Determination of concentration of methotrexate enantiomers in intracellular and extracellular fluids of HepG2 cells by liquid chromatography-tandem mass spectrometry.

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 HepG2 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 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.