人血浆百草枯高效液相色谱-串联质谱法的建立与评价

目的建立测定人血浆百草枯浓度的高效液相色谱-串联质谱法并评价。方法用甲醇蛋白质沉淀法对血浆样品预处理后,以乙腈-水(含200 mmol/L甲酸铵和0.1%甲酸)为流动相梯度进样,流速为0.4 mL/min。通过色谱柱(Waters XBridge BEH HILIC,2.5μm,2.1 mm×100 mm)分离样品。以ESI正离子模式、多反应离子监测(MRM)模式监测百草枯(m/z 186.1→171.1作为定量离子对)。用该方法检测临床患者血浆百草枯浓度,用ROC曲线评价血浆百草枯浓度和百草枯中毒严重指数(SIPP)对临床结局的判断价值。结果血浆百草枯浓度在50～10 000 ng/mL时,线性关系良好(R^2=0.997),最低定量下限为50 ng/mL。低(100 ng/mL)、中(2 000 ng/mL)、高(8 000 ng/mL)3个浓度水平质控品的不精密度均符合要求,且无基质效应。处理后样品室温放置6 h内稳定,样本反复冻融3次对结果无影响。31例中毒患者SIPP为17.76(0.30～90.91)h·mg/L,死亡组SIPP高于存活组(P<0.05)。SIPP的ROC曲线下面积为0.889,最佳临床临界值为11.679 h·mg/L。结论本法灵敏、准确、快速、特异性好,适用于临床检测百草枯中毒患者。

Rapid determination of paraquat in human plasma using liquid chromatography-tandem mass spectrometry

Objective To establish a high performance liquid chromatography-tandem triple quadrupole mass spectrometry method for the detection of human plasma paraquat concentration. Methods The plasma samples were pretreated with methanol to precipitate plasma protein, and then were separated by a Waters XBridge BEH HILIC column (2.5 μm, 2.1 mm × 100 mm) with acetonitrile-water containing 200 mmol/L of ammonium formate and 0.1% of formic acid as mobile phase and 0.4 mL/min of flow rate. The paraquat was monitored by ESI positive ion mode, multi-reaction ion monitoring (MRM) scanning, and m/z 186.1-171.1 as quantitative transition ion-pair. The plasma paraquat concentrations in patients were determined by the established method, and the clinical values of plasma paraquat concentration and severity index of paraquat poisoning (SIPP) were evaluated by the receiver operating characteristic (ROC) curve. Results When the plasma paraquat concentration ranged from 50 to 10 000 ng/mL, the linearity was good ( R^2= 0.997 ), and the lower limit of quantification was 50 ng/mL. The recovery rates and imprecisions of three quality control products at low (100 ng/mL), medium (2 000 ng/mL) and high (8 000 ng/mL) concentration levels all met the requirements, and no matrix effect was found. The pretreated samples were stable at room temperature for 6 hours, and the results were not affected by repeated freezing and thawing for 3 times. The SIPP of 31 poisoned patients was 17.76 (0.30-90.91) h·mg/L. The SIPP in dying patients was significantly higher than that in survival patients ( P <0.05). The area under the ROC curve of SIPP was 0.889, and the optimal cut-off value was 11.679 h·mg/L. Conclusion The established method is sensitive, accurate, rapid and specific, and suitable for the detection of plasma paraquat concentration in patients.