关键词: 百草枯, 高效液相色谱串联质谱法, 方法学评价

Abstract:

Objective To establish high performance liquid chromatography-tandem mass spectrometry（HPLC-MS/MS） for the determination of paraquat in plasma. Methods Phenomenex Kinetex 2.6 μm HILIC （100.0 mm×2.1 mm） was selected as chromatographic column,mobile phase A was 1% formic acid aqueous solution （with 250 mmol/L ammonium formate）,and mobile phase B was acetonitrile. Gradient elution was performed,the flow rate was 0.35 mL/min,the sample volume was 10 μL,and the column temperature was 40 ℃. Electrospray ionization（ESI） source and positive ion multiple reaction monitoring scan mode were used. The mass-to-charge ratio（m/z）of paraquat select ion was 92.7→171.0,and the m/z of ethyl-paraquat selected ion was 107.0→185.1. The methodology（linear range,quantitative limit,accuracy,precision,selectivity,matrix effect and stability） was evaluated. Plasma specimens of 75 patients with acute paraquat poisoning were determined. Receiver operating characteristic（ROC） curve was used to assess plasma paraquat level for diagnosis. Results There was a linearity at the level of 54.28-13 190.00 ng/mL,and the linear equation was Y=0.000 1X+0.011 6（r²=0.998 3）. The within-run and between-run accuracies were 96.31%-120.04% and 104.43% at the quantitative limit levels. The bias between within-run and between-run accuracies and listed value were ±15% at the high,medium or low concentration. The within-run and between-run relative standard deviations（RSD） were <15% at the high,medium and low concentrations or quantitative limit levels. The selectivity and matrix effect of the method were assessed,and 6 blank matrix were selected randomly. The location of the paraquat peaks appeared without interference,and the method was selective. The coefficient of variation of matrix factor,which was normalized by internal standard,was <15% at the high or low concentration,and the matrix effect met related requirements. Specimens can be stable being placed within 1 d at room temperature,within 1 week at 2-8 ℃ refrigerator and within 1 month at -80 ℃ refrigerator and being stable after repeating freezing and thawing for 3 times. The median was 2 820（0-22 200） ng/mL. The median in survival group was higher than that in death group（P<0.05）. The area under ROC curve for plasma level of paraquat predicting outcome was 0.855. The optimal cut-off value was 2 431 ng/mL. Conclusions The established HPLC-MS/MS can be used to clinical routine determination for plasma level of paraquat.

Key words: Paraquat, High performance liquid chromatography-tandem mass spectrometry, Methodology evaluation