高效液相色谱法检测水杨酸的优化

doi:10.11983/CBB25102

摘要/Abstract

摘要： 植物激素水杨酸(SA)促进植物的抗病性, 但抑制植物生长。植物通过动态调控SA的含量以平衡抗病与生长。高效液相色谱-荧光检测器技术是检测SA含量最常用的方法。该研究优化了流动相的成分、离子浓度、pH值以及检测波长和检测程序。优化后的流动相为10%乙腈、100 mmol∙L^-1乙酸钠、pH5.2。优化后的激发光波长为300 nm, 发射光波长为405 nm。优化后的检测程序为: 进样后3.5分钟开始清洗色谱柱, 清洗时间为3.5分钟, 平衡时间为3分钟, 总时长为10分钟。优化后的检测方法显著提高了检测灵敏性、稳定性和高效性。

关键词: 水杨酸, 抗病, 检测方法, 高效液相色谱, 优化

Abstract: INTRODUCTION: The phytohormone salicylic acid (SA) plays multiple important roles in plants, such as disease resistance, seed germination, and leaf senescence. Among these, the role of SA in plant disease resistance is the most studied. Since SA promotes disease resistance at the cost of plant growth, plants need to dynamically regulate the content of SA to balance disease resistance and growth. Therefore, fast and accurate measurement of SA content is a critical basis for plant immunity research. RATIONALE: High-performance liquid chromatography (HPLC)-fluorescence detector is the most popular method for the quantitative measurement of SA. In order to improve the efficiency and sensitivity of current methods, this study optimized the composition, ion concentration, and pH of the mobile phase, as well as the detection wavelength and detection procedure. RESULTS: The baseline of the chromatogram was more stable when using acetonitrile instead of methanol in the mobile phase. When the pH of the mobile phase was 5.2, the retention time of SA was the shortest, without interference peak near the SA peak, which was preferred for minimizing the detection time. The higher concentration of sodium acetate (100 mmol∙L^-1) in the mobile phase was better than that of lower concentration (20-50 mmol∙L^-1). Wavelength scanning revealed that the optimal excitation wavelength was 300 nm and the optimal emission wavelength was 405 nm, under which the highest sensitivity for SA detection was obtained. At a flow rate of 2 mL∙min^-1, it took 3.5 min for elution, 3.5 min for column wash, and 3 min for column balance, shortening the measurement time per sample from 50 min to 10 min. CONCLUSION: These optimizations greatly improved the sensitivity, stability, and efficiency of the SA measurement using HPLC, which will contribute to the plant immunity research.

Optimization of salicylic acid (SA) measurement. (A) SA detection procedure; (B) Chromatogram using the optimized condition. The samples are total SA in Arabidopsis without Psm ES4326 infection. EU: Emission units. The peak labeled in red indicates SA.

Key words: salicylic acid, disease resistance, measurement, high-performance liquid chromatography, optimization

史世肸, 严顺平. 高效液相色谱法检测水杨酸的优化. 植物学报, 2025, 60(5): 846-853.

Shi Shixi, Yan Shunping. Optimization of an High-performance Liquid Chromatography Method for the Determination of Salicylic Acid. Chinese Bulletin of Botany, 2025, 60(5): 846-853.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB25102

https://www.chinbullbotany.com/CN/Y2025/V60/I5/846

图/表 6

图1 使用Zhang等(2017)的方法检测水杨酸(SA) (A) 0.1 μg·mL-1 SA标准样品; (B) 样品为未被Psm ES4326侵染的拟南芥游离态SA。EU: 发射单位。红色的峰指示SA。

Figure 1 Detection of salicylic acid (SA) using methods reported in Zhang et al., 2017 (A) 0.1 μg·mL-1 SA standard sample; (B) The sample is free SA in Arabidopsis without Psm ES4326 infection. EU: Emission units. The peaks labeled in red indicate SA.

图2 流动相pH值对水杨酸(SA)分离的影响 (A)-(C) 样品为被Psm ES4326侵染24小时后的拟南芥总SA; (D)-(F) 样品为未被Psm ES4326侵染的拟南芥总SA。EU同图1。红色的峰指示SA。

Figure 2 Effects of pH of the mobile phase on salicylic acid (SA) separation (A)-(C) The samples are total SA extracted from Arabidopsis treated with Psm ES4326 for 24 h; (D)-(F) The samples are total SA in Arabidopsis without Psm ES4326 infection. EU is the same as shown in Figure 1. The peaks labeled in red indicate SA.

图3 流动相中乙酸钠浓度对水杨酸(SA)分离的影响 (A) 20 mmol∙L-1乙酸钠; (B) 100 mmol∙L-1乙酸钠。样品为未被Psm ES4326侵染的拟南芥总SA。EU同图1。红色的峰指示SA。

Figure 3 Effects of the concentration of sodium acetate in the mobile phase on salicylic acid (SA) separation (A) 20 mmol∙L-1 NaAc; (B) 100 mmol∙L-1 NaAc. The samples are total SA in Arabidopsis without Psm ES4326 infection. EU is the same as shown in Figure 1. The peaks labeled in red indicate SA.

图4 水杨酸(SA)检测波长的优化 (A), (B) 激发光波长为296 nm, 发射光波长为350-450 nm; (C), (D) 激发光波长为280-320 nm, 发射光波长为395 nm; (E), (F) 激发光波长为300 nm, 发射光波长为375-415 nm。(A), (C), (E) 二维信号图; (B), (D), (F) 荧光波长-荧光能量积分图。样品均为1 µg∙mL-1 SA标准样品。EU同图1。

Figure 4 Optimization of detection wavelength for salicylic acid (SA) (A), (B) The excitation wavelength is 296 nm and the emission wavelength is 350-450 nm; (C), (D) The excitation wavelength is 280-320 nm and the emission wavelength is 395 nm; (E), (F) The excitation wavelength is 300 nm and the emission wavelength is 375-415 nm. (A), (C), (E) Two-dimensional signal plots; (B), (D), (F) Fluorescence wavelength-fluorescence energy integration plots. All samples are 1 µg∙mL-1 SA standards. EU is the same as shown in Figure 1.

图5 水杨酸(SA)检测程序的优化 (A) 检测程序图; (B) 检测结果。样品为未被Psm ES4326侵染的拟南芥总SA。EU同图1。红色的峰指示SA。

Figure 5 Optimization of salicylic acid (SA) detection procedure (A) Graph of detection procedure; (B) Detection results. The samples are total SA in Arabidopsis without Psm ES4326 infection. EU is the same as shown in Figure 1. The peak labeled in red indicates SA.

图6 水杨酸(SA)标准曲线 (A) 不同浓度SA标准样品对应的峰面积; (B) 标准曲线(截距为0, R2表示决定系数)

Figure 6 Salicylic acid (SA) standard curve (A) Peak areas corresponding to different concentrations of SA standards; (B) The standard curve (with an intercept of 0, R2 indicates the coefficient of determination)

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