HPLC-HG-AFS法测定亚砷酸治疗的急性早幼粒细胞白血病患者尿液中砷代谢产物

Determination of arsenic metabolites in urine of acute promyelocytic leukemia patients treated with arsenic trioxide by HPLC-HG-AFS

目的:建立高效液相色谱-氢化物发生-原子荧光(HPLC-HG-AFS)法测定亚砷酸治疗的急性早幼粒细胞白血病(APL)患者尿样中四种砷代谢产物(As~Ⅲ,DMA,MMA,AsV)的浓度,探究砷代谢产物在APL患者尿中的形态类别、分布及变化等情况,进而揭示其与疗效及毒副作用的关系。方法:色谱柱:Hamilton PRP-X100柱(4.1 mm×250 mm,10μm);流动相:CH_3COONa(13 mmol·L^(-1))-Na H_2PO_4(3mmol·L^(-1))-KNO_3(4 mmol·L^(-1))-EDTA·2Na(0.2 mmol·L^(-1)),p H=6;流速:1.0 m L·min^(-1);柱温:30℃;进样量:100μL。还原液:3%KBH4(m/v)-0.5%KOH(m/v);载流液:10%HCl(v/v)。主电流:80 m A,辅电流:40 m A;屏蔽气流量:900 m L·min^(-1);载气流量:300 m L·min-1;负高压:300 V;泵转速:65 r·min^(-1)。结果:尿液中四种砷代谢产物在2.0~100.0 ng·m L-1浓度范围内呈良好线性关系(R^2≥0.999 1);加标回收率在82%以上,精密度良好,并用本方法检测了患者尿液中四种砷代谢产物的浓度。结论:所建立的测定方法准确度和灵敏度高,操作简便,可靠性好,成本低,适用于亚砷酸治疗的APL患者尿液中砷代谢产物浓度的检测。

Objective： To establish a method to determinate arsenic metabolites （AsⅢ, DMA, MMA, AsV） in urine of acute promyelocytic leukemia （APL） patients treated with arsenic trioxide by HPLC-HG-AFS, for studying the morphological categories, distributions and change of arsenic metabolites in urine of APL patients and investigating the relationship between the curative effect and side effects. Methods： Chromatographic column： Hamilton PRP-X100 column （4.1 mm × 250 mm, 10 μm）; mobile phase： CH3COONa （13 mmol·L-1）-NaH2PO4 （3 mmol·L-1）-KNO3 （4 mmol·L-1）-EDTA·2Na （0.2 mmol·L-1）, pH = 6; fow rate： 1.0 mL·min-1; column temperature： 30 ℃; injection volume： 100 μL. Reductant： 3% KBH4 （m/v）-0.5% KOH （m/v）, the carrier fuid： 10% HCl （v/v）. AFS conditions： main current： 80 mA, auxiliary current： 40 mA, shielding gas fow rate： 900 mL·min-1; carrier gas fow rate： 300 mL·min-1; negative high voltage： 300 V; pump speed： 65 r·min-1. Results： The concentration of AsⅢ , DMA, MMA, AsV in （2.0 – 100.0） ng·mL-1 showed good linear relationships （R2≥ 0.999 1）. Spiked recovery rates were above 82%, and the precision was good. This method was used to determinate concentration of arsenic metabolites in urine of APL patients treated with arsenious acid. Conclusion： The accuracy and sensitivity of this method are high, the operation is simple, the reliability is good and the cost is low, which is suitable for the determination of arsenic metabolites in urine of APL patients.