反相高效液相-荧光色谱法测定美托洛尔及其代谢物α-羟基美托洛尔血浆尿液药物浓度

Determination of metoprolol and its metabolite, α-hydroxymetoprolol, in human plasma and urine by RP-HPLC-FD

目的建立人血浆及尿液中美托洛尔及α-羟基美托洛尔的高效液相-荧光色谱法。方法以比索洛尔为内标,采用Agilent ZORBA×SB-C18色谱柱(4.6mm×150mm,5μm);流动相:乙腈-0.05mol/LpH3.5磷酸二氢钾三乙胺水溶液(0.3%三乙胺)(90∶10);流速1ml/min;荧光检测器:激发波长(Ex):216nm,发射波长(Em):312nm。结果血浆中美托洛尔在10～400ng/ml范围内(r=0.999),α-羟基美托洛尔在5～360ng/ml范围内(r=0.9991)线性良好。其绝对回收率分别为89%～98%和77%～94%,相对回收率分别为94%～106%和95%～99%。精密度:日内RSD分别<5.8%和9.3%,日间RSD分别<8.0%和9.0%;尿液中美托洛尔在10.0～4000ng/ml范围内(r=0.999),α-羟基美托洛尔在5～2400ng/ml范围内(r=0.9991)线性良好。其绝对回收率分别为90%～95%和79%～92%,相对回收率分别为96%～101%和93%～103%。精密度,日内RSD分别<8.9%和6.6%,日间RSD分别<8.3%和7.7%;均符合药代动力学研究要求。结论用该法研究中国健康成年志愿者口服美托洛尔的体内药物代谢,及研究中国人群CYP2D6基因多态性与美托洛尔代谢的关系灵敏、可靠、实用。

Objective To establish a method for determination of metoprolol and its metabolite, α-hydroxymetoprolol （α-OH-met）, in human plasma and urine by RP-HPLC-FD. Methods Chromatographic conditions included an Agilent ZORBAxSB-C18 column （4.6 mm×150 mm, 5 μm）, a mobile phase of a mixture of 0.05M KH2PO4 （adjusted to pH 3.5 with phosphoric acid） - acetonitrile （90：10, V：V） with a flow rate of 1 ml·min^-1, a fluorescence G1321A detector chosen excitation wavelength （hex） in 216nm and emission wavelength （hem） in 312nm from Agilent Technologies （USA）. Bisoprolol was used as the internal standard. Results The linear ranges of the plasma samples were 10-400.0 ng/ml （r=0.999） for metoprolol and 5-360 ng/ml （r=0.9991） for α-OH-met, with absolute recovery of 89%-95% and 77%-94%, relative recovery of 94%-106% and 95%-99%, respectively. The intra-day relative standard deviation （RSD） was 〈5.8% and 9.3%, and inter-day relative standard deviation （RSD） was 〈8.0% and 9.0%, respectively. The linear ranges of the human urine samples were 10.0-4000 ng/ml （r=0.999） for metoprolol, and 5-2400 ng/mL （r= 0.9991） for α-OH-met, with absolute recovery of 90%-95% and 79%-92%, relative recovery of 96%-101% and 93% -103%, respectively. The intra-day relative standard deviation （RSD） was 〈8.9% and 6.6%, and inter-day relative standard deviation （RSD） was 〈8.3% and 7.7% respectively. Conclusion The current study recognized a method that is sensitive, reliable and practical for in-vivo pharmacokinetic evaluation of oral metoprolol in healthy Chinese adults, and study of CYP2D6 polymorphism in Chinese population associated with metoprolol metabolism.