超高效液相色谱-串联质谱法同时检测人血清中4种内源性皮质激素

Simultaneous Detection of Four Endogenous Corticosteroids in Human Serum by Ultra-performance Liquid Chromatography-Tandem Mass Spectrometry

为建立人血清中醛固酮(ALD)、皮质醇(F)、皮质酮(CORT)、可的松(COR)4种内源性皮质激素的快速分析方法,该文首先对样品前处理方法进行优化,以0.3 mol/L硫酸锌溶液结合叔丁基甲基醚对样品进行蛋白沉淀-液液萃取(PPT-LLE)前处理。采用ACQUITY UPLC BEH C_(18)(2.1 mm×100 mm,1.7μm)色谱柱以0.01 mmol/L氨水-乙腈作为流动相进行梯度洗脱,电喷雾离子源正负快速切换模式检测,基于同位素稀释(ID)内标法采用超高效液相色谱-三重四极杆质谱联用(UPLC-MS/MS)进行定量。结果表明,ALD的线性范围为0.05~20 ng/mL,其他3种内源性皮质激素的线性范围为0.02~20 ng/mL,相关系数均大于0.995。ALD的检出限为20 pg/mL,定量下限为50 pg/mL,其它3种皮质激素的检出限为10 pg/mL,定量下限为20 pg/mL。4种皮质激素的绝对提取回收率为55.5%~90.8%,方法回收率为91.0%~101%,日内相对标准偏差(RSD)不大于13%,日间RSD不大于18%,基质效应为85.9%~108%。该方法能实现对空白血清和人血清样本的准确、快速检测,适用于复杂基质血清中内源性皮质激素的检测。

Corticosteroids are important signaling molecules in organisms.Although the content of corticosteroids in human body is very low,they play an important role in regulating physiological activities such as immune system and stress response.In order to establish a rapid quantitative method for the detection of four endogenous corticosteroids,including aldosterone(ALD),cortisol(F),corticosterone(CORT)and cortisone(COR)in human serum by ultra-performance liquid chromatographytandem mass spectrometry(UPLC-MS/MS).The sample was pretreated by protein precipitation combined with liquid-liquid extraction(PPT-LLE)using 0.3 mol/L zinc sulfate solution and tertbutyl methyl ether.The separation of target compounds were performed on an ACQUITY UPLC BEH C_(18) column(2.1 mm×100 mm,1.7μm)using 0.01 mmol/L ammonia-acetonitrile system as mobile phase by gradient elution.The four endogenous corticosteroids were detected by UPLC-MS/MS under multiple reaction monitoring(MRM)mode in positive or negative fast-switching ESI mode,and quantified by the isotope dilution(ID)internal standard method.The results showed that the calibration curves were linear(r2>0.995)in the range of 0.05-20 ng/mL for ALD,and 0.02-20 ng/mL for the other analytes.The detection of limit(LOD)and quantitation of limit(LQQ)of ALD wase 20,50 pg/mL,respectively,the LODs and LOQs of the other analytes were 10,20 pg/mL,respectively.The absolute extraction recoveries for four corticosteroids were between 55.5%and 90.8%.The method recoveries ranged from 91.0%to 101%,with intra-day relation deviation standards(intra-RSDs)and inter-day RSDs(inter-RSDs)not more than 13%and 18%,respectively.Meanwhile,the matrix effects were in the range of 85.9%-108%.The pretreatment method could purify serum samples significantly and increase the reliability of the detection results.It was easy to realize high-throughput processing without derivatization and complex steps.This method was accurate,rapid,stable and reproducible,and was suitable for the detection of serum endogenous corticosteroids in complex matrices.