纳米纤维固相萃取高效液相色谱荧光检测麻辣烫汤液中喹诺酮类药物

邓思维等
摘要建立了固相萃取高效液相色谱荧光检测麻辣烫汤液中5种喹诺酮类抗生素的分析方法。麻辣烫汤液样品经EDTAMcllvaine缓冲溶液(pH 4)提取后,以HCX固相萃取小柱净化富集,用水淋洗,2%氨化甲醇洗脱。采用高效液相色谱荧光检测器(HPLCFLD),于激发波长280 nm,发射波长450 nm处进行检测,流动相为甲醇水磷酸(25∶75∶0.1, V/V, 三乙胺调至pH 2.8)。麻辣烫汤液样品中氟罗沙星、诺氟沙星、沙拉沙星、环丙沙星、奥比沙星5种喹诺酮类抗生素加标回收率为72.1%~110.3%;日内相对标准偏差
1引言
喹诺酮类(4Quinolones),又称吡酮酸类或吡啶酮酸类,是一类人工合成的含4喹诺酮基本结构抗菌药, 被广泛应用于畜牧养殖业,主要用于治疗疾病,促进生长。然而,近年来畜牧业滥用抗生素现象十分严重\[1\],此类药物不仅残存在动物体内,逐渐增强细菌的耐药性,还会通过食物链的富集作用进入人体内,危害人体健康,损害消化系统、神经系统、免疫等系统\[2\]。
目前,对生物样品中喹诺酮类抗生素残留的分析方法研究较多\[3~9\],还有水、土壤、肥料等环境样品检测也有报道\[10~13\],主要采用C18柱、HLB柱固相萃取及液液萃取等前处理方法。在卫生部已发布的被滥用食品添加剂及非法添加物名单中包括喹诺酮类抗生素。麻辣烫原材料可能含有喹诺酮,不法商家为了防止顾客食用卫生不合格的麻辣烫后可能发生疾病,人为添加的喹诺酮。由于麻辣烫的原材料经烹煮之后汤液的基质成分复杂、油脂含量高,目前尚没有合适的检测方法来进行检测,因此也没有国家标准来对此类食品安全进行监督。国内虽有报道火锅底料中的喹诺酮的检测\[14\],但仅对其中各种食材进行检测,对其中的药物残留的分析方法尚未见报道。
为了克服复杂基质对目标物检测的干扰,本研究采用新型纳米纤维固相萃取技术,结合高效液相色谱法,直接对麻辣烫汤液中5种喹诺酮类药物,包括氟罗沙星(Fleroxacin, FLX)、诺氟沙星(Norfloxacin, NOR)、沙拉沙星(Sarafloxacin, SAR)、环丙沙星(Ciprofloxacin, CIP)和奥比沙星(Orbifloxacin, ORB)进行检测。纳米纤维固相萃取技术是本研究组建立的一种快速样品前处理方法,尤其适合于复杂样品的分析,此前的研究工作所用的纳米纤维多是单组份的纳米纤维\[15~19\],本研究利用复合组分的纳米纤维对复杂食品样品进行前处理。
2实验部分
2.1仪器与试剂
Shimadzu LC20AD高效液相色谱仪(日本岛津公司),包括RF10AXL荧光检测器、LC20AD双泵、SIL20AC自动进样器;台式离心机(上海安亭科学厂);梅特勒AB265S分析天平(杭州汇尔仪器设备有限公司);超声波清洗机(宁波新芝生物科技股份有限公司);雷磁PHSJ3FpH计(上海仪电科学仪器股份有限公司);阵列式固相萃取仪(苏州东奇生物科技有限公司)。
HCX固相萃取柱(磺化聚苯乙烯聚乙烯吡咯烷酮共纺物纤维填充柱,装量10mg,苏州东奇生物科技有限公司);甲醇(色谱纯);三乙胺、EDTA2Na,柠檬酸、乙酸、乙酸乙酯、乙腈等均为分析纯(江苏永华精细化学品有限公司);氟罗沙星、诺氟沙星、沙拉沙星、环丙沙星、奥比沙星标准品(阿拉丁公司);实验用水为三次蒸馏水。
2.2标准溶液的配制
准确称取适量氟罗沙星、诺氟沙星、沙拉沙星、环丙沙星和奥比沙星,加水配制成1 g/L的标准储备液,于4 ℃保存(有效期1个月)。测定前,用水稀释该储备液,制备系列标准溶液。
2.3样品前处理
准确移取麻辣烫汤液1 mL,加0.2 g EDTA2Na,超声10 min,以10000 r/min离心10 min,分取上清液;残渣再加200 μL水超声离心,合并上清液;上清液中加1 mL EDTAMcllvaine缓冲溶液,超声离心;取1 mL上清液加缓冲溶液稀释1倍,过HCX固相萃取柱。
操作阵列式固相萃取仪(图1):固相萃取柱使用前先用100 μL甲醇活化、200 μL蒸馏水冲洗。将样品上清液移入固相萃取柱中,手动操作阵列式固相萃取仪,利用空气加压,使液滴缓慢逐滴被压出。用200 μL水作为淋洗液去除杂质,再用100 μL 2%氨化甲醇洗脱液洗脱目标物。取10 μL洗脱液进行HPLC分析。
3.2固相萃取条件优化
3.2.1提取溶剂的选择[图2HCX柱中纳米纤维扫描电镜图
Fig.2Scanning electron microscopy of nanofibers in HCX column[TS)]麻辣烫汤液基质成分复杂,对HPLC检测的影响很大。本研究分别选取1 mL EDTAMcllvaine缓冲溶液(pH 4)、乙酸乙酯、含1%乙酸的乙腈和含10% HClO4的乙腈作为提取溶剂,先对麻辣烫汤液中喹诺酮类物质进行提取,然后进高效液相色谱检测。实验表明,EDTAMcllvaine缓冲溶液的提取效果最好。继续考察了EDTAMcllvaine缓冲溶液加入量的影响,分别加入0.50, 1.0和2.0 mL进行提取,以各目标峰面积相对于0.50 mLEDTAMcllvaine 缓冲溶液提取所得目标峰面积的比值为纵坐标作图。由图3可见,缓冲溶液缓冲溶液加入量为1.0 mL时提取效果最好。
3.2.2样液的稀释倍数加入EDTAMcllvaine缓冲溶液稀释样液可以减少杂质浓度,提高吸附剂对目标物的吸附效率。以各目标峰面积相对于稀释1倍所得目标峰面积的比值为纵坐标作图。由图4可见,样液稀释1倍能提高目标物的回收率。
