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

KeyWord:corticosteroids  ultra-performance liquid chromatography£­tandem mass spectrometry£¨UPLC£­MS/MS£©  isotope dilution£¨ID£©  protein precipitation£¨PPT£©  liquid£­liquid extraction£¨LLE£©

Abstract:

      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 chromatography£­tandem 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 tert-butyl methyl ether£®The separation of target compounds were performed on an ACQUITY UPLC BEH C18 column£¨2.1 mm ¡Á 100 mm£¬1.7 ¦Ìm£© 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£®