摘要
目的:建立测定人血浆中25羟基维生素D[25(OH)D]的方法,并将其应用于临床。方法:血浆样品经液-液萃取后,采用超高效液相色谱-串联质谱法测定。以氘代25(OH)D3为内标,色谱柱为Zorbax Eclipse Plus C18,流动相为水(含0.1%甲酸)-甲醇(含0.1%甲酸),梯度洗脱,流速为0.35 ml/min,柱温30℃采用电喷雾离子源,以多反应离子监测方式进行正离子扫描;用于定量分析的离子对分别为m/z 413.2→395.2[25(OH)D2]、m/z 401.2→383.2[25(OH)D3]、m/z 404.3→368.2(内标)。结果:25(OH)D2与25(OH)D3血药浓度在2-80 ng/ml范围内线性关系均良好(r分别为0.998 0、0.999 9,n=3),其定量下限均为2 ng/ml,日间、日内RSD为0.78%-9.42%,相对偏差为-2.90%-1.95%。采用该法检测78例健康受试者及98例结核患者体内25(OH)D平均血药浓度分别为(21.53±6.33)、(8.84±4.05)ng/ml,差异有统计学意义(P〈0.01)。结论:该方法准确、可靠、灵敏度高,适用于血浆中25(OH)D的定量分析及人体药动学研究。
OBJECTIVE:To establish the method for the determination of 25-hydroxyvitamin D [25(OH)D] in human plasma,and apply it in the clinic. METHODS:After liquid-liquid extraction,UPLC-MS/MS method was used to determine plasma sample.Using deuteron-25(OH)D3as internal standard,the determination was performed on Zorbax Eclipse Plus C18 column with mobile phase consisted of water(containing 0.1% formic acid)-methanol(containing 0.1% formic acid)in gradient mode at flow rate of0.35 ml/min,and the column temperature was 30 ℃. By ESI,positive ion scanning was conducted in MRM mode;the monitoring transition ion-pair was m/z 413.2→395.2 for 25(OH)D2,m/z 401.2→383.2 for 25(OH)D3and m/z 404.3→368.2 for internal standard. RESULTS:The linear range of 25(OH)D2and 25(OH)D3were both 2-80 ng/ml(r=0.998 0 and 0.999 9,n=3);the limit of quantitation was 2 ng/ml. RSDs of inter-day and intra-day were 0.78%-9.42%,and relative deviation were-2.90%-1.95%,respectively. Average plasma concentration of 25(OH)D in 78 health volunteers and 98 tuberculosis patients were(21.53±6.33)and(8.84±4.05)ng/ml,with statistical significance(P〈0.01). CONCLUSIONS:The method is accurate,reliable and sensitive. It is suitable for quantitative analysis and pharmacokinetic study of 25(OH)D in human plasma.
出处
《中国药房》
CAS
北大核心
2016年第14期1907-1910,共4页
China Pharmacy
基金
北京市医院管理局重点医学专业发展计划项目(No.ZYLX201304)