯甲醚、四氟醚菊酯、四氯苯醌、速灭磷、速灭威、特丁磷砜、特丁硫磷、涕灭威、涕灭威砜、五氯苯甲腈、西拉硫磷、烯啶虫胺、辛硫磷、溴氰菊酯、蚜灭多、蚜灭磷亚砜、亚胺硫磷、氧化乐果、氧环唑、氧异柳磷、乙基谷硫磷、乙基溴硫磷、乙基异柳磷、乙硫苯威、乙硫磷、乙霉威、乙酰甲胺磷、乙氧嘧啶磷、异丙威、异恶隆、抑食肼、茚虫威、蝇毒磷、右旋苯醚菊酯、鱼藤酮、育畜磷、治螟磷、仲丁威、兹克威和唑虫酰胺中的至少一种;
所述杀菌包括(E)-苯氧菌胺、(Z)-苯氧菌胺、百克敏、苯菌灵、苯醚甲环唑、苯噻菌胺、苯霜灵、苯酰菌胺、苯锈啶、比芬诺、吡嘧磷、卞氯三唑醇、丙环唑、丙硫菌唑、丙森锌、赤霉素、春雷霉素、代森联、代森锰锌、代森银锌、稻瘟灵、丁苯吗啉、啶氧菌酯、多菌灵、噁唑菌酮、恶霜灵、二甲嘧酚、粉唑醇、呋氨丙灵、呋菌胺、呋酰胺、氟苯尼考、氟吡菌酰胺、氟啶胺、氟硅唑、氟环唑、氟菌唑、氟喹唑、氟醚唑、氟酰胺、福美双、腐霉利、咯喹酮、硅氟唑、环丙嘧啶醇、环丙唑醇、磺嘧菌灵、己唑醇、甲基立枯磷、甲基硫菌灵、甲霜灵、腈苯唑、腈菌唑、精甲霜灵、井冈霉素、菌核净、糠菌唑、克菌丹、克瘟散、喹氧灵、联氨恶唑酮、联苯三唑醇、邻苯基苯酚、邻碘酰苯胺、硫菌灵、六氯代苯、螺环菌胺、氯苯嘧啶醇、氯硝胺、麦穗宁、咪鲜胺、咪鲜胺锰盐、咪唑菌酮、咪唑嗪、嘧菌胺、嘧菌环胺、嘧菌酯、嘧霉胺、灭菌丹、灭菌磷、灭菌唑、灭锈胺、氰菌胺、噻苯咪唑、噻苯咪唑-5-羟基、噻枯唑、噻唑菌胺、三苯基氢氧化锡、三环唑、三唑醇、三唑酮、十二环吗啉、十三吗啉、双炔酰菌胺、霜霉灭、霜霉威、霜脲氰、土菌灵、萎锈灵、肟菌酯、戊菌隆、戊菌唑、戊唑醇、烯酰吗啉、烯唑醇、新燕灵、亚胺唑、氧化萎锈灵、叶菌唑、乙呋草黄、乙环唑、乙嘧酚、异稻瘟净、异菌脲、抑霉唑、酯菌胺、种菌唑和唑嘧菌胺中的至少一种;
所述杀螨剂包括阿维菌素、苯螨特、虫螨腈、哒螨灵、丁氟螨酯、丁醚脲、啶虫脒、氟啶脲、甲氨基阿维菌素苯甲酸盐、焦磷酸四乙酯、可尼丁、克螨特、喹螨醚、联苯肼酯、螺螨酯、嘧螨醚、噻螨酮、三氯杀螨醇、三氯杀螨砜、三唑锡、杀铃脲、杀螟丹、虱螨脲、双甲脒、霜螨灵、四螨嗪、溴螨酯和唑螨酯中的至少一种;
所述杀鼠剂包括氟乙酸、杀鼠灵、杀鼠醚、溴敌隆、溴鼠灵和溴鼠隆中的至少一种;
所述杀线虫剂包括1,2-二溴乙烷、阿苯达唑、硫线磷和棉隆中的至少一种;
所述植物生长调节剂包括3-吲哚丙酸、3-吲哚丁酸、3-吲哚乙酸、6-苄氨基嘌呤、矮壮素、胺鲜酯、丁酰肼、多效唑、激动素、甲哌翁、抗倒胺、抗倒酯、氯吡脲、萘乙酸、萘乙酰胺、青鲜素、赛苯隆、三丁氯苄膦、缩节胺、甜菜碱、脱落酸、脱叶磷、烯效唑、乙烯利、异恶草松、抑芽唑、玉米素和植酸中的至少一种。
本发明能筛查分析动物性食品中597种农药残留。
本发明具有以下优点:
本发明针对动物性食品中脂质含量高、基质复杂等特点建立了一种动物性食品中农药残留检测的方法。该方法操作简单、灵敏度高、重现性好,可广泛应用于动物性食品中多种农药残留的筛查分析。本发明采用乙腈作为提取剂,EMR-Lipid作为净化剂,配制工作溶液使用外标法定量,实现了动物性食品中多种农药残留的确证及定量分析。并且该方法具有回收率高、精密度好、适用性强、经济快速等特点,各项技术指标均能满足多类动物性食品日常检测分析的要求,为食品安全领域提供了重要的技术支持。
