高效液相色谱法检测鼠饲料中抗菌肽NZ2114

doi:10.16431/j.cnki.1671-7236.2023.11.015

摘要/Abstract

摘要： 【目的】本研究旨在建立鼠粉状饲料中抗菌肽NZ2114固相萃取-高效液相色谱（SPE-HPLC）快速分析方法，用于检测低、中、高浓度（2、10和50 g/kg）的鼠粉状饲料中受试物NZ2114含量，以分析抗菌肽NZ2114是否均匀分布。【方法】试验分别对提取条件、净化柱和净化方法进行优化，并考察了提取剂、提取时间对提取效率的影响，SPE清洗剂、洗脱剂等对回收率的影响。饲料样品经超声提取后离心，上清液经WCX固相萃取柱富集，随后用3 mL甲醇和5 mL 20%氨化甲醇进行净化洗脱。HPLC检测方法以ZORBAX Eclipse Plus C18作为固定相，0.1%甲酸-乙腈和0.1%甲酸-水作为流动相，柱温35 ℃，进样量20 μL，以1.0 mL/min流速，按照梯度洗脱的方式洗脱，在波长276 nm下采用外标法定量检测饲料样品中NZ2114含量，并对该方法的线性、检测限与定量限、基质效应、回收率、日内与日间精密度进行考察。【结果】SPE-HPLC分析方法在62.5~4 000 μg/mL浓度范围内线性关系良好，线性方程为y＝0.9353x-32.721，相关系数（R²）为0.9996，低、中、高浓度下该方法的添加回收率范围在88.05%~111.23%，日内精密度为0.58%~4.45%，日间精密度为3.18%~6.37%，125、500和2 000 μg/mL浓度下基质效应分别为1.02、1.01和0.99。【结论】本研究建立了鼠饲料中抗菌肽NZ2114的SPE-HPLC检测方法，符合方法学考察要求，具有灵敏度和精密度高、重现性好、快速等优点，能够实现3个浓度水平饲料样品中NZ2114的精准定量。

关键词: 高效液相色谱; 固相萃取; 抗菌肽NZ2114; 鼠饲料

Abstract: 【Objective】 The aim of this study was to establish a rapid method for the determination of antimicrobial peptide NZ2114 in rat powder feed by solid phase extraction and high performance liquid chromatography (SPE-HPLC), which was used to detect the content of the test substance NZ2114 in rat powder feed samples at low, medium and high concentrations (2, 10 and 50 g/kg), and to analyze whether the antimicrobial peptide NZ2114 was evenly distributed.【Method】 The experiment mainly optimized extraction conditions, purification column and purification method, which investigated the influence of extraction agent and extraction time on extraction efficiency, the effect of SPE cleaning agent and elutriation agent on recovery rate.Feed samples were extracted by ultrasonic centrifugation, and the supernatant was enriched via WCX solid-phase extraction column, followed by purification and elution with 3 mL methanol and 5 mL of 20% ammoniated methanol.ZORBAX Eclipse Plus C18 was used as the stationary phase, and 0.1% formic acid-acetonitrile and 0.1% formic acid-water were used as mobile phases.The column temperature was 35℃, the injection volume was 20 μL, and the flow rate was 1.0 mL/min.The content of NZ2114 in feed samples was quantitatively determined by external standard method at a wavelength of 276 nm, and the linearity, limit of detection and limit of quantification, matrix effect, recovery rate, intra-day and inter-day precision of the method were investigated.【Result】 The SPE-HPLC method had a good linear relationship in the concentration range of 62.5-4 000 μg/mL, and the linear equation was y=0.9353x-32.721, whose correlation coefficient (R²) was 0.9996.The recovery rate of the method was 88.05%-111.23% at low, medium and high concentrations.The intra-day and inter-day precision were 0.58%-4.45%, 3.18%-6.37%, respectively.And the matrix effects at 125, 500 and 2 000 μg/mL concentrations were 1.02, 1.01 and 0.99, respectively.【Conclusion】 In this study, the SPE-HPLC method for the determination of antimicrobial peptide NZ2114 in rat feed was established, which met the requirements of methodological investigation, with the advantages of high sensitivity and precision, good reproducibility and rapid analysis.Thus, the method could achieve accurate quantification of NZ2114 in feed samples at three concentration levels.

Key words: high performance liquid chromatography; solid phase extraction; antimicrobial peptide NZ2114; rat feed

中图分类号:

S816

冯亚楠, 杨娜, 毛若雨, 郝娅, 马炫炫, 滕达, 王建华. 高效液相色谱法检测鼠饲料中抗菌肽NZ2114[J]. 中国畜牧兽医, 2023, 50(11): 4463-4472.

FENG Yanan, YANG Na, MAO Ruoyu, HAO Ya, MA Xuanxuan, TENG Da, WANG Jianhua. Determination of Antimicrobial Peptide NZ2114 in Rat Feed by High Performance Liquid Chromatography[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(11): 4463-4472.

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