反相高效液相色谱法测定大鼠脑组织吗啡-3-葡萄糖醛酸苷含量

目的:建立一种反相高效液相色谱(RP-HPLC)检测法,用于测定大鼠脑组织中吗啡-3-葡萄糖醛酸苷(M3G)的浓度。方法:微粒体酶体外反应体系,经孵育和处理后,采用HPLC分析定量,并进行方法学考察。RP-HPLC测定采用DiamonsilTMC18色谱柱(250mm×4.6mm,5μm),流动相为10mmol.L-1磷酸二氢钾缓冲液(含1mmol.L-1十二烷基磺酸钠,磷酸调pH=2.15):乙腈(79.5∶20.5),流速:1.5ml.min-1,紫外检测波长:210nm,进样量50μl。结果:本实验条件下各物质分离良好,M3G线性范围50-600μg.L-1。M3G最低检测限为50μg.L-1。M3G的方法回收率在90%-105%范围内,提取回收率在75%以上。结论:本法可用于对大鼠脑微粒体体外孵育生成吗啡-3-葡萄糖醛酸苷含量的测定。

DETERMINATION OF MORPHINE-3-GLUCURONIDE IN RAT BRAIN IN VITRO BY REVERSED-PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Objective:To establish a reversed-phase high performance liquid chromatography(RP-HPLC)method for the determination of morphine's metabolite-M3G in rat brain. Methods:Chromatographic separation was achieved with a Diamonsil~(TM)C_(18) analytical column(250 mm× 4.6 mm, 5 μm). The mobile phase was 10 mmol·L~(-1) potassium phosphate and 1 mmol ·L~(-1) sodium dodecyl sulfate(pH=2.15 adjusted by phosphoric acid)- acetonitrile(79.5∶20.5),and flow rate was 1.5 ml · min~(-1). The drug concentrations were measured by UV at 210 nm. Results:The calibration curves were linear in the concentration range of 50-600 μg · L~(-1) for M3G. The limit of quantitation(LOQ)was 50 μg· L~(-1) for M3G. The method recovery of the sample was within 90%-105%,the absolute recovery was above 75%. Conclusion:This method is simple and rapid,especially for quantitative analysis of M3G in rat brain in vitro.