Abstract: INTRODUCTION: The phytohormone salicylic acid (SA) plays various important roles in plants, such as disease resistance, seed germination, and leaf senescence. Among them, the roles of SA in plant disease resistance are most well-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, measurement of SA content is an important aspect in plant immunity study. 
RATIONALE: High-performance liquid chromatography (HPLC)-fluorescence detector is the most commonly used 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, and the detection wavelength and detection procedure. 
RESULTS: The baseline of the chromatogram was more stable when acetonitrile instead of methanol was used in the mobile phase. When the pH of the mobile phase was 5.2, the retention time of SA was short, and there was no interference peak near the SA peak, which was favorable to shorten 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, which improved the sensitivity of the detection of SA. 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 of one sample to 10 min. 
CONCLUSION: These optimizations greatly improved the sensitivity, stability, and efficiency of the SA measurement, which will contribute to the plant immunity research.

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