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

• Published in: Cell biochemistry and biophysics 2013, Vol.67 (3), p.1343-1351
• Main Authors: Wang, Rong, Guo, Lifang, Xie, Hua, Zhang, Juanhong, Li, Xiaoyun, Li, Wenbing, Wang, Jianfeng, Wu, Xiaoyu, Jia, Zhengping
• Format: Article
• Language: English
• Subjects: Biochemistry; Biological and Medical Physics; Biomedical and Life Sciences; Biophysics; Biotechnology; Cell Biology; Chromatography, High Pressure Liquid; Extracellular Fluid - chemistry; Hep G2 Cells; Humans; Life Sciences; Methotrexate - analysis; Methotrexate - chemistry; Original Paper; Pharmacology/Toxicology; Spectrometry, Mass, Electrospray Ionization; Stereoisomerism; Tandem Mass Spectrometry; Temperature
• ISSN: 1085-9195; 1559-0283
• DOI: 10.1007/s12013-013-9666-9

A rapid and sensitive liquid chromatography–tandem mass spectrometry assay (LC–MS/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 HepG 2 cells. The analytes were extracted from homogenates using organic solvent to precipitate proteins. The extracted samples were analyzed by LC–MS/MS, operating in multiple reactions monitoring (MRM) mode. The condition of HPLC included the following: Gemini column (3 μm, 3.0 × 75 mm) 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 0.4 mL min −1 . The gradient was as follows: 0–7.0 min 10–90 % B, 7.0–10 min 90 % B followed by 3 min. The column temperature was maintained at 40 °C. The condition of MS included using electrospray ionization source; MRM mode with the transitions of m/z 455.2 →  m/z 308.1 was used to quantify MTX enantiomers. The linear calibration curve was obtained in the concentration range of 10.0 to 10,000 ng mL −1 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 HepG 2 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 HepG 2 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.