附图说明
图1为利用LC-QToF-MS方法筛查分析动物性食品中136种农药残留的提取离子流色谱(EIC)叠加图。
图2为使用不同提取溶剂时,136种农药残留回收率的统计直方图。
图3为使用不同提取盐时,136种农药残留回收率的统计直方图。
图4为使用不同净化方式时,136种农药残留回收率的统计直方图。
图5为不同稀释或浓缩倍数时,136种农药残留回收率的统计直方图。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中,1、仪器:LC-QToF-MS(美国Agilent 1290-6545);涡旋仪(德国IKA公司);氮气吹干仪(美国Zymark公司);Milli.Q超纯水器(美国Millipore公司);匀浆机(瑞典Electrolux公司);电子天平(德国Sartorius公司);高速离心机(美国Thermo公司)。
2、试剂:农药残留标准品采购于美国Chemtek公司(纯度96.2%-99.9%);净化剂(PSA、GCB、C18、EMR-Lipid)采购于美国Agilent公司。乙腈:色谱纯;丙酮:色谱纯;甲醇:色谱纯;甲酸(纯度≥99.0%):色谱纯;乙酸(纯度≥99.0%):色谱纯;甲酸铵(纯度≥99.0%):质谱纯;MgSO4:质谱纯;NaCl:质谱纯;柠檬酸钠:质谱纯;柠檬酸二钠盐:质谱纯;乙酸钠:质谱纯。
3、标准工作溶液的配制:
配制储备液:分别准确称取5mg标准品,用5mL色谱级的乙腈溶解,配制成1000μg/mL的储备液,0~4℃冰箱避光、密封保存,保存期为6个月。
配制工作溶液:准确移取适量储备液,使用时用体积比95:5的水和乙腈的混合溶液稀释配制成不同浓度工作溶液,现配现用。
实施例1、
1、仪器工作条件:
色谱条件:
流动相A为含5mmol/L甲酸铵和0.1%甲酸的水溶液;流动相B为含5mmol/L甲酸铵和0.1%甲酸的乙腈溶液;梯度洗脱程序为:0min:5%B,0.5min:5%B,3.5min:50%B,17min:100%B,20min:100%B,20.1min:5%B,后运行2min;流速为0.4mL/min;柱温:35℃;进样量:2μL。
质谱条件:
Agilent 6545 LC-QToF-MS质谱仪的离子源喷雾电压:3500V;离子源温度:450℃;干燥气流量6L/min;鞘流气流速11L/min;鞘流气温度为350℃;喷雾气压35psig;全扫描质荷比范围为50~1700,并使用内标参比溶液对仪器质量精度进行实时校正,通过AgilentMassHunter Workstation Software对质谱检测进行数据采集与处理。136种农药残留的分子式、质荷比、保留时间等信息如表1所示。
表1 136种农药残留的基本信息及检出限、定量限
2、样品前处理
(1)提取
