超高效液相色谱-串联质谱法同时测定畜禽排泄物中15种喹诺酮类药物

doi:10.3969/j.issn.1004-1524.20230345

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (7): 1519-1529.DOI: 10.3969/j.issn.1004-1524.20230345

超高效液相色谱-串联质谱法同时测定畜禽排泄物中15种喹诺酮类药物

裘丞军¹(), 侯轩¹, 陈凯¹, 吴望君¹, 周炜¹, 段友刚²^,^*()

1.浙江省动物疫病预防控制中心,浙江杭州 311199
2.开化县农业事务管理中心,浙江开化 324300

收稿日期:2023-03-15 出版日期:2024-07-25 发布日期:2024-08-05
作者简介:裘丞军(1984—),男,浙江杭州人,学士,畜牧师,主要研究方向为饲料、兽药及畜产品安全检测。E-mail: 282934553@qq.com
通讯作者: *段友刚,E-mail: kh661628@sohu.com
基金资助:
浙江省重点研发计划项目(2020C02032);浙江省农业重大技术协同推广计划(2021XTTGXM03)

Simultaneous determination of 15 quinolones in livestock and poultry excrement by UPLC-MS/MS

QIU Chengjun¹(), HOU Xuan¹, CHEN Kai¹, WU Wangjun¹, ZHOU Wei¹, DUAN Yougang²^,^*()

1. Zhejiang Center of Animal Disease Control, Hangzhou 311199, China
2. Kaihua County Center of Agricultural Affairs Management, Kaihua 324300, Zhejiang, China

Received:2023-03-15 Online:2024-07-25 Published:2024-08-05

摘要/Abstract

摘要：

建立了应用超高效液相色谱-串联质谱法(UPLC-MS/MS)同时测定畜禽排泄物中15种喹诺酮类药物残留量的分析方法。样品经冷冻干燥后粉碎过筛,N,N-二甲基甲酰胺(DMF)和0.1 mol·L^-1盐酸溶液混合提取,HLB固相萃取柱净化,UPLC-MS/MS检测,基质匹配校准外标法定量。结果表明:15种喹诺酮在2~50 ng·mL^-1浓度范围内线性良好,决定系数(R²)>0.99,检出限为5 μg·kg^-1,定量限为10 μg·kg^-1,添加量为10、20、500 μg·kg^-1浓度时,平均回收率为76.2%~109.0%,相对标准偏差(RSD)为1.3%~12.4%(n=6)。该方法实现了较低的检测限、定量限,较高回收率和稳定性,可应用于畜禽排泄物中喹诺酮类药物残留量的监测,为畜禽排泄物在种植业中的科学施用提供准确指导,为间接监测养殖过程中各喹诺酮类药物的含量提供技术方法,指导养殖企业合理使用抗菌药物。

关键词: 超高效液相色谱-串联质谱(UPLC-MS/MS), 喹诺酮类, 畜禽排泄物

Abstract:

An analytical method for the simultaneous determination of 15 quinolones residues in livestock and poultry excrement by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established. The sample was crushed and sieved after freeze-drying, extracted by mixing N, N-dimethylformamide (DMF) and 0.1 mol·L^-1 hydrochloric acid solution, purified by HLB solid phase extraction column, detected by UPLC-MS/MS, and quantified by matrix matching calibration external standard method. The results showed that the linearity of 15 quinolones was good in the concentration range of 2-50 ng·mL^-1, the coefficient of determination (R²)>0.99, and the detection limit was 5 μg·kg^-1, limit of quantitation was 10 μg·kg^-1, the average recovery rate was 76.2%-109.0%, relative standard derivation (RSD) was 1.3%-12.4% (n=6) at the concentration of 10, 20 and 500 μg·kg^-1. This method has achieved lower detection limit, quantitative limit, higher recovery rate and stability. It can be applied to the monitoring of quinolones residues in livestock and poultry excrement, provide accurate guidance for the scientific application of livestock and poultry excrement in the planting industry, provide technical methods for indirect monitoring of the content of quinolones in the breeding process, and guide the rational use of antibiotics by breeding enterprises.

Key words: UPLC-MS/MS, quinolones, livestock and poultry excrement

中图分类号:

S859.8

裘丞军, 侯轩, 陈凯, 吴望君, 周炜, 段友刚. 超高效液相色谱-串联质谱法同时测定畜禽排泄物中15种喹诺酮类药物[J]. 浙江农业学报, 2024, 36(7): 1519-1529.

