酶水解-HPLC法同时测定尿中三唑仑及其代谢物

Determination of triazolam and its metabolites in urine by enzyme-hydrolysis and high performance liquid chromatography

目的 :用酶水解 HPLC方法同时测定尿中三唑仑及其主要代谢物α 羟基三唑仑。方法 :尿样 2mL ,加 β 葡萄糖醛酸苷酶 0 2 5mL ,5 5℃酶解 2h ,用正己烷 二氯甲烷 (5∶3,v/v) 5mL提取 ,挥干提取溶剂后用 5 0 μL甲醇溶解 ,10 μL进样 ,HPLC检测。结果 :本法对三唑仑和α 羟基三唑仑的最低检测限均为 4ng·mL-1;绝对回收率分别为 93 12 %和 6 7 70 % ;日内、日间RSD分别在 6 0 8%和 11 0 5 %以下 ;线性范围均为 12 5～ 375 0ng·mL-1。结论 :酶水解提高了α 羟基三唑仑的检出率 ,从而使本法能同时测定尿中三唑仑及α 羟基三唑仑 ,此法重现性好、灵敏、可靠 。

AIM: To improve sensitivity of high performance liquid chromatography (HPLC) photodiode array detector that determines triazolam and its metabolites α hydroxytriazolam. METHODS: To add 0 25mL β glucuronidase and 0 1 mL NaAc buffer to 2 mL urine. The urine was heated at 55℃ for 2 h, then extracted with 5 mL n hexane dichloromethane(5:3, V/V). The extract from urine was evaporated to dryness in water bath at 50 ℃. The residue was dissolved in 50 μL methanol and injected 10 μL onto HPLC system. RESULTS: The LOD of this method for triazolam and α hydroxytriazolam was 4 ng·mL -1 . The absolute recovery is 93 12% and 67 70% for triazolam and α hydroxytriazolam, respectively. Their linearity range was from 12 5 to 375 ng·mL -1 in urine. CONCLUSION: This method improves sensitivity of HPLC to detect triazolam and α hydroxytriazolam. This method has good recovery and reliability.