在50mL离心管中准确称取5g(±0.05g)匀浆后的动物性食品样品(具体为鸡肉),并加入10mL乙腈,充分混合后涡旋10s,使样品与提取溶液充分接触。
在含有鸡肉的样品和乙腈的离心管中加入4g无水MgSO4、1g NaCl、1g柠檬酸钠和0.5g柠檬酸二钠盐,再将混合物在机械摇床上摇匀1min,4℃下4000×g离心5min后,取上清液备用。
(2)净化
称取400mg新型分散固相萃取材料(英文名称为Enhanced Matrix Removal-Lipid,简称EMR-Lipid),并加入5mL水,涡旋10s进行活化。再取5mL提取所得的上清液,与活化后的EMR-Lipid混合涡旋1min,4℃下4000×g离心5min。取5mL上清液,与400mg NaCl、1600mg MgSO4充分混合后涡旋1min,4℃下4000×g离心5min。取800μL上清液,加入200μL的超纯水,混合均匀后,经0.22μm滤膜过滤,得到待测样品溶液a,待上机检测。
3、实验结果
(1)提取步骤的优化
1)提取溶剂的优化
农药类化合物可溶于多种有机试剂,实验中对乙腈、乙腈(1%甲酸)、乙腈(1%乙酸)、甲醇、丙酮五种提取溶剂进行了比较,结果见表2。以回收率的范围为横坐标,以在该范围内农药的个数为纵坐标,制作使用五种不同提取溶剂时,136种农药残留回收率的统计直方图,如图2所示。当甲醇作为提取溶剂时,提取溶液中基质成分复杂,不利于净化,且回收率较差。乙腈、乙腈(1%甲酸)、乙腈(1%乙酸)的提取效果相似,但当乙腈作为提取溶剂时,各农药残留的回收率更好,更利于动物性食品中农药残留的筛查分析。另外,直接用乙腈作为提取溶剂可简化实验步骤。因此,实验优选乙腈为提取溶剂。
表2不同提取溶剂对回收率的影响
2)提取盐的优化
无水MgSO4常用于除去基质中的水分,由于乙腈和水互溶,加入NaCl等电解质盐有助于乙腈和水的分层,而柠檬酸钠和柠檬酸二钠盐作为缓冲剂,利于维持PH的稳定。实验中对比了4g MgSO4、1g NaCl、1g柠檬酸钠、0.5g柠檬酸二钠盐(标记为第1组),以及6g MgSO4、1.5g乙酸钠(标记为第2组)的提取效率,发现第1组的提取效果高于第2组(结果见表3和图3)。因此,实验选择4g MgSO4、1g NaCl、1g柠檬酸钠和0.5g柠檬酸二钠盐为提取盐。
表3不同提取盐对回收率的影响
(2)净化方式的优化
动物性食品中脂质、蛋白质等含量高,这些成分复杂的基质会对农药残留的检测分析产生干扰,影响定量准确性,也可能对分析色谱柱造成伤害。因此,净化剂的选择至关重要。
本发明对常见的吸附剂及新型吸附剂进行组合,并配合不同剂量的除水剂,分成以下5组:
1、400mg EMR-Lipid(新型吸附剂)、400mg NaCl、1600mg MgSO4
2、400mg PSA、1200mg MgSO4
3、400mg PSA、400mg C18、45mg GCB、1200mg MgSO4
4、400mg PSA、400mg C18、1200mg MgSO4
5、400mg C18、1200mg MgSO4