QIU Chengjun, HOU Xuan, CHEN Kai, WU Wangjun, ZHOU Wei, DUAN Yougang. Simultaneous determination of 15 quinolones in livestock and poultry excrement by UPLC-MS/MS[J]. Acta Agriculturae Zhejiangensis, 2024, 36(7): 1519-1529.

图/表 8

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序号 Number	化合物 Compound	母离子 Precursor ion (m/z)	子离子 Product ions (m/z)	锥孔电压 Cone voltage/V	碰撞能量 Collision energy/eV	相对保留时间 Retention time/min
1	氟甲喹Flumequine(FLU)	262.1	244.1*/202.0	56	16/30	6.25
2	恶喹酸Oxolinic acid(OXO)	262.1	244.1*/216.0	56	17/27	5.37
3	诺氟沙星Norfloxacin(NOR)	320.2	302.1*/276.2	56	18/16	3.74
4	依诺沙星Enoxacin(ENO)	321.2	303.1*/232.2	56	18/32	3.65
5	环丙沙星Ciprofloxacin(CIP)	332.2	314.1*/288.1	56	19/16	3.88
6	培氟沙星Pefloxacin(PEF)	334.3	316.1*/290.2	56	18/16	3.64
7	洛美沙星Lomefloxacin(LOM)	352.2	265.1*/308.1	56	21/16	4.09
8	达氟沙星Danofloxacin(DAN)	358.2	340.1*/314.2	56	22/16	4.03
9	恩诺沙星Enrofloxacin(ENR)	360.3	316.1*/245.1	56	18/25	3.99
10	氧氟沙星Ofloxacin(OFL)	362.2	318.1*/261.2	56	18/26	3.61
11	马波沙星Marbofloxacin(MAR)	363.3	320.1*/345.1	56	14/17	3.30
12	沙拉沙星Sarafloxacin(SAR)	386.2	342.1*/299.1	56	17/26	4.37
13	司帕沙星Sparfloxacin(SPA)	393.3	349.2*/292.1	56	18/23	4.83
14	奥比沙星Orbifloxacin(ORB)	396.2	352.1*/295.1	56	17/24	4.18
15	双氟沙星Difloxacin(DIF)	400.2	356.1*/299.1	56	18/28	4.22

序号 Number	化合物 Compound	母离子 Precursor ion (m/z)	子离子 Product ions (m/z)	锥孔电压 Cone voltage/V	碰撞能量 Collision energy/eV	相对保留时间 Retention time/min
1	氟甲喹Flumequine(FLU)	262.1	244.1*/202.0	56	16/30	6.25
2	恶喹酸Oxolinic acid(OXO)	262.1	244.1*/216.0	56	17/27	5.37
3	诺氟沙星Norfloxacin(NOR)	320.2	302.1*/276.2	56	18/16	3.74
4	依诺沙星Enoxacin(ENO)	321.2	303.1*/232.2	56	18/32	3.65
5	环丙沙星Ciprofloxacin(CIP)	332.2	314.1*/288.1	56	19/16	3.88
6	培氟沙星Pefloxacin(PEF)	334.3	316.1*/290.2	56	18/16	3.64
7	洛美沙星Lomefloxacin(LOM)	352.2	265.1*/308.1	56	21/16	4.09
8	达氟沙星Danofloxacin(DAN)	358.2	340.1*/314.2	56	22/16	4.03
9	恩诺沙星Enrofloxacin(ENR)	360.3	316.1*/245.1	56	18/25	3.99
10	氧氟沙星Ofloxacin(OFL)	362.2	318.1*/261.2	56	18/26	3.61
11	马波沙星Marbofloxacin(MAR)	363.3	320.1*/345.1	56	14/17	3.30
12	沙拉沙星Sarafloxacin(SAR)	386.2	342.1*/299.1	56	17/26	4.37
13	司帕沙星Sparfloxacin(SPA)	393.3	349.2*/292.1	56	18/23	4.83
14	奥比沙星Orbifloxacin(ORB)	396.2	352.1*/295.1	56	17/24	4.18
15	双氟沙星Difloxacin(DIF)	400.2	356.1*/299.1	56	18/28	4.22

