伏立康唑在新西兰白兔眼组织中的浓度测定及眼组织分布研究

Determination of voriconazole and its distribution of ocular tissues in New Zealand white rabbits by LC-MS/MS

目的建立新西兰白兔眼组织中伏立康唑浓度的测定方法,并应用于玻璃体腔注射伏立康唑的眼组织分布研究。方法采用高效液相色谱-串联质谱(HPLC-MS/MS)法测定兔眼组织中伏立康唑浓度。以甲苯磺丁脲为内标,色谱柱为Waters X-Bridge BEH C_(18)(50 mm×2.1 mm,2.5μm),流动相A:0.2%甲酸-水溶液;流动相B:0.2%甲酸-乙腈溶液;梯度洗脱程序:0~0.30 min,30%B;0.30~1.81 min,30%→90%B;1.81~2.30 min,90%B;2.30~2.31 min,90%→30%B;2.31~2.60 min,30%B;体积流量为0.5 mL·min-1,柱温30℃,进样量1μL。使用电喷雾离子源,以多重反应监测方式进行正离子扫描,用于定量分析的离子对分别为m/z 350.1→281.1(伏立康唑)、m/z 271.1→155.0(内标)。新西兰白兔按照体质量、性别随机区间分成5组,每组6只,雌雄各半。无菌条件下,新西兰白兔眼部散瞳,im氯胺酮12 mg·kg^(-1)、赛拉嗪3 mg·kg^(-1)麻醉动物,玻璃体腔注入伏立康唑眼用注射剂,分别在行玻璃体腔注射术后的0.5、1.0、4.0、24.0、48.0 h,过量麻醉后放血处死试验兔。冰浴条件下分离兔眼组织(结膜、角膜、房水、虹膜、晶体、玻璃体、视网膜、脉络膜、巩膜),采用建立的HPLC-MS/MS测定各组织中伏立康唑浓度。结果伏立康唑质量浓度在0.5~500.0 ng·mL^(-1),线性关系良好(R^(2)>0.98),定量下限为0.5 ng·mL^(-1)。批内精密度(RSD)和准确度(RE)均小于15%,伏立康唑各眼组织的提取回收率均大于85%;新西兰白兔单眼单次给予伏立康唑后,其主要在眼后段分布,且视网膜浓度相对玻璃体、脉络膜更高。结论建立的伏立康唑测定方法分析时间短,能够快速检测眼组织中的药物,准确度和灵敏度高,适用于伏立康唑在眼组织的分布研究。

Objective To establish a method for determining the concentration of voriconazole in the ocular tissue of white rabbit in New Zealand,and to apply it to the study of the distribution of ocular tissue of intravitreal injection of voriconazole.Methods Determination by high performance liquid chromatography-tandem mass spectrometry.Tolbutamide was used as the internal standard,the chromatographic column was Waters X-Bridge BEH C_(18)(50 mm×2.1 mm,2.5μm),the mobile phase was gradient elution with 0.2%formic acid-acetonitrile(A)and 0.2%formic acid-water(B),gradient elution procedure 0—0.30 min,30%B,0.30—1.81 min,30%→90%B,1.81—2.30 min,90%B,2.30—2.31 min,90%→30%B,2.31—2.60 min,30%B,the flow rat was 0.5 mL·min^(-1),and 1μL was injected.The sample size,the column temperature was 30°C,and the electrospray ion source was used for positive ion scanning in the multiple reaction monitoring mode.The ion pairs used for quantitative analysis were m/z 350.1→281.1(voriconazole),m/z 271.1→155.0(internal standard).New Zealand white rabbits were randomly divided into five groups according to body weight and gender,with six rabbits in each group,half male and half male.Under sterile conditions,the eyes of New Zealand white rabbits were dilated,and the animals were anesthetized by intramuscular injection ketamine 12 mg·kg^(-1)and xylazine 3 mg·kg^(-1),and voriconazole ophthalmic injection was injected into the vitreous cavity.At 0.5,1.0,4.0,24.0,and 48.0 h,the rabbits were sacrificed by bleeding after excessive anesthesia.Rabbit ocular tissues(conjunctiva,cornea,aqueous humor,iris,lens,vitreous,retina,choroid,sclera)were separated under ice bath conditions,and the concentration of voriconazole in each tissue was determined by established HPLC-MS/MS.Results Voriconazole had a good linear relationship between 0.5—500.0 ng·mL^(-1)(R^(2)>0.98),the lower limit of quantification was 0.5 ng·mL^(-1).The intra-batch precision(RSD)and accuracy(RE)were less than 15%,and the extraction recoveries of voriconazole in all ocular tissues were greater than 85%.The drug concentration in rabbit ocular tissues was successfully detected by this method after single dose unilateral administration of voriconazole in New Zealand white rabbits,which was mainly distributed in the posterior segment of the eye,and the retinal concentration was higher compared to the vitreous and choroid.Conclusion The method has short analysis time,high accuracy and sensitivity,and can rapidly detect the drug in ocular tissues,which is suitable for the distribution study of voriconazole in ocular tissues.