Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada.
J Pharm Biomed Anal. 2010 Nov 2;53(3):617-22. doi: 10.1016/j.jpba.2010.04.018. Epub 2010 Apr 21.
A high performance liquid chromatographic (HPLC) assay was developed for the simultaneous quantitation of midazolam (MDZ) and ketoconazole (KTZ) in plasma. MDZ, KTZ and diazepam (internal standard) were extracted from 100 microL or 500 microL plasma from rat or human, respectively, using liquid-liquid extraction with diethyl ether in the presence of 0.1N NaOH. After vortexing, centrifugation and freezing, the organic layer was transferred to clean tubes and evaporated. The dried residue was reconstituted in mobile phase and injected into the HPLC through a C18 column. The mobile phase consisted of acetonitrile:15 mM potassium dihydrogen orthophosphate (45:55, v/v), pumped at 1 mL/min and measured at lambda=220 nm. The method was tested in a pharmacokinetic study involving orally dosed KTZ 40 mg/kg in 1% methylcellulose followed by intravenous dosing of 5mg/kg MDZ to rats 1.5h latter. The components eluted within 10 min and were baseline resolved with no interferences from endogenous substances in plasma. The calibration curves were linear (r(2)=0.999) over the range of 25-25,000 and 5-10,000 ng/mL of KTZ and MDZ in rat and human plasma, respectively. The intraday and interday CV% were <15% and <6% for KTZ and <7% and <4% for MDZ and the mean error was <13% for both drugs in rat plasma. In human plasma the intraday CV% and % error of the mean were <11% and <10% for KTZ, respectively; both values were <13% for MDZ. The validated lower limit of quantitation was 25 and 5 ng/mL for both drugs based on 100 muL rat plasma and 500 microL human plasma, respectively. In rats, plasma concentrations of MDZ and KTZ were simultaneously measured up to 8 and 9.5h, respectively. In conclusion, the assay was shown to be rapid, sensitive and appropriate for use in drug-drug interaction studies involving MDZ and KTZ in rat, and potentially in humans.
建立了一种高效液相色谱（HPLC）分析方法，用于同时定量测定人血浆中的咪达唑仑（MDZ）和酮康唑（KTZ）。MDZ、KTZ 和地西泮（内标）分别从大鼠或人 100 μL 或 500 μL 血浆中用二乙醚在 0.1N NaOH 存在下进行液-液萃取提取。涡旋、离心和冷冻后，将有机层转移到清洁管中并蒸发。干燥残留物在流动相中重新溶解，通过 C18 柱注入 HPLC。流动相由乙腈：15 mM 磷酸二氢钾（45：55，v/v）组成，以 1 mL/min 的速度泵入，在 lambda=220nm 处测量。该方法在一项药代动力学研究中进行了测试，研究对象是口服给予 1%甲基纤维素中的 KTZ 40mg/kg，1.5 小时后静脉给予大鼠 5mg/kg MDZ。在 10 分钟内洗脱的组分与血浆中的内源性物质基线分离，无干扰。大鼠和人血浆中 KTZ 和 MDZ 的校准曲线在 25-25000 和 5-10000ng/mL 范围内均呈线性（r（2）=0.999）。日内和日间 CV%分别小于 15%和小于 6%用于 KTZ，小于 7%和小于 4%用于 MDZ，大鼠血浆中两种药物的平均误差均小于 13%。在人血浆中，KTZ 的日内 CV%和平均误差%分别小于 11%和小于 10%；MDZ 的这两个值均小于 13%。基于 100μL 大鼠血浆和 500μL 人血浆，两种药物的验证下限定量分别为 25 和 5ng/mL。在大鼠中，同时测定了 MDZ 和 KTZ 的血浆浓度，分别可达 8 和 9.5 小时。总之，该测定方法快速、灵敏，适用于大鼠中涉及 MDZ 和 KTZ 的药物相互作用研究，并可能适用于人类。