3.3.2回收率与精密度取加标麻辣烫汤液1 mL,参照2.3节方法进行样品前处理,每个添加水平平行测定6次,计算回收率和日内相对标准偏差;连续测定6 d,计算回收率和日间相对标准偏差。结果见表2。
3.3.3可能的萃取机理喹诺酮类药物是一类极性相对较大的化合物,
本实验结果表明,HCX固相萃取柱在优化条件下能有效富集麻辣烫中喹诺酮类抗生素,该柱的填料为磺化聚苯乙烯聚乙烯吡咯烷酮共纺物纤维,装量仅为10 mg,就可以满足复杂样液中目标物的提取,并且操作中试剂用量较常见方法显著减少,省略了加热、氮吹、复溶等步骤,简化了操作环节,且对目标物有富集、浓缩、净化作用。这些优点都得益于纳米纤维填料优异的吸附/脱附特性。本研究根据HCX固相萃取柱填料的成分,推测其提取喹诺酮类抗生素机理如图6所示,其吸附目标物的作用可能如下: ①磺化后的聚苯乙烯聚乙烯吡咯烷酮共纺物亲水性增加,利于与极性较大的喹诺酮分子相互作用,增加了纤维的吸附效率;②聚苯乙烯聚乙烯吡咯烷酮上的羰基可与喹诺酮上的羧基形成氢键,加强吸附剂与目标分子相互间的吸引;③目标物在弱酸性条件下吸附效率高的事实说明此时喹诺酮分子上的氨基为阳离子时有利于固相萃取,而纤维上磺酸基的存在显然是该相互作用(离子交换)发生的必需基团; ④喹诺酮类药物与聚苯乙烯聚乙烯吡咯烷酮上均有苯环,相互间的ππ作用加强了吸附作用。因此多重相互作用是提取效率提高的保障,而静电纺丝显然是制备具有多重相互作用官能团新材料的一种有效而方便的技术。
3.3.4与其它固相萃取方法比较本方法与文献已报道的固相萃取方法相比(表3),所用纳米纤维装量少,操作时间短,消耗的试剂少,回收率指标也符合要求。因此本方法更加快速、简便且绿色环保。
3.4样品分析
以本方法对市场上数份样品进行检测,都未检出目标成分,取一份样品按照2.3节所述方法
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曹 鹏, 牟 妍, 高 飞, 耿金培, 张禧庆, 隋 涛, 梁君妮, 沙美兰, 关丽丽. 色谱, 2013, 31(9): 862-868
15Kang X J, Pan C, Zhang Y Y , Gu Z Z . Anal. Chim. Acta, 2007, 587(1): 75-81
16SUN Jing, MA YuQin, KANG XueJun. Environment Chemistry, 2010, 29(1): 105-108
孙 静, 马玉琴, 康学军. 环境化学, 2010, 29(1): 105-108
17QU ZiJian, KANG XueJun, WU WeiWei, CHEN BaoAn, GU ZhongZe. Chinese J. Anal. Chem.,2011, 39(3): 312-316
曲子健, 康学军, 吴玮玮, 陈宝安, 顾忠泽. 分析化学, 2011, 39(3): 312-316
18CHEN LiQin, KANG XueJun, YAN Yan, GU ZhongZe, LU ZuHong. Chinese J. Anal. Chem.,2012, 40(1): 124-128
陈利琴, 康学军, 晏 嫣, 顾忠泽, 陆祖宏. 分析化学, 2012, 40(1): 124-128
19Yan Y, Deng J J, Deng S W, Wang Y, Wang F, Xiao M, Kang X J. Journal of Southeast University ( English Edition),2012, 28(4): 464-468
20ZHAO SiJun, LI Cun, JIANG HaiYang, LI BingYu, SHEN JianZhong. Chinese J. Anal. Chem., 2007, 35(3): 786-790
赵思俊, 李 存, 江海洋, 李炳玉, 沈建忠. 分析化学, 2007, 35(3): 786-790
AbstractAn analytical method for simultaneous determination of five quinolones in spicy soup was developed. Spicy soup samples were firstly extracted by EDTAMcllvaine buffer at pH 4, then purified and concentrated by a novel Packed fiber solid phase extraction (PFSPE) coulumn. The extracted liquid supernatant was loaded onto the column, rinsed with water, and then eluted with 2% ammoniated methanol. The mobile phase was methanolwaterphosphoric acid (25∶75∶0.1, V/V, adjusting the pH to 2.8 with triethylamine). These analytes were quantified by high performance liquid chromatographyfluorimetric detector(HPLCFLD) at excitation and emission wavelength of 280 nm