反相高效液相色谱法测定兔房水中乙酰唑胺的含量

Determination of acetazolamide levels in rabbit aqueous humor by reverse phasehigh performance liquid chromatography

目的应用反相高效液相色谱（ＲＰ－ＨＰＬＣ）技术，建立兔房水中乙酰唑胺的定量分析方法。方法乙酰唑胺房水样品经固相萃取后，进行ＲＰ－ＨＰＬＣ检测。检测条件：分析柱为Ｃ１８不锈钢柱（２００ｍｍ×４．６ｍｍ，５μｍ）；流动相为０．０１ｍｏｌ／Ｌ乙酸铵－甲醇（９５∶５）；流速为１．５ｍｌ／ｍｉｎ；柱温为４０℃；紫外检测器波长为２６５ｎｍ。结果兔房水的药物峰与杂质峰分离良好，在０．１～６．４μｇ／ｍｌ范围内，峰面积与浓度呈良好的线性关系，ｒ＝０．９９７；最低检测限为２ｎｇ／ｍｌ；在０．４、１．６、４．０μｇ／ｍｌ３种浓度的平均回收率为９８．５１±０．２９％，日内和日间ＲＳＤ均小于１．７％。结论建立了简便、敏感的ＲＰ－ＨＰＬＣ检测法。

ObjectiveA reverse phase high performance liquid chromatographic(RP HPLC) assay has been developed for the determination of acetazolamide levels in rabbits aqueous humor.MethodsUsing the solid phase extraction technique,acetazolamide was extracted from the aqueous humor,and detected by RP HPLC.The analytical column was a C 18 stainless steel column(200 mm× 4.6 mm, 5 μm); the mobile phase consisted of a mixture of 0.01 mol/L ammonium acetate methanol(95∶5);the flow rate was 1 5 ml/min and the column temperature was 40℃;the wavelength of the UV vis detector was 265 nm.ResultsThe drug peak was separated well without interference from the aqueous humor.The relationship between the peak area and concentration of the drug in aqueous humor was linear within the range of 0.1～6.4 μg/ml,with a correlation coefficient of 0.9997.The minimal detectable limit was 2 ng/ml.The average recovery in samples of 0.4,1.6,4.0 μg/ml was 98.68±0.54%,and the RSD for the inter and intra day were all less than 1.7%. ConclusionRP HPLC is an simple and sensitive assay,which may be used in ocular pharmacokinetic studies of acetazolamide.