Rapid Determination of Clopidol in Animal Feed by Solid-Phase Extraction Coupled with Liquid Chromatography-Tandem Mass Spectrometry (SPE-LC-MS/MS)

doi:10.11924/j.issn.1000-6850.casb2025-0373

Abstract

Abstract:

To address the problems of low sensitivity, cumbersome pretreatment, large matrix interference, and easy missed detection of low concentration of clopidol in feed, this study aimed to establish a solid-phase extraction- liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) method for the rapid determination of clopidol content in feed, in order to meet the requirements of accurate quantification and safety supervision. Clopidol in feed samples was extracted using a 0.2% ammonia-methanol solution, purified through an alkaline alumina solid-phase extraction (SPE) cartridge, and separated on a C₁₈ column with gradient elution using ammonium formate solution and methanol as the mobile phase. Detection was performed by LC-MS/MS in positive electrospray ionization (ESI⁺) mode, and quantification was conducted using matrix-matched external standard calibration. The results showed that clopidol exhibited a good linearity within the concentration range of 0.2-100 μg/L, with a correlation coefficient (r²) greater than 0.999. The method detection limits (LOD) and quantification limits (LOQ) were 0.05 mg/kg and 0.1 mg/kg, respectively. Method accuracy was evaluated in various types of feed, yielding recoveries of 90%-102%, with a relative standard deviation (RSD) below 7%. This analytical method is simple to perform, highly sensitive, accurate, and reproducible, and significantly improves the detection limit and qualitative reliability compared with the national standard method. It meets the methodological requirements for the determination of clopidol in feed and provides a reliable technical approach for its precise quantification.

Key words: feeds, clopidol, liquid chromatography-tandem mass spectrometry, solid-phase extraction, anticoccidial drugs, matrix effect

CLC Number:

O657.63

PAN Fei, LIN Xuexian, LIU Zhenni, DING Chenhong, WANG Weili. Rapid Determination of Clopidol in Animal Feed by Solid-Phase Extraction Coupled with Liquid Chromatography-Tandem Mass Spectrometry (SPE-LC-MS/MS)[J]. Chinese Agricultural Science Bulletin, 2026, 42(9): 163-170.

Figures/Tables 8

References 31

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化合物	监测离子对/(m/z)	Q1 电压/V	碰撞能量/eV	Q3 电压/V
氯羟吡啶	192.0>101.1^a	-10	27	-18
氯羟吡啶	192.0>87.1	-10	32	-30

化合物	监测离子对/(m/z)	Q1 电压/V	碰撞能量/eV	Q3 电压/V
氯羟吡啶	192.0>101.1^a	-10	27	-18
氯羟吡啶	192.0>87.1	-10	32	-30

饲料	线性范围/(µg/L)	回归方程	相关系数(r²)	检出限/(mg/kg)	定量限/(mg/kg)
配合饲料	0.2~100	Y = 49647.6X + 7496.20	0.9993	0.05	0.1
浓缩饲料	0.2~100	Y = 51013.3X + 56.2521	0.9994	0.05	0.1
添加剂预混合饲料	0.2~100	Y = 58060.5X + 709.803	0.9996	0.05	0.1
精料补充料	0.2~100	Y = 53164.6X + 123.706	0.9994	0.05	0.1
微生物混合饲料添加剂	0.2~100	Y = 57480.2X + 771.404	0.9993	0.05	0.1
鸡肉粉	0.2~100	Y = 51783.6X - 190.775	0.9991	0.05	0.1

饲料	线性范围/(µg/L)	回归方程	相关系数(r²)	检出限/(mg/kg)	定量限/(mg/kg)
配合饲料	0.2~100	Y = 49647.6X + 7496.20	0.9993	0.05	0.1
浓缩饲料	0.2~100	Y = 51013.3X + 56.2521	0.9994	0.05	0.1
添加剂预混合饲料	0.2~100	Y = 58060.5X + 709.803	0.9996	0.05	0.1
精料补充料	0.2~100	Y = 53164.6X + 123.706	0.9994	0.05	0.1
微生物混合饲料添加剂	0.2~100	Y = 57480.2X + 771.404	0.9993	0.05	0.1
鸡肉粉	0.2~100	Y = 51783.6X - 190.775	0.9991	0.05	0.1

饲料	加标浓度/(mg/kg)	实测值/(mg/kg)						回收率/%	相对标准偏差/%
配合饲料	0.1	0.0943	0.0937	0.0899	0.0907	0.0915	0.0938	92.3	2.0
	1	0.910	0.900	1.018	0.924	1.018	1.024	96.6	6.2
	10	9.66	9.53	10.27	10.17	9.96	9.31	98.2	3.9
	250	258	249	255	261	247	259	102.0	2.2
浓缩饲料	0.1	0.0939	0.0925	0.0896	0.0934	0.0880	0.0911	91.4	2.5
	1	0.994	0.947	0.952	1.040	0.972	1.018	98.7	3.8
	10	9.62	9.43	9.75	9.84	9.63	10.34	97.7	3.2
	250	245	254	255	246	235	230	97.6	4.0
添加剂预混合饲料	0.1	0.0890	0.0930	0.0910	0.0909	0.0932	0.0896	91.1	1.9
	1	0.996	1.005	0.960	1.025	0.999	0.939	98.7	3.2
	10	9.87	10.43	10.46	10.34	9.65	9.54	100.5	4.1
	250	249	239	264	243	272	232	99.9	6.1
精料补充料	0.1	0.0887	0.0941	0.0947	0.0945	0.0948	0.0935	93.4	2.5
	1	1.048	0.948	0.914	0.937	1.016	0.967	97.2	5.2
	10	9.39	10.16	9.72	10.28	10.13	9.22	98.2	4.5
	250	260	261	252	248	250	257	101.8	2.1
微生物混合饲料添加剂	0.1	0.0945	0.0906	0.0916	0.0902	0.0883	0.0894	90.8	2.4
	1	0.994	1.049	0.975	0.915	0.991	0.925	97.5	5.1
	10	10.41	10.02	10.49	9.23	10.06	9.58	99.7	4.9
	250	253	235	240	230	270	237	97.6	6.1
鸡粉	0.1	0.0909	0.0936	0.0948	0.0886	0.0917	0.0946	92.4	2.6
	1	0.971	0.917	0.909	0.966	1.037	0.970	96.2	4.8
	10	9.38	10.19	9.54	9.84	9.62	9.41	96.6	3.2
	250	232	239	240	230	233	256	95.3	3.9
兔配合饲料	0.1	0.0880	0.0905	0.0914	0.0919	0.0884	0.0899	90.0	1.7
	1	0.954	0.988	0.975	0.923	1.042	0.973	97.6	4.0
	10	10.19	9.24	10.36	9.46	9.82	10.31	99.0	4.7
	250	233	246	252	237	242	238	96.5	2.8