Liquid-phase Microextraction - Photochemical Fluorescence Method In Vivo Drug Analysis

OBJECTIVE To develop a method for determining the concentration of methotrexate in biological matrices by liquid–phase microextraction combined with photochemical fluorescence high performance liquid chromatography. METHODS By liquid-phase microextraction with back extraction apparatus self-made, methotrexate were extracted from biological sample made acidify in 0.05 mol/L HCL. The unionized analytes were extracted into n-butanol impregnated in the pores of the hollow fibre, and into an alkalizing solution inside the hollow fibre. The extracts were diluted with the equal volume 0.2 mol/L hydrochloric acid,after irradiation below the ultraviolet light for 45 minutes, And then, let the sample flow through a C18 column of HPLC with methanol-phosphate (pH=6.6)as the mobile phase( V V=15:85) .The C18 column is used with flow rate at 0.8 ml/min and temperature at 25℃.sample of 20μl were injected into the HPLC instrument and determined with fluorescence detector: excitation wavelength at 275nm and emission wavelength at 370nm.RESULTS Efficient sample clean-up was seen for methotrexate. The enrich factor was 20~24,the linear range and the detection limit (S/N=3) were 0.05～50μg/ml and 10ng/ml in plasma ,and 0.01~50μg/ml and 5 ng/ml in urine for this method,respectively, the relative standard deviation was below 9.31%. With the method ,some endogenous substances in matrices which interfered the determination of methotrexate were eliminated efficiently, and the higher selectivity was obtained. CONCLUSION The method was validated and successfully applied to determine methotrexate in plasma and urine from rabbit treated with methotrexate, indicating that liquid–phase microextraction with back extraction combined with photochemical fluorescence high performance liquid chromatography is a promising combination for analysis of drugs present in low concentrations in biological matrices.