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目的:建立高效液相色谱质谱联用技术测定耐药真菌细胞内氟康唑的浓度并观察羽苔素E对细胞内氟康唑聚集的影响。方法:将白念耐药株菌悬液与氟康唑单独培养或氟康唑和羽苔素E联合培养24h后,经离心、皂化、提取等过程得到菌体细胞内聚集的氟康唑。样品以Phenomenex-C18柱(250mm×4.6mm,5μm)色谱柱进行分离,流动相为醋酸铵-乙腈(3∶7,v/v),流速0.8mL·min-1。正离子方式检测,多反应监测(MRM)方式测定样品浓度,监测离子对分别为m/z307.4→220.3(氟康唑)和m/z531.2→489.3(内标酮康唑)。通过测定氟康唑的浓度,观察不同浓度羽苔素E对细胞内氟康唑浓度的影响。结果:高效液相色谱质谱联用法可用于真菌细胞内氟康唑浓度的测定。氟康唑在1～100μg·L-1浓度范围内线性关系良好(r=0.9956),最低定量限为1μg·L-1。低、中、高3种浓度质控样品的日内、日间精密度小于6.4%,方法回收率94.1%～98.3%。并且羽苔素E能显著增加耐药株细胞内氟康唑的浓度,呈剂量依赖性。结论:羽苔素E能显著提高耐药株菌体细胞内氟康唑的浓度,是羽苔素E和氟康唑合用产生协同作用的重要机理,提示羽苔素E可能具有逆转氟康唑耐药的作用。
OBJECTIVE: To establish a high performance liquid chromatography mass spectrometry technique for the determination of fluconazole in drug-resistant fungal cells and to observe the effect of typhoid E on the intracellular fluconazole accumulation. METHODS: The fluconazole accumulated in the cells was obtained by centrifugation, saponification, and extraction after 24 hours of combined culture of fluconazole alone or fluconazole and lignin. The sample was separated on a Phenomenex-C18 column (250 mm×4.6 mm, 5 μm). The mobile phase was ammonium acetate-acetonitrile (3:7, v/v) and the flow rate was 0.8 mL·min-1. Positive ion detection and multiple reaction monitoring (MRM) were used to determine the sample concentration. The monitored ion pairs were m/z 307.4→220.3 (fluconazole) and m/z 531.2→489.3 (internal standard ketoconazole). By measuring the concentration of fluconazole, the effects of different concentrations of cyanodine E on the concentration of fluconazole in the cells were observed. Results: High performance liquid chromatography mass spectrometry can be used to determine fluconazole concentration in fungal cells. Fluconazole had good linearity (r=0.9956) in the concentration range of 1～100μg·L-1, and the lowest limit of quantification was 1μg·L-1. The intra-day and inter-day precisions of the low, medium, and high concentrations of quality control samples were less than 6.4%, and the method recoveries were between 94.1% and 98.3%. In addition, the concentration of fluconazole in the resistant strain cells was significantly increased in a dose-dependent manner. Conclusion: Litulin E can significantly increase the concentration of fluconazole in the bacterial cells of drug-resistant strains. It is an important mechanism for the synergistic effect of the combination of lignan E and fluconazole, suggesting that yukonin E may have the potential to reverse fluconazole. The role of drug resistance.