Yang Yongsheng, Faustino Patrick J, Pine P Scott, Davis Hirsch, Grunberg Neil, Phillips Jennifer, Lyon Robbe C, Yu Lawrence X, Ciavarella Anthony B, Del Grosso Alfred V, Hanig Joseph P
Food and Drug Administration, Center for Drug Evaluation and Research/Division of Product Quality Research, 10903 New Hampshire Avenue, Silver Spring, MD 20993, USA.
J Pharm Biomed Anal. 2005 Feb 7;37(1):157-63. doi: 10.1016/j.jpba.2004.10.002.
An isocratic reversed-phase high-performance liquid chromatographic method was established and validated according to FDA's Guidance for Industry, "Bioanalytical Method Validation", for the determination of isotretinoin in plasma and brain tissue from mice following single and multiple oral doses of Accutane. Plasma sample preparation included deproteination with acetonitrile-perchloric acid followed by centrifugation. Brain tissue was homogenized and extracted with acetonitrile-perchloric acid followed by centrifugation. The supernatants were analyzed by high-performance liquid chromatography (HPLC). Benz[alpha]anthrancene-7,12-dione was used as the internal standard. Chromatographic separation was achieved on a C18 column using an acetonitrile-aqueous 0.5% acetic acid (85:15, v/v) elution. The average extraction efficiency was >95% for plasma and >82% for brain. The lower limit of quantification was 30 ng/mL for plasma and was 30 ng/0.1g for brain tissue, respectively. The linear range for plasma was 30-600 ng/mL, and 15-300 ng/0.1g for brain. Maximum concentrations of isotretinoin in both plasma and brain were observed at 1h after single oral dosing (25 mg/kg). The maximum concentrations in plasma and brain were 2.36 microg/mL and 0.34 microg/g, respectively. The mean area under curve (AUC) in plasma was 6.13 microg h/mL. The mean eliminate half-life in plasma was estimated as 46 min.
建立了一种等度反相高效液相色谱法，并根据美国食品药品监督管理局（FDA）的行业指南《生物分析方法验证》进行了验证，用于测定单次和多次口服异维甲酸后小鼠血浆和脑组织中的异维甲酸。血浆样品制备包括用乙腈 - 高氯酸进行蛋白沉淀，然后离心。脑组织匀浆后用乙腈 - 高氯酸提取，随后离心。上清液通过高效液相色谱（HPLC）分析。苯并[a]蒽 - 7,12 - 二酮用作内标。在C18柱上进行色谱分离，使用乙腈 - 0.5%乙酸水溶液（85:15，v/v）洗脱。血浆的平均提取效率>95%，脑组织的平均提取效率>82%。血浆的定量下限为30 ng/mL，脑组织的定量下限为30 ng/0.1g。血浆的线性范围为30 - 600 ng/mL，脑组织的线性范围为15 - 300 ng/0.1g。单次口服给药（25 mg/kg）后1小时观察到血浆和脑组织中异维甲酸的最大浓度。血浆和脑组织中的最大浓度分别为2.36 μg/mL和0.34 μg/g。血浆中的平均曲线下面积（AUC）为6.13 μg h/mL。血浆中的平均消除半衰期估计为46分钟。
