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目的:建立大鼠下丘脑微透析样品中5-HIAA的柱前衍生化高效液相检测方法,为动态研究药物对下丘脑内神经递质的影响提供方法。方法:将微透析探针植入大鼠下丘脑,用林格氏液恒速灌流,每30 min收集微透析液一次,接收三次后灌胃给予氟西汀,继续收集微透析液,共收集7 h。以苄胺柱前衍生化5-HIAA标准液建立检测方法并进行方法学考察,最终确定检测条件,甲醇-20mmol/L醋酸盐缓冲液(体积比70:30)为流动相,在C18(250 mm×4.6 mm,5μm)柱上等度洗脱,荧光检测,λex=345nm,λem=480 nm。运用该方法检测上述下丘脑微透析液中的5-HIAA的动态变化。结果:5-HIAA在0.98～62.50 ng/ml浓度范围内线性关系良好,检测限低于0.49 ng/ml,衍生化产物在12 h内基本稳定。微透析样品中5-HIAA可以得到完全分离,无杂峰干扰。采用该方法观察到了灌胃给予氟西汀后大鼠下丘脑5-HIAA动态降低的变化过程。结论:该方法准确、灵敏、重现性好,适用于动态观察大鼠下丘脑中5-HIAA的变化。
OBJECTIVE: To establish a pre-column derivatization HPLC method for the determination of 5-HIAA in rat hypothalamic microdialysis samples and to provide a method for dynamically studying the effects of drugs on neurotransmitters in the hypothalamus. Methods: The microdialysis probe was implanted into the hypothalamus of rat and perfused with Ringer’s solution at a constant speed. Micro-dialysate was collected every 30 min. After receiving three times, fluoxetine was given intragastrically to continue the collection of microdialysis fluid. 7 h. The pre-derivatized 5-HIAA standard was established by benzylamine column and the methodological study was carried out. The final detection conditions were as follows: methanol-20mmol / L acetate buffer (volume ratio 70:30) 250 mm × 4.6 mm, 5 μm) with isocratic elution, fluorescence detection, λex = 345 nm, λem = 480 nm. The method was used to detect the dynamic changes of 5-HIAA in hypothalamic microdialysis solution. Results: The linearity of 5-HIAA ranged from 0.98 to 62.50 ng / ml. The detection limit was lower than 0.49 ng / ml. The derivatized products were stable within 12 h. 5-HIAA in microdialysis samples can be completely separated without interference of perturbation. Using this method, we observed the dynamic change of hypothalamus 5-HIAA in rats after oral administration of fluoxetine. Conclusion: The method is accurate, sensitive and reproducible. It is suitable for the dynamic observation of 5-HIAA in rat hypothalamus.