通过对比可以发现,当选用EMR-Lipid作为净化剂时,回收率结果最优(结果见表4和图4)。因此实验选择EMR-Lipid为净化剂。
表4不同净化方式对回收率的影响
(3)浓缩和稀释的影响
浓缩和稀释会对农药残留的回收率产生影响。实验设立了5组稀释和浓缩实验的对比,分别为原液、稀释1.25倍、稀释2倍、氮吹浓缩1.25倍和氮吹浓缩2倍。优化结果所示,当稀释1.25,即取800μL原液与200μL的超纯水混合时,基质效应最小,回收率最优(结果见表5和图5)。因此,实验将方法最终离心获得的上清液取800μL与200μL超纯水混合稀释后,过滤备用。
表5稀释、浓缩倍数对回收率的影响
(4)方法学考察
采用外标法定量,以目标农药残留的峰面积对相应的质量浓度,绘制标准曲线,线性相关系数均大于0.99,表明该筛查分析方法中具有良好的线性。根据3倍信噪比确定方法检出限,10倍信噪比确定方法定量限,动物性食品中136种农药残留的检出限和定量限详见表1。
取空白动物性食品(不含有农药残留的动物性食品)分别添加0.05、0.1、0.2mg/kg工作溶液,进行3个水平上6次平行加标的回收实验)。由表6可见,在高、中、低三个水平中,动物性食品中加标回收率在80%-120%范围内的农药分别为125、125和124个,方法准确性较好。所有农药的日内和日间相对标准偏差RSD值均小于20%,表明方法重复性较好。因此,该方法具有良好的准确性和重复度,符合食品卫生和农产品质量安全检测的要求。
表6不同添加浓度对应的回收率
本发明建立了一种基于QuEChERS的前处理方法,并利用LC-QToF-MS筛查分析动物性食品中的多种农药残留。本方法采用乙腈作为提取剂,EMR-Lipid作为净化剂,配制标准溶液校准曲线后利用外标法定量,实现了动物性食品中136种农药残留的确证及定量分析。并且该方法具有回收率高、重复性好、适用性强、简单快速等特点,各项技术指标均能满足动物性食品日常检测分析的要求,同时为食品安全领域提供了重要的技术支持。
上述虽然结合附图对本发明的具体实施方式进行了描述,但并非对本发明保护范围的限制,在本发明的技术方案的基础上,本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围以内。
Claims (1)
1.一种动物性食品中农药残留的LC-QToF-MS筛查分析方法,包括如下步骤:
(1)提取:将动物性食品试样均质后与提取溶剂混合,经振荡提取得到提取液,然后将所述提取液经除水、离心后,取上清液;
所述动物性食品包括鸡肉、猪肉、牛肉、羊肉、鸭肉、鹅肉、鸡肝、猪肝和羊肝中的至少一种;
所述提取溶剂为乙腈、含有1%甲酸的乙腈或含有1%乙酸的乙腈;
所述动物性食品试样的质量与所述提取溶剂的体积比为1 g:2~4 mL;
所述振荡提取的时间为10~60 s;
步骤(1)中,对所述提取液进行除水,在提取液中加入过量的无水MgSO4、干燥的NaCl,以及质量比为2:1的柠檬酸钠和柠檬酸二钠盐的混合物,所述除水时间为1~5 min;
