UPLC-MS/MS法测定游离棉酚及其代谢产物棉酚酮在大鼠体内的组织分布

Tissue distribution of free gossypol and its metabolite gossypolone in rat determined by UPLC-MS/MS

目的采用超高效液相色谱-串联质谱(UPLC-MS/MS)法测定大鼠体内各组织器官中游离棉酚及其代谢产物棉酚酮的含量。方法分别在饲养验周期为1、3、4、5个月取大鼠的心脏、肝脏、脾脏等组织器官,按质量与体积比1∶1加生理盐水,样品经匀浆、蛋白沉淀后,采用Ostro样品制备板去除磷脂,UPLC-MS/MS法进行含量测定。结果棉酚和棉酚酮浓度范围在0.01~10.00μg/mL内线性关系良好,检出限分别为2.56ng/mL和1.32ng/mL,精密度、回收率和基质效应均符合生物样品分析方法要求。喂食5个月大鼠体内各组织器官中游离棉酚的含量由大到小依次为:肝脏(0.648 1 mg/mL)、血浆(0.156 9 mg/mL)、脾脏(0.107 5 mg/mL)、肺脏(0.097 3mg/mL)、心脏(0.081 1mg/mL)、睾丸(0.071 2mg/mL)、肾脏(0.039 7mg/mL)、肌肉(未检出)。棉酚的代谢产物棉Tissue dis酚酮仅在饲养5个月的大鼠肝脏中检出,且含量较低。结论本方法简便可靠,适用于生物样品中游离棉酚及其代谢产物棉酚酮的含量测定。

Objective To establish an analytical method for determination of the free gossypol and its metabolite gossypolone in different rat tissues based on ultra pressure liquid chromatography tandem mass spectrometry(UPLC-MS/MS).Methods The rats were sacrificed in the feeding period for 1,3,4,5 months and the heart,liver,spleen and other tissues of them were taken.Tissue samples were added to saline and homogenated.After the protein precipitation,the phospholipids were removed by Ostro sample preparation plate.The free gossypol and its metabolite gossypolone in the tissue of rats were analysized by UPLC-MS/MS.Results The calibration curves of gossypol and gossypolone were linear in the concentration range of 0.01~10μg/mL and showed good correlation.The detection limit of gossypol and gossypolone were separately 2.56,1.32 ng/mL.The precisions,recoveries and matrix effects were compliance with biological sample analysis requirements.The content of gossypol in rat tissues after feeding at 5 months from high to low was liver(0.648 1 g/mL),plasma(0.156 9 g/mL),spleen(0.107 5 g/mL),lung(0.097 3 g/mL),heart(0.081 1 g/mL),testis(0.071 2 g/mL),kidney(0.039 7 g/mL)and muscle(not detected).The gossypolone was only detected in rat liver after feeding at 5 months with a low content.Conclusion This mehod is simple and reliable.It can be used for the determination of free gossypol and its metabolite gossypolone in biological samples.