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氟喹诺酮类抗生素(fluoroquinolones,FQs)是目前使用量较大的一类合成抗菌药,基本结构为4-氟喹诺酮酸[1]。FQs具有抗菌谱广、杀菌能力强、口服效果吸收率好、与其他抗菌药无交叉耐药性等特点,已成为治疗细菌性疾病最常用药物之一[2-3]。目前,我国每年生产的喹诺酮类抗生素约为3.57×104 t,是应用最广泛的抗生素之一,尤其在畜牧养殖和水产养殖行业的应用更为广泛[4-8]。近年来,随着FQs生产量和使用量的不断增加,环境中检出FQs的报道越来越多。胶州湾海洋沉积物中检测出8种FQs残留,质量分数为0.478~47.545 ng·g−1[9];白洋淀沉积物中检出6种FQs残留,质量分数为nd~52.90 ng·g−1[10];闽江河口区域沉积物中检出6种FQs残留,质量分数为0.03~15.60 ng·g−1[11]。FQs已逐渐成为海洋沉积物中重要的污染物之一[7]。海洋沉积物中残留的FQs通过迁移转化进入到生态系统中,对生态环境及人体健康构成严重威胁[12-13]。因此,FQs在环境中的残留及潜在风险受到国内外专家学者的广泛关注[14-15]。
随着海水养殖技术的日益进步及海水养殖产品需求的不断上涨,我国海水养殖产业得到了迅猛发展。2020年我国海水养殖面积为1.99×106 hm2,海水养殖产品年产量达2.14×107 t,稳居世界第一[16]。随着海产品年产量的不断增加,FQs的投放量不断增加,仅有20%~30%的FQs被利用或吸收,其余均以原形或代谢物的形式排入海洋环境中,通过吸附、沉降作用汇集于底部沉积物[15],造成海洋 FQs 污染[17-19]。目前,已有很多学者对环境中 FQs 开展了研究。这些研究主要集中在水环境[20-28]、饲料[29-30]、食品[31-32]、水产品[33-34]等领域,而对海洋沉积物中FQs及相关化合物的研究较少,很难满足监测监管的需求。
本研究针对海水养殖沉积物中可能存在的13种 FQs,基于SPE-LC-MS/MS技术,建立了一种快速、准确的测定方法,实现了海洋沉积物中13种FQs的同时检测;并对广州某湾区海洋沉积物进行实地检测,以期揭示该地区海水养殖业FQs污染的分布规律,为维护海水安全提供基础数据,为渔用FQs的监管提供参考。
用固相萃取-高效液相色谱-三重四极杆串联质谱法测定海洋沉积物中13种氟喹诺酮类抗生素
Determination of 13 fluoroquinolones antibiotics in marine sediments using LC-MS/MS coupled with solid phase extraction
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摘要: 为进一步探索海洋沉积物中氟喹诺酮类抗生素的污染情况,基于固相萃取-高效液相色谱-三重四极杆串联质谱(SPE-LC-MS/MS)技术,建立了海洋沉积物中 13 种FQs的测定方法;采用高效液相色谱-三重四极杆串联质谱多反应监测离子模式(MRM)对FQs进行分离检测。结果表明:在优化实验条件下,13种FQs的质量浓度为0.50~100 μg·L−1,目标化合物峰面积与内标物质峰面积之比与质量浓度的线性关系良好(R2>0.99),方法检出限为0.003~0.03 μg·kg−1;在加标量为1 μg·kg−1和10 μg·kg−1时,空白加标的平均回收率为73.5%~124.6%和67.5%~118.5%,相对标准偏差(RSD)为1.0%~9.7%(n=7);以海洋沉积物为基质,13种目标物的加标回收率为67.7%~142.4%,RSD小于10.2%(n=6);使用该方法对广州某湾区海洋沉积物中 13 种 FQs 的残留量进行了实地检测,培氟沙星质量分数最高,为1.6 μg·kg−1,氧氟沙星、环丙沙星和恩诺沙星质量分数次之,为0.7 μg·kg−1。该方法实现了对海洋沉积物中 13种 FQs 的同时检测,具有快速、准确等优点,适用于海洋沉积物中13种FQs的测定。本研究成果可为海洋生态环境保护提供数据基础及技术支撑。
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关键词:
- 固相萃取-高效液相色谱-三重四极杆串联质谱 /
- 海洋沉积物 /
- 氟喹诺酮类抗生素 /
- 方法优化
Abstract: In order to explore the pollution status of FQs in marine sediments, a method was developed for the determination of 13 target fluoroquinolones antibiotics (FQs) in marine sediments by liquid chromatography-triple quadruple tandem mass spectrometry coupled with solid phase extraction (SPE-LC-MS/MS). All FQs were isolated and quantified by LC-MS/MS under multi-reaction monitoring (MRM) mode. Results showed that under the optimal conditions, the mass concentrations of all 13 targets were 0.5~100 μg·L−1, and a good linear relation occurred between above concentration and the peak area ratio of target compound to internal control standard compound with correlation coefficients higher than 0.99, the detection limit (MDL) of this method was 0.003~0.03 μg·kg−1.The average recovery rates of blank spiking were in the ranges of 73.5%~124.6% and 67.5%~118.5% with the relative standard deviations (RSDs) from 1.0% to 9.7% (n=7) with two spiked levels of 1.0 and 10 μg·kg−1. The average recoveries of 13 FQs with spiking in the marine