羽苔素E对白念珠菌细胞内氟康唑浓度的影响研究

HPLC-MS determination concentration of fluconazole and the impact of plagiochin E in Candida albicans

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 develop a bioanalytical method using HPLC-MS for detecting the concentration of fluconazole （FLC） in Candida albican （C.albicans） cells and consequently evaluating the impact of plagiochin E （PLE） on the accumulation of FLC in FLC-resistant C.albicans cells.Methods：FLC-resistant C.albicans cells were treated with FLC or FLC with PLE together.Samples of C.albicans thallus that were obtained by centrifuging after 24 h incubation saponified and centrifuged to extract FLC.The HPLC separation was performed on a Phenomenex-C18（250 mm×4.6 mm,5 μm）column,using ammonium acetate-acetonitrile （3∶7,v/v） as mobile phase,with a flow rate of 0.8 mL·min^-1.The sample was analyzed in the positive-ion mode.Then multiple reaction monitoring mode with the transition of m/z 307.4→220.3 and m/z 531.2 →489.3 were used to quantify fluconazole and ketoconazole （IS）,respectively.Results：A good linearity of FLC was obtained in the concentration range of 1^-100 μg·L^-1 （r=0.9956）.The lower quantitative limit was 1 μg L^-1.The inter-and intra-day RSDs were less than 6.4%,the method recoveries were in the range of 94.1%-98.3%.Results showed that PLE facilitated the accumulation of FLC in FLC-resistant C.albicans cells significantly in a dose-dependent manner.Conclusion：PLE can increase the concentration of FLC in FLC-resistant C.albicans cells markedly.This can be one of the important mechanisms attributed to the synergetic effect of PLE in combination with FLC against the resistant strains.