犬血浆中特比萘芬的高效液相色谱检测方法

Development of HPLC method for the determination of Terbinafine in dog plasma

关键词:  特比萘芬  高效液相色谱法  血药浓度测定  稳定性

Key words:  terbinafine  HPLC  determination of blood concentration  stability

摘要:

本试验旨在建立并优化一种简单、快速、灵敏、高选择性的HPLC方法测定犬血浆中特比萘芬血药浓度,并考察方法的稳定性。特比萘芬血浆样品经提取剂乙腈-异丙醇(用磷酸调pH至3.0)(40:60)液-液萃取后,提取液经40℃氮气吹干,用流动相复溶后离心过滤,取上清液20 μL进样分析,对血样处理后及标准溶液保存不同时间后的稳定性进行了考察。色谱柱Agilent 5 HC-C18(2)柱(250 mm×4.6 mm,5 μm)；流动相甲醇-0.1%磷酸(59:41)；流速0.8 mL/min；紫外检测波长223 nm；柱温35℃。色谱峰分离良好,无干扰。特比萘芬最低检测浓度为0.01 μg/mL,线性范围是0.02 ~ 5 μg/mL,相关回归方程:y = 356.28 x -2.0311(r2 = 0.99989)。低、中、高血浆质控样品日内RSD均小于5%,日间RSD均小于7%,方法平均回收率分别为108.9%,100.4%,102.1%。血样处理后8 h内样品能保持较好稳定性,血样处理后3次循环冻融能保持较好稳定性。血样处理后于-20℃条件下冻存能够保持稳定2个月,标准溶液有效保存期同样为2个月。本研究建立的特比萘芬血样浓度测定方法有较高的准确度和灵敏度,适用于该候选化合物的体内药代动力学研究。

Abstract:

To establish and improve a simple, rapid,sensitive and high selective high-performance liquid chromatographic (HPLC) method for the determination of terbinafine in canine plasma, and study the stability of terbinafine in different condition. The plasma sample containing terbinafine was extracted by the extracting agent acetonitrile/isopropanol (phosphoric acid adjusting to pH 3.0, 40:60), and dried by nitrogen at 40℃, then dissolved in mobile phase and centrifuging, and 20 μL of the upper phase was injected into the sampler. The stability of the samples in different condition were studied. Separation was performed on a reversed-phase Agilent 5 HC-C18(2) column (250 mm×4.6 mm, 5 μm). The isocratic mobile phase was methyl alcohol and 0.1% phosphoric acid with the rate of (59:41), and the run rate was 1 mL.min-1. The detective UV wavelength was 223 nm and the column temperature was 35℃. The chromatography was good and not interfered by the components of the plasma. The limit of quantity was 0.01 μg.mL-1. The HPLC method had a linearity over the range of 0.02 ~ 5 μg.mL-1. The linear equation was y = 356.28 x -2.0311(r2 = 0.99989). The intra-assay precision did not exceed 5% and inter-assay precision did not exceed 7% for low, medium, high quality samples, respectively. The average recovery of the described method was 108.9%, 100.4% and 102.1%, respectively. The samples in 8 h or 3 times of freezing and thawing cycles after processing can maintain good stability. The samples after processing and the standard solution of terbinafine were stablity for 2 months. A sensitive and accurate method for determining terbinafine has been established, which may be used in future in vivo pharmacokinetic studies of this antifungal drugs.