sediments in the Pearl River estuary samples were 67.7%~142.4% with RSDs < 10.2%(n=6). This method was used to detect 13 kinds of FQs residues in marine sediment of a bay area in Guangzhou, pefloxacin showed the highest mass fraction of 1.6 μg·kg−1, ofloxacin, ciprofloxacin and enrofloxacin were following with the mass fraction of 0.7 μg·kg−1. The method could rapidly and accurately detect 13 kinds of FQs in marine sediments at the same time, and was suitable for the determination of 13 FQs in marine sediments. The research results can provide data basis and technical support for marine ecological environment protection. -
表 1 目标化合物、替代物及内标物的多离子反应监测条件
Table 1. Multiple ion reaction monitoring conditions for target compound, surrogate and internal standard
化合物 质荷比(m/z) 锥孔电压/V 母离子碰撞
电压/V定量(定性)
离子碰撞电压/V定量内标 母离子 定量离子 定性离子 氟甲喹 262.1 202 244.1 56 27 45 13C3-氟甲喹 诺氟沙星 320.2 276.2 233.1 101 25 35 环丙沙星d8 依诺沙星 321.1 303.1 232.1 61 29 49 环丙沙星d8 环丙沙星 332.1 314.1 288.1 81 29 27 环丙沙星d8 培氟沙星 334.2 316 290.1 76 29 27 环丙沙星d8 达氟沙星 358.2 340.2 82.1 76 33 73 恩诺沙星-d5 恩诺沙星 360.2 316.1 245.1 76 27 37 恩诺沙星-d5 那氟沙星 361.2 343.2 283.1 85 35 50 13C3-氟甲喹 氧氟沙星 362.2 318.2 261.1 76 27 39 恩诺沙星-d5 马波沙星 363.1 72.1 320.1 80 46 23 环丙沙星d8 氟罗沙星 370.1 326.1 269.1 76 27 37 恩诺沙星-d5 沙拉沙星 386.1 342.1 299.1 106 27 39 恩诺沙星-d5 二氟沙星 400.2 356.2 299.1 81 29 39 恩诺沙星-d5 诺氟沙星-d5 325.2 281.2 238.1 86 25 35 环丙沙星d8 恩诺沙星-d5 365.2 321.2 347.2 81 29 31 — 环丙沙星-d8 340.2 322.1 296.1 91 31 27 — 13C3-氟甲喹 265.1 247.1 205.1 46 25 45 — 表 2 13 种 FQs 化合物的石英砂加标回收率(n=7)及相对标准偏差
Table 2. Recovery rate (n=7)and RSD of 13 fluoroquinolones antibiotics in quartzite
化合物 添加FQs质量分数/
(μg·kg−1)平均
回收率/%RSD/% 化合物 添加FQs质量分数/
(μg·kg−1)平均
回收率/%RSD/% 氟罗沙星 1.0 80.9 4.5 达氟沙星 1.0 91.3 6.3 氟罗沙星 10 84.9 1.6 达氟沙星 10 88.3 1.5 氧氟沙星 1.0 90.7 2.3 马波沙星 1.0 94.4 2.4 氧氟沙星 10 92.8 2.1 马波沙星 10 91.1 1.8 培氟沙星 1.0 88.6 2.5 二氟沙星 1.0 91.2 8.5 培氟沙星 10 79.3 1.0 二氟沙星 10 86.6 3.1 依诺沙星 1.0 73.5 8.6 沙拉沙星 1.0 85.3 5.9 依诺沙星 10 67.5 1.3 沙拉沙星 10 86.6 2.0 诺氟沙星 1.0 88.9 3.7 氟甲喹 1.0 79.6 5.3 诺氟沙星 10 83.4 2.1 氟甲喹 10 86.0 1.3 环丙沙星 1.0 88.7 6.2 那氟沙星 1.0 88.2 9.7 环丙沙星 10 87.07 4.5 那氟沙星 10 96.7 3.5 恩诺沙星 1.0 85.0 3.5 恩诺沙星 10 89.4 3.0 表 3 13 种 FQs 化合物的沉积物加标回收率(n = 6)及相对标准偏差
Table 3. Recovery rate(n = 6)and RSD of 13 fluoroquinolones antibiotics in deposit sediment
化合物 背景样品残留
质量分数/(μg·kg−1)添加质量分数/
(μg·kg−1)平均回收率/% RSD/% 氟罗沙星 0.19 1.0 142.4 2.9 氟罗沙星 0.19 50 140.2 4.4 氧氟沙星 20.23 1.0 139.6 2.9 氧氟沙星 20.23 50 113.7 5.1 培氟沙星 0.25 1.0 79.7 9.2 培氟沙星 0.25 50 116.9 2.8 依诺沙星 0 1.0 90.0 10.2 依诺沙星 0 50 67.7 4.3 诺氟沙星 11.50 1.0 101.3 7.5 诺氟沙星 11.50 50 91.6 4.1 环丙沙星 3.45 1.0 97.0 7.9 环丙沙星 3.45 50 97.7 4.0 恩诺沙星 3.01 1.0 97.2 5.0 恩诺沙星 3.01 50 95.8 1.8 达氟沙星 0.17 1.0 99.7 5.3 达氟沙星 0.17 50 94.6 5.1 马波沙星 0.07 1.0 84.8 8.2 马波沙星 0.07 50 107.4 4.4 二氟沙星 0 1.0 126.7 2.2 二氟沙星 0 50 136.1 1.5 沙拉沙星 0 1.0 139.7 2.8 沙拉沙星 0 50 114.2 3.3 氟甲喹 0.19 1.0 97.8 3.3 氟甲喹 0.19 50 100.8 1.4 那氟沙星 0.11 1.0 104.0 7.4 那氟沙星 0.11 50 88.8 2.3 -
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