Federal University of Alfenas, Laboratory of Toxicological Analysis, Alfenas, MG, Brazil.
J Pharm Biomed Anal. 2010 Jan 5;51(1):170-7. doi: 10.1016/j.jpba.2009.07.017. Epub 2009 Jul 19.
A three-phase, liquid-phase microextraction using a hollow fibre (HF-LPME) combined with high performance liquid chromatography-fluorescence detection (HPLC-FL) was developed for the analysis of fluoxetine (FLX) and its active metabolite, norfluoxetine (NFLX), in human plasma. An HF-LPME system using a disposable 7-cm polypropylene porous hollow fibre, 5 mL of alkaline plasma solution (donor phase), n-hexyl ether (extraction solvent) and 20 mM hydrochloric acid (acceptor phase) was used in the extraction. The method was validated after optimisation of several parameters that influence LPME efficiency. A reverse-phase LiChrospher 60 RP-Select B column (125 mm x 4 mm, 5 microm particle size) was used with 0.005 M sodium acetate buffer (pH 4.5) and acetonitrile at a 50:50 (v/v) as the mobile phase at a flow rate of 0.6 mL min(-1). In these conditions satisfactory chromatographic resolution and efficiency for the analytes were obtained. Fluorescence detection at 230 nm excitation wavelength and 290 nm emission wavelength was performed. Linearity over a range of 5-500 ng mL(-1), with determination coefficients (R(2)) of 0.9999 and 0.9962 for FLX and NFLX, respectively, was established. Venlafaxine was used as the internal standard for both analytes. Extraction recoveries from plasma samples were 70.9% for FLX and 59.7% for NFLX. The intra-day coefficients of variation (CVs) were below 5.4%, and inter-day CVs were below 13.0%, for both analytes at concentrations of 20, 80 and 160 ng mL(-1). HF-LPME extraction followed by HPLC-FL detection for FLX and NFLX analyses demonstrated excellent sample clean-up and selectivity. This method was simple, cheap, and easy to perform, yielding substantial analytes enrichment. The method was applied to the analysis of samples from 12 patients under fluoxetine treatment and proved suitable for routine therapeutic drug monitoring for this antidepressant.
建立了一种采用中空纤维液相微萃取（HF-LPME）与高效液相色谱-荧光检测（HPLC-FL）联用的方法，用于分析人血浆中的氟西汀（FLX）及其活性代谢物去甲氟西汀（NFLX）。萃取采用的是 7 cm 长的一次性聚丙烯多孔中空纤维，萃取溶剂为正己醚，碱性血浆溶液（供体相）为 5 mL，20 mM 盐酸（受体相）。通过优化影响 LPME 效率的几个参数，对方法进行了验证。采用反相 LiChrospher 60 RP-Select B 柱（125 mm×4 mm，5 µm 粒径），以 0.005 M 乙酸钠缓冲液（pH 4.5）和乙腈（50：50，v/v）为流动相，流速为 0.6 mL min(-1)。在此条件下，可获得对分析物满意的色谱分辨率和效率。荧光检测采用 230 nm 激发波长和 290 nm 发射波长。FLX 和 NFLX 的线性范围分别为 5-500 ng mL(-1)，相关系数（R(2)）分别为 0.9999 和 0.9962。文拉法辛被用作两种分析物的内标。FLX 和 NFLX 从血浆样品中的提取回收率分别为 70.9%和 59.7%。在 20、80 和 160 ng mL(-1)浓度下，两种分析物的日内 CV 均低于 5.4%，日间 CV 均低于 13.0%。HF-LPME 萃取后采用 HPLC-FL 检测，用于 FLX 和 NFLX 的分析，显示出出色的样品净化和选择性。该方法简单、廉价且易于操作，可实现大量分析物的富集。该方法已应用于接受氟西汀治疗的 12 名患者样本的分析，证明适用于该抗抑郁药的常规治疗药物监测。
