RESUMO
Aim To rapidly prepare and purify hydrogen sulfide specific fluorescent probe (WSP-5), establish and optimize the fluorescent probe method for the determination of hydrogen sulfide in animal tissues, and verify the applicability of the method in the model of malignant pleural effusion. Methods The preparation solvent of fluorescent probe reaction solution, DMSO addition volume, pH, reaction solution solvent and reaction solution volume, sample pretreatment temperature, grinding times, and standing time after grinding were investigated. The mouses model of malignant pleural effusion was established with S-180 ascites tumor cells, and the concentration of hydrogen sulfide in various organs and tissues of the model animal was measured. Results After optimization, silica gel and dextran gel were used as stationary phases, dichloromethane methanol formic acid (60: 1: 0.1, V/V/V) and dichloromethane methanol (1: 1, V/V) were used as eluents for separation and purification, and the first eluting component was taken to prepare WSP-5 with a purity of more than 700 mg. Animal tissue samples and sodium hydrosulfide standard solution were added with 5 times of cold reaction solution, after low temperature vibration grinding, highspeed centrifugation, the supernatant was incubated in dark for 12 hours, the fluorescence intensity was measured by fluorescent microplate reader. Hydrogen sulfide concentration was calculated according to the standard curve. The LOD of this method was about 0. 6 |JLmol • L