高效液相色谱法对绿脓菌素的表达分析

Expression Analysis of Pyocyanin by High Performance Liquid Chromatography

目的 采用高效液相色谱法建立铜绿假单胞菌代谢物绿脓菌素测定方法,并对绿脓菌素含量表达进行分析。方法 采用Inertsil ODS-SP(4.6 mm×250 mm,5μm)色谱柱分离,柱温40℃,流速为1.0 mL/min,进样量20μL,流动相为0.01%甲酸水—乙腈梯度洗脱,检测波长为268 nm。结果 绿脓菌素在0.20~10.00μg/mL范围内线性关系良好;绿脓菌素方法检出限为0.10μg/mL,方法定量限为0.20μg/mL;在0.22~0.66μg/mL和1.10~3.30μg/mL的添加范围内,绿脓菌素的回收率(n=3)为94.85%~113.67%,方法精密度(RSD)小于0.61%。通过该方法初步测定,发现较低浓度左氧氟沙星对铜绿假单胞菌分泌绿脓菌素起促进作用,可能与其耐药机制有关。结论 该方法简便、准确,精密度和回收率良好,可用于绿脓菌素的检测,对进一步研究铜绿假单胞菌耐药机制和绿脓菌素分泌量的关系具有一定的指导意义。

Objective A method for the determination of Pseudomonas aeruginosa metabolite was established by high performance liquid chromatography,and the expression of pyocyanin content was analyzed.Method Using Inertsil ODS-SP(4.6 mm×250 mm,5μm)Chromatographic column separation,column temperature 40℃,flow rate 1.0 mL/min,sample injection amount 20μL.The mobile phase was 0.01%formic acid water acetonitrile gradient elution with a detection wavelength of 268 nm.Results Pseudomonas aeruginosa had a detection limit of 0.10μg and a quantitative limit of 0.20μg under optimized conditions.The method's precision(RSD)was less than 0.80%in the addition range of 0.22-0.66μg/mL and 1.10-3.30μg/mL,and the target compound was linearly good in the linear range of 0.20-10.00μg/mL.Preliminary tests using this method show that low quantities of levofloxacin promote Pseudomonas aeruginosa's secretion of pyocyanin,which may be connected to the bacterium's drug resistance mechanism.Conclusion The method is simple,accurate,with good precision and recovery,and can be used for the detection of pyocyanin.It has a certain guiding significance for further research on the relationship between the drug resistance mechanism of Pseudomonas aeruginosa and the secretion of pyocyanin.