Determination of Concentration of Methotrexate Enantiomers in Intracellular and Extracellular Fluids of HepG2 Cells by Liquid Chromatographyandem Mass Spectrometry

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• 作者：Rong Wang (1) (2)
  Lifang Guo (1) (2)
  Hua Xie (1)
  Juanhong Zhang (1)
  Xiaoyun Li (1)
  Wenbing Li (1)
  Jianfeng Wang (1)
  Xiaoyu Wu (1)
  Zhengping Jia (1) (2)
  
• 关键词：Methotrexate ; Enantiomers ; LCS/MS ; HepG2 ; Intracellular ; Extracellular
• 刊名：Cell Biochemistry and Biophysics
• 出版年：2013
• 出版时间：December 2013
• 年：2013
• 卷：67
• 期：3
• 页码：1343-1351
• 全文大小：
• 作者单位：Rong Wang (1) (2)
  Lifang Guo (1) (2)
  Hua Xie (1)
  Juanhong Zhang (1)
  Xiaoyun Li (1)
  Wenbing Li (1)
  Jianfeng Wang (1)
  Xiaoyu Wu (1)
  Zhengping Jia (1) (2)
  
  1. Department of Pharmacy, Lanzhou General Hospital of PLA, Lanzhou, 730050, Gansu, People Republic of China
  2. School of Pharmacy, Lanzhou University, Lanzhou, Gansu, People Republic of China
  
• ISSN：1559-0283

文摘

A rapid and sensitive liquid chromatographyandem mass spectrometry assay (LCS/MS) with electrospray ionization was developed and validated for the quantitative determination of the concentration of methotrexate (MTX) enantiomers in intracellular and extracellular fluids of HepG2 cells. The analytes were extracted from homogenates using organic solvent to precipitate proteins. The extracted samples were analyzed by LCS/MS, operating in multiple reactions monitoring (MRM) mode. The condition of HPLC included the following: Gemini column (3, 3.075mm) with chromatographic column was used, and the mobile phase consisting of gradient elution utilized 0.1% formic acid as solvent A and acetonitrile as solvent B at a flow rate of 0.4mLmin1. The gradient was as follows: 0.0min 100% B, 7.00min 90% B followed by 3min. The column temperature was maintained at 40. The condition of MS included using electrospray ionization source; MRM mode with the transitions of m/z 455.2m/z 308.1 was used to quantify MTX enantiomers. The linear calibration curve was obtained in the concentration range of 10.0 to 10,000ngmL1 for MTX enantiomers in intracellular and extracellular fluids. The inter- and intraday precision was less than 15%. The mean recovery of (+)-MTX and ()-MTX in the extracellular fluid of HepG2 cells were 95.30 and 96.53%, respectively, and the mean recovery of (+)-MTX and ()-MTX in the intracellular fluid of HepG2 cells were 93.53 and 94.12%, respectively. This method was successfully used to detect the concentration of MTX enantiomers in the intracellular and extracellular fluids of HepG2 cells and that the concentration of (+)-MTX in intracellular fluid was twice higher than the concentration of ()-MTX in intracellular fluid. The inhibitory effect of (+)-MTX and ()-MTX was (+)-MTX>()-MTX. It is a simple, precise method that can effectively explain the difference in pharamocological effect of MTX enantiomers in vitro.