液相色谱-串联质谱法检测猪血浆中除虫脲的方法学研究

doi:10.16431/j.cnki.1671-7236.2022.07.033

摘要/Abstract

摘要： 【目的】考察口服灭蝇蛆药除虫脲（diflubenzuron，DFB）预混剂灌服给药后，在猪体内的药代动力学特征，建立猪血浆中除虫脲浓度的液相色谱-串联质谱（LC-MS/MS）检测方法。【方法】以除虫脲-¹³C₆（DFB-¹³C₆）为内标，血浆样品经甲醇沉淀蛋白，离心后取上清液减压浓缩至干，甲醇-水（1∶1）复溶后进行分析。色谱柱为ZORBAX Eclipse Plus C18柱；流动相为甲醇-0.1%甲酸水（含5 mmol/L甲酸铵），进行梯度洗脱；流速为0.4 mL/min；柱温为35 ℃；进样量为10 μL。电喷雾离子源，正离子模式，多离子反应监测模式检测，DFB及内标的定量离子对分别为m/z 311.1→158.1和m/z 317.1→158.1。对色谱和质谱条件优化后，考察该方法的专属性、定量下限、基质效应、线性范围、准确度、精密度和样品稳定性等。【结果】所建立的方法灵敏度高，检测限为0.5 ng/mL，定量限为1 ng/mL；基质效应对样品的检测影响较小；标准溶液在1~1 000 ng/mL的浓度范围内，线性关系良好（R²≥0.998）；在定量下限1 ng/mL及低（2 ng/mL）、中（100 ng/mL）、高浓度（800 ng/mL）的添加水平下，平均准确度在97.78%~115.06%之间，批内、批间变异系数均<10%，符合方法学要求。在考察的条件下，标准溶液、空白血浆加标样品及处理后的样品，稳定性良好；但应避免样品的反复冻融。【结论】建立的LC-MS/MS检测方法简便、专属性强、灵敏度高、准确可靠，可用于猪血浆中除虫脲浓度的检测，进而应用于除虫脲预混剂在猪体内的药代动力学研究。

关键词: 猪; 血浆; 除虫脲(DFB); 液相色谱-串联质谱法; 内标法

Abstract: 【Objective】 To investigate the pharmacokinetics character of the diflubenzuron (DFB) premix in swine after drenching administration,a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to determine diflubenzuron in swine plasma.【Method】 DFB-¹³C₆ was employed as internal standard.Plasma samples were processed by protein precipitation of methanol.After centrifugation,the supernatant was vacuum evaporation to dry,and the residue was resolved by the solvent of methanol-water (1∶1).The column of ZORBAX Eclipse Plus C18 was used to separate the analyte.Mobile phase was methanol-buffer of 0.1% formic acid and 5 mmol/L ammonium formate with flow rate of 0.4 mL/min under gradient elution.The column temperature was 35 ℃ and injection volume was 10 μL.Determination of DFB was performed using LC-MS/MS with an electrospray ionization source interface operated in the positive-ion scan mode under the multiple reaction monitoring mode.Product ions 311.1→158.1 and 317.1→158.1 were employed as quantifier ion of DFB and DFB-¹³C₆,respectively.The conditions of LC and MS were optimized.The proposed method was validated through specificity,matrix effects,quantitative lower limits,lineral range,precision,accuracy and samples stability.【Result】 The established method had high sensitivity,the limit of detection (LOD) was 0.5 ng/mL and the limit of quantitative (LOQ) was 1 ng/mL.Matrix effects had little influence on the detection of samples.The standard solution had a good linear relationship in the concentration range of 1-1 000 ng/mL (R²≥0.998).The average accuracy was during 97.78% to 115.06% and the intra-assay and inter-assay precisions were both less than 10% when the spiked levels of DFB in plasma were LOQ (1 ng/mL),low (2 ng/mL),medium (100 ng/mL) and high concentration (800 ng/mL).These results could meet the methodological requirements.The standard solution,spiked samples and processed samples had good stability when they stored at the specific conditions except freeze-thaw cycles.【Conclusion】 The established LC-MS/MS detection method was simple,specific,sensitive,accurate and reliable.It could be used to detect the concentration of diflubenzuron in pig plasma,and then applied to the pharmacokinetics of diflubenzuron premix in swine.

Key words: swine; plasma; diflubenzuron (DFB); LC-MS/MS; internal standard method

中图分类号:

S859.79⁺5

徐玉珍, 刘希望, 秦哲, 李世宏, 白莉霞, 范丽萍, 魏彦明, 李剑勇, 杨亚军. 液相色谱-串联质谱法检测猪血浆中除虫脲的方法学研究[J]. 中国畜牧兽医, 2022, 49(7): 2746-2756.

XU Yuzhen, LIU Xiwang, QIN Zhe, LI Shihong, BAI Lixia, FAN Liping, WEI Yanming, LI Jianyong, YANG Yajun. Methodology Study on Determination of Diflubenzuron in Swine Plasma by Liquid Chromatography-tandem Mass Spectrometry[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(7): 2746-2756.

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