Simultaneous determination of isoniazid and acetylisoniazid in human serum by HPLC-MS/MS
Objective To establish a high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)method for the simultaneous determination of isoniazid and acetylisoniazid in human serum,and to detect the concentration of isoniazid in the blood of tuberculosis patients.Methods The serum samples were achieved on an Agilent RRHD SB-Aq(50.0 mm ×3.0 mm,1.8 μm),whose column temperature was 40 ℃.The mobile phase consisted of purified water and methanol with a gradient program at a flow rate of 0.25 mL·min-1.Both isoniazid and acetyl-isoniazid mass spectrometry detection methods were electrospray positive ion mode,multiple reaction monitoring.The specificity,lower limit of quantitation,standard curve,precision and recovery,matrix effect,stability and residual effect of the method were investigated.Results The retention times of isoniazid and acetylisoniazid were 1.37 and 1.24 min,respectively.Isoniazid showed a good linear relationship in the range of 0.2-20.0 μg·mL-1.The regression equation of isoniazid was y=0.29x+3.05 × 10-3(r2=0.999 6),and the lower limit of quantitation was 200 ng·mL-1.The regression equation of acetyl isoniazid was y=0.29x-3.21 × 10-3(r2=0.999 7),and the lower limit of quantitation was 200 ng·mL-1.The relative standard deviation(RSD)of intra-lot and inter-lot precision was less than 4.00％.The recovery rates were 100.34％-106.22％.Internal standard normalized matrix effect factor(MF)≤ 3.79％.The samples have good freeze-thaw cycle stability and long-term stability.The method has been successfully used to analyze serum samples of 72 patients with tuberculosis in our hospital.Conclusion This method is simple,rapid and accurate,and is suitable for simultaneous determination of isoniazid and acetylisoniazid in the blood of tuberculosis patients.
isoniazidacetyl-isoniazidhigh performance liquid chromatography-tandem mass spectrometryclinical application
万方数据
维普
