Lindegårdh N, Hanpithakpong W, Wattanagoon Y, Singhasivanon P, White N J, Day N P J
Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand.
J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Nov 1;859(1):74-83. doi: 10.1016/j.jchromb.2007.09.018. Epub 2007 Sep 19.
A bioanalytical method for the analysis of oseltamivir (OP) and its metabolite oseltamivir carboxylate (OC) in human plasma, saliva and urine using off-line solid-phase extraction and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. OP and OC were analysed on a ZIC-HILIC column (50 mm x 2.1 mm) using a mobile phase gradient containing acetonitrile-ammonium acetate buffer (pH 3.5; 10mM) at a flow rate of 500 microL/min. The method was validated according to published FDA guidelines and showed excellent performance. The lower limit of quantification for OP was determined to be 1, 1 and 5 ng/mL for plasma, saliva and urine, respectively and for OC was 10, 10 and 30 ng/mL for plasma, saliva and urine, respectively. The upper limit of quantification for OP was determined to be 600, 300 and 1500 ng/mL for plasma, saliva and urine, respectively and for OC was 10,000, 10,000 and 30,000 ng/mL for plasma, saliva and urine, respectively. The within-day and between-day precisions expressed as R.S.D., were lower than 5% at all tested concentrations for all matrices and below 12% at the lower limit of quantification. Validation of over-curve samples ensured that it would be possible with dilution if samples went outside the calibration range. Matrix effects were thoroughly evaluated both graphically and quantitatively. No matrix effects were detected for OP or OC in plasma or saliva. Residues from the urine matrix (most likely salts) caused some ion suppression for both OP and its deuterated internal standard but had no effect on OC or its deuterated internal standard. The suppression did not affect the quantification of OP.
已开发并验证了一种生物分析方法，该方法采用离线固相萃取和液相色谱-正离子串联质谱联用技术，用于分析人血浆、唾液和尿液中的奥司他韦（OP）及其代谢产物奥司他韦羧酸盐（OC）。OP和OC在ZIC-HILIC柱（50 mm×2.1 mm）上进行分析，流动相梯度为含乙腈-醋酸铵缓冲液（pH 3.5；10 mM），流速为500微升/分钟。该方法根据美国食品药品监督管理局（FDA）发布的指南进行验证，表现出优异的性能。OP在血浆、唾液和尿液中的定量下限分别确定为1、1和5纳克/毫升，OC在血浆、唾液和尿液中的定量下限分别为10、10和30纳克/毫升。OP在血浆、唾液和尿液中的定量上限分别确定为600、300和1500纳克/毫升，OC在血浆、唾液和尿液中的定量上限分别为10000、10000和30000纳克/毫升。以相对标准偏差（R.S.D.）表示的日内和日间精密度在所有测试浓度下对所有基质均低于5%，在定量下限处低于12%。超曲线样品的验证确保了如果样品超出校准范围，可以通过稀释进行分析。对基质效应进行了全面的图形和定量评估。在血浆或唾液中未检测到OP或OC的基质效应。尿液基质中的残留物（很可能是盐类）对OP及其氘代内标均造成了一定程度的离子抑制，但对OC及其氘代内标没有影响。这种抑制不影响OP的定量分析。
