目的 建立新西蘭白兔眼組織中伏立康唑濃度的測定方法，并應用于玻璃體腔注射伏立康唑的眼組織分布研究。方法 采用高效液相色譜-串聯(lián)質(zhì)譜（HPLC-MS/MS）法測定兔眼組織中伏立康唑濃度。以甲苯磺丁脲為內(nèi)標，色譜柱為Waters X-Bridge BEH C₁₈（50 mm×2.1 mm，2.5 μm），流動相A：0.2%甲酸-水溶液；流動相B：0.2%甲酸-乙腈溶液；梯度洗脫程序：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；體積流量為0.5 mL·min^-1，柱溫30℃，進樣量1 μL。使用電噴霧離子源，以多重反應監(jiān)測方式進行正離子掃描，用于定量分析的離子對分別為m/z 350.1→281.1（伏立康唑）、m/z 271.1→155.0（內(nèi)標）。新西蘭白兔按照體質(zhì)量、性別隨機區(qū)間分成5組，每組6只，雌雄各半。無菌條件下，新西蘭白兔眼部散瞳，im氯胺酮12 mg·kg^-1、賽拉嗪3 mg·kg^-1麻醉動物，玻璃體腔注入伏立康唑眼用注射劑，分別在行玻璃體腔注射術后的0.5、1.0、4.0、24.0、48.0 h，過量麻醉后放血處死試驗兔。冰浴條件下分離兔眼組織（結(jié)膜、角膜、房水、虹膜、晶體、玻璃體、視網(wǎng)膜、脈絡膜、鞏膜），采用建立的HPLC-MS/MS測定各組織中伏立康唑濃度。結(jié)果 伏立康唑質(zhì)量濃度在0.5～500.0 ng·mL^-1，線性關系良好（R²＞0.98），定量下限為0.5 ng·mL^-1。批內(nèi)精密度（RSD）和準確度（RE）均小于15%，伏立康唑各眼組織的提取回收率均大于85%；新西蘭白兔單眼單次給予伏立康唑后，其主要在眼后段分布，且視網(wǎng)膜濃度相對玻璃體、脈絡膜更高。結(jié)論 建立的伏立康唑測定方法分析時間短，能夠快速檢測眼組織中的藥物，準確度和靈敏度高，適用于伏立康唑在眼組織的分布研究。

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 C₁₈ (50 mm×2.1 mm, 2.5 μm), 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 rate was 0.5 mL·min^-1, and 1 μL was injected. The sample size, the column temperature was 30 ℃, 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 (R² > 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.

R917

江蘇省新藥一站式高效非臨床評價公共服務平臺建設（BM2021002）