Kirresh Tatian, Tuteja Sony, Russo D, Brophy P D, Murry D J
College of Pharmacy, University of Iowa, Iowa City, IA, United States.
College of Medicine, University of Iowa, Iowa City, IA, United States.
J Pharm Biomed Anal. 2017 Mar 20;136:32-37. doi: 10.1016/j.jpba.2016.12.034. Epub 2016 Dec 29.
A sensitive and robust LC-MS/MS method has been developed and validated to determine the concentrations of tacrolimus and its major metabolite 13-O-desmethyl tacrolimus (13-ODMT) in kidney tissue from rats who received tacrolimus intra-peritoneally at doses of 0.5mg/kg and 2mg/kg. The samples were prepared by a liquid-liquid extraction procedure using ethyl ether as the extraction solvent and ascomycin as the internal standard. Chromatographic separation was achieved using Phenomenex Kinetex column (2.6μm C18 100Å, 100×2.1mm, Phenomenex, Torrance CA) and a gradient mobile phase of water and methanol-acetonitrile (50:50, v/v) both containing 0.1% formic acid. The limit of quantification was 0.25ng/ml and the calibration curves covered a concentration range from 0.25 to 50ng/ml. Intra-and inter-assay precision and accuracy for both tacrolimus and 13-ODMT were all within FDA guidelines for bioanalysis. Extraction efficiency for tacrolimus ranged from 67.00 to 74.90% and from 66.70 to 78.40% for 13-ODMT. Several challenges interfering with the performance of the method such as phospholipid build-up have also been addressed. Kidney tissue samples from six rats receiving either 0.5 or 2mg/kg dose were analyzed and resulted in a median concentration of 11.54 and 0.72ng/ml for tacrolimus and 13-ODMT, respectively, for the lower dose level, and a median concentration of 8.89ng/ml and 1.50ng/ml for tacrolimus and 13-ODMT, respectively, at the higher dose level.
已开发并验证了一种灵敏且稳健的液相色谱-串联质谱法，用于测定腹腔注射剂量为0.5mg/kg和2mg/kg他克莫司的大鼠肾脏组织中他克莫司及其主要代谢物13-O-去甲基他克莫司（13-ODMT）的浓度。样品采用液-液萃取法制备，以乙醚为萃取溶剂，以子囊霉素为内标。使用菲罗门Kinetex柱（2.6μm C18 100Å，100×2.1mm，菲罗门，加利福尼亚州托伦斯）和水与甲醇-乙腈（50:50，v/v）的梯度流动相进行色谱分离，二者均含有0.1%甲酸。定量限为0.25ng/ml，校准曲线涵盖的浓度范围为0.25至50ng/ml。他克莫司和13-ODMT的批内和批间精密度及准确度均在FDA生物分析指南范围内。他克莫司的萃取效率为67.00%至74.90%，13-ODMT的萃取效率为66.70%至78.40%。还解决了一些干扰该方法性能的挑战，如磷脂堆积问题。对六只接受0.5或2mg/kg剂量的大鼠的肾脏组织样本进行了分析，低剂量水平下他克莫司和13-ODMT的中位浓度分别为11.54和0.72ng/ml，高剂量水平下他克莫司和13-ODMT的中位浓度分别为8.89ng/ml和1.50ng/ml。
