Determination of the concentrations of phenacetin and its metabolites in human serum and urine by LC-MS/MS method

Objective:To establish a liquid chromatography-tandem mass spectrometry(LC-MS/MS) method for determination of the concentrations of phenacetin and its metabolites paracetamol in human serum and urine.Methods:Phenacetin and its metabolites in human serum and urine were extracted with ethyl acetate and measured by LC-MS/MS method.A Restek Allure PFPP column(100 mm×2.1 mm,5 μm) was used.The mobile phase was acetonitrile-0.1% formic acid water solution(30∶70,V/V) with the flow rate of 0.3 mL/min.Sulfamonomethoxine was used as internal standard(IS).Phenacetin,paracetamol and IS were monitored by multiple reaction monitoring(MRM) mode with ion transitions of m/z 180→138,152→110 and 281→156,respectively.Thirteen healthy volunteers were orally administered 1.25 g phenacetin and the concentrations of phenacetin and its metabolites in their sera and urine were determined with the described LC-MS/MS method.Results: The retention time of phenacetin,paracetamol and IS were 4.1,1.4 and 2.8 min,respectively.In the range of 0.2-50 μg/mL,the concentration and peak area of phenacetin and paracetamol showed a good correlation(r0.999 0).The accuracy of phenacetin and paracetamol was 87.1%-101.5% and 89.5%-107.3%,the precision was 0.85%-7.83% and 2.53%-8.74%,respectively.The extraction recovery ranged from 58.25% to 69.85%,and matrix effect was 99.0% to 103.6%.No interference was found with conventional drugs,except vancomycin and methylprednisolone.The average concentrations of phenacetin and paracetamol were(5.94±4.79) and(8.81±2.91) μg/mL in serum,(3.25±1.10) and(570.45±187.13) μg/mL in urine in the 13 healthy volunteers.Conclusion: This method is proved to be simple,sensitive and practicable in clinical practice.