动物营养学报 >

2022 , Vol. 34 >Issue 11: 7411 - 7420

DOI: https://doi.org/10.3969/j.issn.1006-267x.2022.11.058

研究论文 RESEARCH PAPER

基于配体交换柱的高效液相色谱同时检测肠道内容物中8种短链有机酸浓度

  • 张亚伟 ,
  • 田博雅 ,
  • 王月红 ,
  • 刘强 ,
  • 张元庆
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  • 山西农业大学动物科学学院, 晋中 030801
张亚伟(1989—),男,河南扶沟人,讲师,博士,主要从事反刍动物营养与胃肠道微生物(组)学研究。E-mail:ywzhang@sxau.edu.cn

收稿日期: 2022-05-10

  网络出版日期: 2022-11-14

基金资助

山西省高等学校科技创新项目（2021L150）；山西农业大学科技创新基金项目（2020BQ68）；优秀博士来晋奖励项目（SXBYKY2021014）；谷子高粱产业技术体系（CARS-06-14.5-A30）

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Simultaneous Determination of 8 Short-Chain Organic Acids Concentration in Gastrointestinal Content by High Performance Liquid Chromatography with a Ligand-Exchange Column

  • ZHANG Yawei ,
  • TIAN Boya ,
  • WANG Yuehong ,
  • LIU Qiang ,
  • ZHANG Yuanqing
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  • College of Animal Science, Shanxi Agricultural University, Jinzhong 030801, China

Received date: 2022-05-10

  Online published: 2022-11-14

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摘要

本试验旨在建立一种同时检测肠道内容物中乳酸等8种短链有机酸浓度的液相色谱外标分析方法。待测样品使用25%的偏磷酸溶液进行前处理以去除其中的蛋白质等生物大分子后，使用高效液相色谱仪进行分析，色谱条件为：柱温60℃，流动相使用0.005 mol/L的硫酸溶液，流速0.7 mL/min，紫外检测器检测波长210 nm。结果显示：各有机酸基于峰面积和浓度所得到的标准曲线的决定系数均不低于0.999 9，线性关系良好；乳酸、甲酸、乙酸、丙酸、异丁酸、丁酸、异戊酸和戊酸浓度的线性范围分别为0.043 9~7.029 4 mmol/L、0.022 7~9.076 3 mmol/L、0.554 7~88.748 0 mmol/L、0.510 1~81.622 8 mmol/L、0.041 3~6.606 2 mmol/L、0.167 3~26.763 7 mmol/L、0.035 6~5.699 8 mmol/L和0.044 5~7.121 8 mmol/L，而检测限处于0.005 8~0.194 1 mmol/L；混合标准品溶液中各有机酸72 h内峰面积的相对标准偏差均小于3.30%，保留时间的标准差均不大于0.056 min，3 d内稳定性良好。供试瘤胃液样品重复性试验的峰面积相对标准偏差均小于3.20%，回收率试验中所有有机酸的加标回收率处于80.562 8%~111.496 0%，表明方法的精密度和准确度良好。综上所述，本试验所建方法，所需仪器配置较低，前处理和流动相相对简单，系统适应性、线性和稳定性良好，精密度和准确度高，可为研究营养物质在动物肠道中的消化吸收以及肠道甲烷生成乃至代谢性疾病等方面提供技术支撑。

关键词： 乳酸; 挥发性脂肪酸; 外标法; 配体交换; 高效液相色谱

本文引用格式

张亚伟 , 田博雅 , 王月红 , 刘强 , 张元庆 . 基于配体交换柱的高效液相色谱同时检测肠道内容物中8种短链有机酸浓度[J]. 动物营养学报, 2022 , 34(11) : 7411 -7420 . DOI: 10.3969/j.issn.1006-267x.2022.11.058

Abstract

An external standard calibration method based on high performance liquid chromatography (HPLC) equipped with a ligand-exchange column was developed in this study to determine simultaneously eight organic acids including lactic acid concentration in animal gastrointestinal content. The samples were pre-treated with 25% metaphosphoric acid to remove biological macromolecules including proteins. Then the targeted organic acid concentrations were analyzed using HPLC at column temperature of 60℃, with 0.005 mol/L sulfuric acid solution as mobile phase at a flow rate of 0.7 mL/min, and with an Ultraviolet Detectors at the wavelength of 210 nm. Results showed that the determination coefficient (R2) of calibration curves based on values of peak area and molar concentration of each organic acid was not less than 0.999 9, suggesting a great linear relationship. The linear concentration range of lactic acid, formic acid, acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid was 0.043 9 to 7.029 4 mmol/L,0.022 7 to 9.076 3 mmol/L,0.554 7 to 88.748 0 mmol/L,0.510 1 to 81.622 8 mmol/L,0.041 3 to 6.606 2 mmol/L,0.167 3 to 26.763 7 mmol/L,0.035 6 to 5.699 8 mmol/L and 0.044 5 to 7.121 8 mmol/L, respectively, and the detection concentration limits of the organic acids ranged from 0.005 8 to 0.194 1 mmol/L. The relative standard deviation (RSD) of the peak area of each organic acid within 72 h in standard mix was less than 3.30% while the standard deviation of retention time of each organic acid was less than 0.056 min, suggesting a good stability within 3 days. The RSD of peak area of each organic acid in employed rumen fluid sample was less than 3.20%, and the recovery rates of each organic acid at three different concentrations of standards were 80.562 8% to 111.496 0%, suggesting that the method has a good accuracy and precision. In conclusion, the method developed in this study has low requirements for instrument configuration, relatively simple pre-treatment strategy and mobile phases, good system adaptability, linearity, and stability, and high precision and accuracy, which can provide technical support for the study of nutrient digestion and absorption, methanogenesis and metabolic diseases in animal gastrointestinal tract.

Key words： lactic acid; volatile fatty acids; external standard calibration method; ligand-exchange; high performance liquid chromatography

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