and 450 nm respectively. Recoveries of spiked quinolone antibiotics in spicy soup were from 72.1% to 110.3% with intraday relative standard deviation (RSD) between 1.6% and 4.3% and interday RSD from 2.0% to 4.3%. Limit of detection(LOD) and limit of quantitation(LOQ) were from 1.2 to 5.4 μg/L and from 3.9 to 18 μg/L, respectively. The method could be applied to determine the quinolones in spicy soup.
KeywordsSpicy soup; Quinolones; Packed fiber solid phase extraction; High performance liquid chromatography; Fluorimetric detection
曹 鹏, 牟 妍, 高 飞, 耿金培, 张禧庆, 隋 涛, 梁君妮, 沙美兰, 关丽丽. 色谱, 2013, 31(9): 862-868
15Kang X J, Pan C, Zhang Y Y , Gu Z Z . Anal. Chim. Acta, 2007, 587(1): 75-81
16SUN Jing, MA YuQin, KANG XueJun. Environment Chemistry, 2010, 29(1): 105-108
孙 静, 马玉琴, 康学军. 环境化学, 2010, 29(1): 105-108
17QU ZiJian, KANG XueJun, WU WeiWei, CHEN BaoAn, GU ZhongZe. Chinese J. Anal. Chem.,2011, 39(3): 312-316
曲子健, 康学军, 吴玮玮, 陈宝安, 顾忠泽. 分析化学, 2011, 39(3): 312-316
18CHEN LiQin, KANG XueJun, YAN Yan, GU ZhongZe, LU ZuHong. Chinese J. Anal. Chem.,2012, 40(1): 124-128
陈利琴, 康学军, 晏 嫣, 顾忠泽, 陆祖宏. 分析化学, 2012, 40(1): 124-128
19Yan Y, Deng J J, Deng S W, Wang Y, Wang F, Xiao M, Kang X J. Journal of Southeast University ( English Edition),2012, 28(4): 464-468
20ZHAO SiJun, LI Cun, JIANG HaiYang, LI BingYu, SHEN JianZhong. Chinese J. Anal. Chem., 2007, 35(3): 786-790
赵思俊, 李 存, 江海洋, 李炳玉, 沈建忠. 分析化学, 2007, 35(3): 786-790
AbstractAn analytical method for simultaneous determination of five quinolones in spicy soup was developed. Spicy soup samples were firstly extracted by EDTAMcllvaine buffer at pH 4, then purified and concentrated by a novel Packed fiber solid phase extraction (PFSPE) coulumn. The extracted liquid supernatant was loaded onto the column, rinsed with water, and then eluted with 2% ammoniated methanol. The mobile phase was methanolwaterphosphoric acid (25∶75∶0.1, V/V, adjusting the pH to 2.8 with triethylamine). These analytes were quantified by high performance liquid chromatographyfluorimetric detector(HPLCFLD) at excitation and emission wavelength of 280 nm and 450 nm respectively. Recoveries of spiked quinolone antibiotics in spicy soup were from 72.1% to 110.3% with intraday relative standard deviation (RSD) between 1.6% and 4.3% and interday RSD from 2.0% to 4.3%. Limit of detection(LOD) and limit of quantitation(LOQ) were from 1.2 to 5.4 μg/L and from 3.9 to 18 μg/L, respectively. The method could be applied to determine the quinolones in spicy soup.