对所述提取液进行离心的温度为0~4 ℃,转速为2500~5000 ×g,时间为5~10 min;
(2)净化:将步骤(1)得到的上清液与净化剂混合进行净化,收集上清液,然后对得到的所述上清液依次进行加水稀释、过滤,得到待测样液a;
所述净化剂包括EMR-Lipid;
步骤(2)中还包括对净化后收集的所述上清液加入过量的无水硫酸镁和干燥的氯化钠进行干燥的步骤;
步骤(2)中还包括将所述净化剂与水混合活化5~15 s的步骤;所述净化剂的质量与所述水的体积比为0.4 g:5~10 mL;
步骤(1)得到的所述上清液的体积与所述净化剂的质量比为5 mL:0.4~0.8 g;
所述净化后采用离心收集上清液;所述离心的温度为0~4 ℃,转速为2500~5000 ×g,时间为5~10 min;
稀释时,步骤(2)中收集的所述上清液与所述水的体积比为4:1~3;所述过滤采用0.22μm滤膜;
(3)色谱质谱检测:对所述待测样液a采用反相液相色谱法对农药残留进行分离;然后采用带有ESI离子源的QToF-MS筛查分析所述农药残留,外标法进行定量,即得到动物性食品中的农药残留含量;
所述反相液相色谱法采用的色谱柱为C18反相液相色谱柱;
所述反相液相色谱法的色谱条件如下:流动相A为含5 mmol/L甲酸铵和0.1%甲酸的水溶液;流动相B为含5 mmol/L甲酸铵和0.1%甲酸的乙腈溶液;梯度洗脱程序为:0 min:5%B,0.5 min:5%B,3.5 min:50%B,17 min:100%B,20 min:100%B,20.1 min:5%B,后运行2 min;流速:0.4 mL/min;柱温:35 ℃;进样量:2 μL;
所述带有ESI离子源的QToF-MS的质谱条件如下:QToF-MS质谱仪的离子源喷雾电压:3500 V;离子源温度:450 ℃;干燥气流量6 L/min;鞘流气流速11 L/min;鞘流气温度为350℃;喷雾气压35 psig;全扫描质荷比为50~1700,并采用内标参比溶液对仪器质量精度进行实时校正,通过Agilent MassHunter Workstation Software对质谱检测进行数据采集与处理;
所述农药残留包括除草剂、杀虫剂、杀菌剂、杀螨剂、杀鼠剂、杀线虫剂和植物生长调节剂;
所述除草剂包括2,4-D、(Z)-嘧草醚、阿特拉通、胺苯磺隆、胺唑草酮、稗草胺、稗草畏、苯草醚、苯草酮、苯达松、苯磺隆、苯嗪草酮、苯噻草胺、苯酮唑、苯氧乙酸、吡草胺、吡草酮、吡氟草胺、吡氟甲禾灵、吡氟氯禾灵、吡嘧磺隆、吡唑草胺、吡唑解草酯、吡唑特、苄草丹、苄草隆、苄草唑、苄磺隆、丙苯磺隆、丙草胺、草不隆、草达津、草净津、除草丹、除草定、除草醚、哒草特、达草止代谢产物、得杀草、敌稗、敌草胺、敌草净、敌草隆、敌乐胺、地乐胺、地散磷、叠氮净、丁草胺、丁草特、丁嗪草酮、啶嘧磺隆、毒草安、毒莠定、恶草灵、恶唑隆、二丙烯草胺、二甲吩草胺、二甲戊灵、二氯苯甲酰胺、二氯吡啶酸、二氯喹啉酸、非草隆、砜嘧磺隆、呋草酮、呋嘧醇、伏草隆、氟胺磺隆、氟吡甲禾灵、氟丙嘧草酯、氟草敏、氟