化合物 Compound	线性范围 Linear range/ (ng·mL^-1)	线性方程 Linearity	决定系数 R²	检测限 Limit of detection/ (μg·kg^-1)	定量限 Limit of quantification/ (μg·kg^-1)
氟甲喹 Flumequine	2~50	Y=7 981.72X+1 253.69 Y=10 034.2X+5 070.04	0.996 949 0.996 796	5	10
恶喹酸 Oxolinic acid	2~50	Y=11 939.8X-242.496 Y=12 801.1X-514.299	0.999 801 0.999 759	5	10
诺氟沙星 Norfloxacin	2~50	Y=4 532.16X+243.862 Y=4 846.96X-626.433	0.997 820 0.999 032	5	10
依诺沙星 Enoxacin	2~50	Y=7 833.85X+3 459.95 Y=8 985.48X-191.619	0.997 384 0.999 732	5	10
环丙沙星 Ciprofloxacin	2~50	Y=4 277.85X+2 162.18 Y=4 334.14X+308.043	0.995 421 0.999 806	5	10
培氟沙星 Pefloxacin	2~50	Y=7 037.72X+552.375 Y=8 063.58X+577.823	0.999 038 0.999 521	5	10
洛美沙星 Lomefloxacin	2~50	Y=5 126.07X+396.715 Y=6 559.55X-111.743	0.999 188 0.999 491	5	10
达氟沙星 Danofloxacin	2~50	Y=6 465.66X+188.032 Y=5 986.14X-749.459	0.998 742 0.999 440	5	10
恩诺沙星 Enrofloxacin	2~50	Y=5 224.72X+2071.06 Y=5 260.01X+1 379.62	0.997 524 0.998 913	5	10
氧氟沙星 Ofloxacin	2~50	Y=6 692.8X+132.333 Y=8 506.42X-421.105	0.998 558 0.999 717	5	10
马波沙星 Marbofloxacin	2~50	Y=2 633.96X-190.033 Y=2 836.17X+259.807	0.999 142 0.999 513	5	10
沙拉沙星 Sarafloxacin	2~50	Y=1 884.08X+74.961 2 Y=2 382.54X+128.562	0.996 013 0.999 389	5	10
司帕沙星 Sparfloxacin	2~50	Y=5 849.4X+472.192 Y=8 643.36X+487.064	0.998 898 0.999 389	5	10
奥比沙星 Orbifloxacin	2~50	Y=5 029.7X+887.703 Y=7 078.64X+516.124	0.998 814 0.999 481	5	10
双氟沙星 Difloxacin	2~50	Y=3 822.18X+402.002 Y=4 383.89X-168.748	0.998 843 0.999 578	5	10

化合物 Compound	线性范围 Linear range/ (ng·mL^-1)	线性方程 Linearity	决定系数 R²	检测限 Limit of detection/ (μg·kg^-1)	定量限 Limit of quantification/ (μg·kg^-1)
氟甲喹 Flumequine	2~50	Y=7 981.72X+1 253.69 Y=10 034.2X+5 070.04	0.996 949 0.996 796	5	10
恶喹酸 Oxolinic acid	2~50	Y=11 939.8X-242.496 Y=12 801.1X-514.299	0.999 801 0.999 759	5	10
诺氟沙星 Norfloxacin	2~50	Y=4 532.16X+243.862 Y=4 846.96X-626.433	0.997 820 0.999 032	5	10
依诺沙星 Enoxacin	2~50	Y=7 833.85X+3 459.95 Y=8 985.48X-191.619	0.997 384 0.999 732	5	10
环丙沙星 Ciprofloxacin	2~50	Y=4 277.85X+2 162.18 Y=4 334.14X+308.043	0.995 421 0.999 806	5	10
培氟沙星 Pefloxacin	2~50	Y=7 037.72X+552.375 Y=8 063.58X+577.823	0.999 038 0.999 521	5	10
洛美沙星 Lomefloxacin	2~50	Y=5 126.07X+396.715 Y=6 559.55X-111.743	0.999 188 0.999 491	5	10
达氟沙星 Danofloxacin	2~50	Y=6 465.66X+188.032 Y=5 986.14X-749.459	0.998 742 0.999 440	5	10
恩诺沙星 Enrofloxacin	2~50	Y=5 224.72X+2071.06 Y=5 260.01X+1 379.62	0.997 524 0.998 913	5	10
氧氟沙星 Ofloxacin	2~50	Y=6 692.8X+132.333 Y=8 506.42X-421.105	0.998 558 0.999 717	5	10
马波沙星 Marbofloxacin	2~50	Y=2 633.96X-190.033 Y=2 836.17X+259.807	0.999 142 0.999 513	5	10
沙拉沙星 Sarafloxacin	2~50	Y=1 884.08X+74.961 2 Y=2 382.54X+128.562	0.996 013 0.999 389	5	10
司帕沙星 Sparfloxacin	2~50	Y=5 849.4X+472.192 Y=8 643.36X+487.064	0.998 898 0.999 389	5	10
奥比沙星 Orbifloxacin	2~50	Y=5 029.7X+887.703 Y=7 078.64X+516.124	0.998 814 0.999 481	5	10
双氟沙星 Difloxacin	2~50	Y=3 822.18X+402.002 Y=4 383.89X-168.748	0.998 843 0.999 578	5	10