KeywordsSpicy soup; Quinolones; Packed fiber solid phase extraction; High performance liquid chromatography; Fluorimetric detection
曹 鹏, 牟 妍, 高 飞, 耿金培, 张禧庆, 隋 涛, 梁君妮, 沙美兰, 关丽丽. 色谱, 2013, 31(9): 862-868
15Kang X J, Pan C, Zhang Y Y , Gu Z Z . Anal. Chim. Acta, 2007, 587(1): 75-81
16SUN Jing, MA YuQin, KANG XueJun. Environment Chemistry, 2010, 29(1): 105-108
孙 静, 马玉琴, 康学军. 环境化学, 2010, 29(1): 105-108
17QU ZiJian, KANG XueJun, WU WeiWei, CHEN BaoAn, GU ZhongZe. Chinese J. Anal. Chem.,2011, 39(3): 312-316
曲子健, 康学军, 吴玮玮, 陈宝安, 顾忠泽. 分析化学, 2011, 39(3): 312-316
18CHEN LiQin, KANG XueJun, YAN Yan, GU ZhongZe, LU ZuHong. Chinese J. Anal. Chem.,2012, 40(1): 124-128
陈利琴, 康学军, 晏 嫣, 顾忠泽, 陆祖宏. 分析化学, 2012, 40(1): 124-128
19Yan Y, Deng J J, Deng S W, Wang Y, Wang F, Xiao M, Kang X J. Journal of Southeast University ( English Edition),2012, 28(4): 464-468
20ZHAO SiJun, LI Cun, JIANG HaiYang, LI BingYu, SHEN JianZhong. Chinese J. Anal. Chem., 2007, 35(3): 786-790
赵思俊, 李 存, 江海洋, 李炳玉, 沈建忠. 分析化学, 2007, 35(3): 786-790
AbstractAn analytical method for simultaneous determination of five quinolones in spicy soup was developed. Spicy soup samples were firstly extracted by EDTAMcllvaine buffer at pH 4, then purified and concentrated by a novel Packed fiber solid phase extraction (PFSPE) coulumn. The extracted liquid supernatant was loaded onto the column, rinsed with water, and then eluted with 2% ammoniated methanol. The mobile phase was methanolwaterphosphoric acid (25∶75∶0.1, V/V, adjusting the pH to 2.8 with triethylamine). These analytes were quantified by high performance liquid chromatographyfluorimetric detector(HPLCFLD) at excitation and emission wavelength of 280 nm and 450 nm respectively. Recoveries of spiked quinolone antibiotics in spicy soup were from 72.1% to 110.3% with intraday relative standard deviation (RSD) between 1.6% and 4.3% and interday RSD from 2.0% to 4.3%. Limit of detection(LOD) and limit of quantitation(LOQ) were from 1.2 to 5.4 μg/L and from 3.9 to 18 μg/L, respectively. The method could be applied to determine the quinolones in spicy soup.
KeywordsSpicy soup; Quinolones; Packed fiber solid phase extraction; High performance liquid chromatography; Fluorimetric detection
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