高效液相二极管阵列检测法测定人血浆中伏立康唑和N-氧化伏立康唑浓度及其对儿童白血病的用药指导

High Performance Liquid Chromatography Coupling with Photo-diode Array Detector Method for Monitoring Voriconazole and Its Main Metabolite N-oxidized Voriconazole and Its Application to Medication Guidance for Childhood Leukemia

目的建立同时测定伏立康唑及其主要代谢物N-氧化伏立康唑血药浓度方法,并用于监测2~12岁儿童白血病中伏立康唑和N-氧化伏立康唑的血药浓度。方法采用Agilent ZORBAX SB-C18柱(4. 6 mm×250 mm,5μm),流动相为乙腈、甲醇与醋酸铵缓冲液(10 mmol·L^-1,用醋酸调节pH值至3. 0)=27∶23∶50,流速为1 mL·min^-1,波长为262nm。以卡马西平为内标,样品处理采用乙酸乙酯液液萃取。结果伏立康唑和N-氧化伏立康唑的血浆浓度在0. 1~8. 0μg·mL^-1内呈良好的线性,定量下限均为0. 1μg·mL^-1,批内、批间精密度(RSD)均在3%~10%以内。准确度和方法回收率均符合要求。此次监测29例儿童白血病患者,共100个血样。伏立康唑浓度、N-氧化伏立康唑及N-氧化伏立康唑与伏立康唑的比值中位数值分别为0. 87μg·mL^-1(IQR,0. 32~1. 65μg·mL^-1)、1. 15μg·mL^-1(IQR,0. 55~1. 67μg·mL^-1)和1. 10 (IQR,0. 66~1. 22)。伏立康唑浓度与N-氧化伏立康唑浓度存在相关性(r=0. 603 1,P <0. 000 1)。结论本方法操作简单、准确、专属性高,满足临床同时监测伏立康唑和N-氧化伏立康唑浓度,可用于伏立康唑药动学研究及临床上治疗药物监测。

OBJECTIVE To establish a method for simultaneous determination of voriconazole and its main metabolite N-oxidized voriconazole in blood,and monitor the levels of voriconazole and N-oxidized voriconazole in leukemia children aged 2-12 years.METHODS Agilent ZORBAX SB-C18 column( 4. 6 mm × 250 mm,5 μm) was used,and the mobile phase was acetonitrile,methanol and ammonium acetate buffer( 10 mmol·L^-1,p H adjusted with acetic acid to 3. 0)=27∶ 23∶ 50,the flow rate was 1 mL·min^-1,and the wavelength was set at 262 nm. The sample was extracted with ethyl acetate. Carbamazepine was used as the internal standard.RESULTS The calibration curves of voriconazole and N-oxidized voriconazole were linear in the range of 0. 1-8. 0 μg·mL^-1,the lower limit of quantitation was 0. 1 μg·mL^-1,and the intra-assay and inter-assay precision RSDs were between 2% and 10%. The accuracy,method recovery and stability all met the requirements. Twenty-nine patients were monitored with a total of 100 blood samples. The medians( IQRs) of voriconazole concentration,N-oxidized voriconazole concentration,and ratio of N-oxidized voriconazole to voriconazole were 0. 87 μg·mL^-1( 0. 32-1. 65 μg·mL^-1) and 1. 15 μg·mL-1( 0. 55-1. 67 μg·mL^-1) and 1. 10( 0. 66-1. 22),respectively.There was a correlation relationship between the concentrations of voriconazole and N-oxidized voriconazole( r = 0. 603 1,P < 0. 000 1).CONCLUSION The method is simple,accurate,highly specific,and can simultaneously monitor the concentrations of voriconazole and N-oxidized voriconazole,thus can be used for the pharmacokinetic research of voriconazole and clinical drug monitoring.