草烟、氟草烟异辛酯、氟哒嗪草酯、氟丁酰草胺、氟啶酮、氟咯草酮、氟禾灵、氟磺胺草醚、氟乐灵、氟硫草定、氟氯氢菊脂、氟噻草胺、氟烯草酸、禾草敌、禾草灵、禾草畏、环丙津、环丙嘧磺隆、环草隆、环草特、环庚草醚、环磺酮、环嗪酮、环莠隆、磺草胺唑、磺草灵、磺草酮、磺乐灵、磺噻隆、磺酰草吡唑、甲磺胺磺隆、甲磺隆、甲基苯噻隆、甲基碘磺隆钠盐、甲基氟嘧磺隆、甲基咪草烟、甲羧除草醚、甲酰胺磺隆、甲氧丙净、甲氧隆、甲氧咪草烟、甲氧醚隆、解草酮、解毒喹、精吡氟禾草灵、精恶唑禾草灵、精喹禾灵、卡草胺、克草猛、枯草隆、喹草酸、喹禾糖酯、利谷隆、亮氨酸、另丁津、隆草特、绿谷隆、绿麦隆、氯苯胺灵、氯吡嘧磺隆、氯草敏、氯氟草醚乙酯、氯磺隆、氯嘧磺隆、氯酯磺草胺、麦草伏甲酯、麦草伏异丙酯、麦草畏、咪草酸、咪草烟、咪唑磺隆、咪唑烟酸、咪唑乙烟酸、醚苯磺隆、醚黄隆、嘧苯胺磺隆、灭草喹、灭草隆、灭藻醌、牧草胺、萘丙胺、脲嘧啶、哌草磷、扑草净、扑灭津、扑灭通、嗪草酸甲酯、氰氟草酯、炔草酯、乳氟禾草灵、噻草定、噻吩草胺、噻氟隆、噻磺隆、噻酮磺隆、赛克津、三氟羧草醚、杀草胺、杀草丹、双苯恶唑酸、双苯酰草胺、双氟磺草胺、特草定、特草克、特丁净、特丁噻草隆、特丁通、甜菜安、甜菜宁、肟草酮、戊炔草胺、戊酰苯草胺、西草净、西玛津、西玛通、烯草酮、烯禾啶、酰嘧磺隆、辛酰溴苯腈、新燕灵、溴苯腈、溴丁酰草胺、溴谷隆、溴杀草敏、烟嘧磺隆、野草枯、野麦畏、乙草胺、乙羧氟草醚、乙氧苯草胺、乙氧氟草醚、乙氧嘧磺隆、异丙草胺、异丙甲草胺、异丙净、异丙乐灵、异丙隆、异恶草胺、异恶唑草酮、异戊乙净、抑草磷、抑草生、莠灭净、莠去津、仲丁通、唑草酮和唑嘧磺草胺;
所述杀虫剂包括3-酮呋喃丹、3-羟基克百威、S-氰戊菊酯、Z-甲基毒虫畏、Z-氯氰菊酯、胺丙畏、胺菊酯、巴丹、倍硫磷、倍硫磷砜、倍硫磷亚砜、苯胺磷砜、苯线磷、苯线磷亚砜、苯氧菊酯、苯氧威、吡丙醚、吡虫啉、吡螨胺、吡蚜酮、吡唑硫磷、避蚊胺、苄呋菊酯、丙虫磷、丙硫克百威、丙硫特普、丙烯菊酯、丙烯酰胺、丙溴磷、残杀威、虫酰肼、除虫脲、哒嗪硫磷、氘代克百威、敌百虫、敌敌畏、敌蝇威、地安磷、地虫硫磷、丁二酸二丁酯、丁基嘧啶磷、丁硫克百威、丁酮砜威、丁酮威、丁烯磷、啶虫丙醚、毒虫畏、毒杀芬、毒死蜱、对硫磷、对氧磷、多杀菌素、恶虫酮、恶虫威、恶唑磷、二甲基二硫醚、二嗪农、发硫磷、伐灭磷、酚线磷、丰索磷、砜吸磷、砜吸磷亚砜、呋虫胺、呋喃虫酰肼、呋线威、伏杀磷、氟胺氰菊酯、氟苯嘧啶醇、氟虫腈、氟虫脲、氟环脲、氟铃脲、氟氯苯菊酯、氟乙酰胺、氟蚁腙、高效氯氰菊酯、庚烯磷、谷硫磷、环虫酰肼、磺吸磷、混灭威、加普胺、甲胺磷、甲拌磷、甲丙硫磷、甲基吡啶磷、甲基毒死蜱、甲基对硫磷、甲基对氧磷、甲基嘧啶磷、甲硫威、甲硫威砜、甲氯磷、甲萘威、甲氰菊酯、甲氧虫酰肼、久效磷、久效威、抗蚜威、克百威、克敌菊酯、克来范、喹硫磷、乐果、联苯菊酯、莨菪碱、邻苯二甲酸丁苄酯、邻苯二甲二丁酯、邻苯二甲酸二环己酯、磷酸三苯酯、硫环磷、硫磷嗪、硫双威、氯氟氰菊酯、氯甲亚胺硫磷、氯菊酯、氯氰菊酯、氯烯炔菊酯、氯辛硫磷、氯唑磷、马拉