化合物 Compound	添加浓度 Additive concentration/ (μg·kg^-1)	鸡粪Chicken manure		猪粪Pig manure
化合物 Compound	添加浓度 Additive concentration/ (μg·kg^-1)	平均回收率 Average recovery/%	相对标准偏差 RSD/%	平均回收率 Average recovery/%	相对标准偏差 RSD/%
氟甲喹	10	106.0	1.9	106.0	1.9
Flumequine(FLU)	20	102.0	4.6	102.0	4.6
	500	96.9	2.7	96.9	2.7
恶喹酸	10	89.8	9.3	106.0	3.1
Oxolinic acid(OXO)	20	102.0	2.9	101.0	3.1
	500	93.3	1.3	96.5	3.5
诺氟沙星	10	85.1	8.0	96.1	9.4
Norfloxacin(NOR)	20	91.9	5.3	101.0	4.2
	500	88.1	2.7	100.0	3.8
依诺沙星	10	76.2	7.9	94.9	1.7
Enoxacin(ENO)	20	81.3	2.8	109.0	5.1
	500	87.8	1.9	98.8	2.8
环丙沙星	10	97.0	9.5	89.4	5.1
Ciprofloxacin(CIP)	20	82.6	9.1	91.6	4.0
	500	90.7	2.1	96.7	4.1
培氟沙星	10	91.4	9.0	105.0	5.7
Pefloxacin(PEF)	20	94.7	6.7	104.0	5.4
	500	90.4	2.4	104.0	2.7
洛美沙星	10	83.9	6.8	99.9	8.0
Lomefloxacin(LOM)	20	88.6	5.8	100.0	2.7
	500	93.7	1.8	101.0	2.2
达氟沙星	10	78.2	11.1	91.4	4.1
Danofloxacin(DAN)	20	80.0	6.5	92.9	6.8
	500	92.5	2.6	100.0	4.7
恩诺沙星	10	104.0	8.0	91.4	6.2
Enrofloxacin(ENR)	20	81.9	6.4	91.5	7.9
	500	93.4	2.4	103.0	2.7
氧氟沙星	10	89.9	8.2	95.1	3.1
Ofloxacin(OFL)	20	97.4	4.6	96.5	6.7
	500	96.8	2.3	104.0	2.9
马波沙星	10	85.5	4.5	92.4	8.4
Marbofloxacin(MAR)	20	87.7	6.0	93.6	12.4
	500	96.3	2.4	101.0	2.4
沙拉沙星	10	97.2	7.1	104.0	8.6
Sarafloxacin(SAR)	20	101.0	8.7	90.8	9.7
	500	93.7	3.7	94.5	4.0
司帕沙星	10	90.8	5.3	108.0	6.5
Sparfloxacin(SPA)	20	99.5	4.0	106.0	7.0
	500	96.3	2.2	109.0	2.4
奥比沙星	10	92.0	2.5	103.0	4.8
Orbifloxacin(ORB)	20	97.1	3.3	107.0	6.8
	500	92.6	1.8	107.0	2.4
双氟沙星	10	78.6	12.1	102.0	7.0
Difloxacin(DIF)	20	91.3	7.9	102.0	8.7
	500	93.5	2.5	104.0	4.7

超高效液相色谱-串联质谱法同时测定畜禽排泄物中15种喹诺酮类药物

Simultaneous determination of 15 quinolones in livestock and poultry excrement by UPLC-MS/MS

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