代氧气磷、马拉松、猛杀威、醚菊酯、嘧啶磷、灭多威、灭害威、灭克磷、灭蚜磷、灭蝇胺、内吸磷、内吸硫磷、氰戊菊酯、炔丙菊酯、噻虫啉、噻虫嗪、噻嗪酮、噻唑磷、赛硫磷、三氟氯氰菊酯、三硫磷、三溴磷、三唑磷、杀虫丹砜、杀虫磺、杀虫脒、杀虫畏、杀扑磷、杀线威、莎稗磷、生物苄呋菊酯、生物丙烯菊酯、双硫磷、水胺硫磷、顺式氯氰菊酯、四氟氯甲醚、四氟醚菊酯、四氯苯醌、速灭磷、速灭威、特丁磷砜、特丁硫磷、涕灭威、涕灭威砜、五氯苯甲腈、西拉硫磷、烯啶虫胺、辛硫磷、溴氰菊酯、蚜灭多、蚜灭磷亚砜、亚胺硫磷、氧化乐果、氧环唑、氧异柳磷、乙基谷硫磷、乙基溴硫磷、乙基异柳磷、乙硫苯威、乙硫磷、乙霉威、乙酰甲胺磷、乙氧嘧啶磷、异丙威、异恶隆、抑食肼、茚虫威、蝇毒磷、右旋苯醚菊酯、鱼藤酮、育畜磷、治螟磷、仲丁威、兹克威和唑虫酰胺;
所述杀菌包括(E)-苯氧菌胺、(Z)-苯氧菌胺、百克敏、苯菌灵、苯醚甲环唑、苯噻菌胺、苯霜灵、苯酰菌胺、苯锈啶、比芬诺、吡嘧磷、卞氯三唑醇、丙环唑、丙硫菌唑、丙森锌、赤霉素、春雷霉素、代森联、代森锰锌、代森银锌、稻瘟灵、丁苯吗啉、啶氧菌酯、多菌灵、噁唑菌酮、恶霜灵、二甲嘧酚、粉唑醇、呋氨丙灵、呋菌胺、呋酰胺、氟苯尼考、氟吡菌酰胺、氟啶胺、氟硅唑、氟环唑、氟菌唑、氟喹唑、氟醚唑、氟酰胺、福美双、腐霉利、咯喹酮、硅氟唑、环丙嘧啶醇、环丙唑醇、磺嘧菌灵、己唑醇、甲基立枯磷、甲基硫菌灵、甲霜灵、腈苯唑、腈菌唑、精甲霜灵、井冈霉素、菌核净、糠菌唑、克菌丹、克瘟散、喹氧灵、联氨恶唑酮、联苯三唑醇、邻苯基苯酚、邻碘酰苯胺、硫菌灵、六氯代苯、螺环菌胺、氯苯嘧啶醇、氯硝胺、麦穗宁、咪鲜胺、咪鲜胺锰盐、咪唑菌酮、咪唑嗪、嘧菌胺、嘧菌环胺、嘧菌酯、嘧霉胺、灭菌丹、灭菌磷、灭菌唑、灭锈胺、氰菌胺、噻苯咪唑、噻苯咪唑-5-羟基、噻枯唑、噻唑菌胺、三苯基氢氧化锡、三环唑、三唑醇、三唑酮、十二环吗啉、十三吗啉、双炔酰菌胺、霜霉灭、霜霉威、霜脲氰、土菌灵、萎锈灵、肟菌酯、戊菌隆、戊菌唑、戊唑醇、烯酰吗啉、烯唑醇、新燕灵、亚胺唑、氧化萎锈灵、叶菌唑、乙呋草黄、乙环唑、乙嘧酚、异稻瘟净、异菌脲、抑霉唑、酯菌胺、种菌唑和唑嘧菌胺;
所述杀螨剂包括阿维菌素、苯螨特、虫螨腈、哒螨灵、丁氟螨酯、丁醚脲、啶虫脒、氟啶脲、甲氨基阿维菌素苯甲酸盐、焦磷酸四乙酯、可尼丁、克螨特、喹螨醚、联苯肼酯、螺螨酯、嘧螨醚、噻螨酮、三氯杀螨醇、三氯杀螨砜、三唑锡、杀铃脲、杀螟丹、虱螨脲、双甲脒、霜螨灵、四螨嗪、溴螨酯和唑螨酯;
所述杀鼠剂包括氟乙酸、杀鼠灵、杀鼠醚、溴敌隆、溴鼠灵和溴鼠隆;
所述杀线虫剂包括1,2-二溴乙烷、阿苯达唑、硫线磷和棉隆;
所述植物生长调节剂包括3-吲哚丙酸、3-吲哚丁酸、3-吲哚乙酸、6-苄氨基嘌呤、矮壮素、胺鲜酯、丁酰肼、多效唑、激动素、甲哌翁、抗倒胺、抗倒酯、氯吡脲、萘乙酸、萘乙酰胺、青鲜素、赛苯隆、三丁氯苄膦、缩节胺、甜菜碱、脱落酸、脱叶磷、烯效唑、乙烯利、异恶草松、抑芽唑、玉米素和植酸。
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