Thippani Ramesh, Pothuraju Nageswara Rao, Ramisetti Nageswara Rao, Shaik Saida
Department of Chemistry, National Institute of Technology, Warangal, India.
Analytical Chemistry Division, IICT, Hyderabad, AP, India.
J Pharm Anal. 2013 Dec;3(6):434-439. doi: 10.1016/j.jpha.2013.07.003. Epub 2013 Aug 2.
A novel isocratic reverse phase high performance liquid chromatography (RP-HPLC) with photo diode array (PDA) detection method for the determination of dobutamine (DBT) in rat plasma was developed and validated after optimization of various chromatographic conditions and other experimental parameters. Homoveratrylamine was used as an internal standard. Methanol was used as the extracting solvent for the preparation of plasma samples. Samples were separated on a Symmetry C18 (250 mm×4.6 mm i.d., 5 μm) analytical column. Acetonitrile and 15 mM potassium dihydrogen phosphate (pH 5.0 with 0.3% TEA) (20:80, v/v) was used. The column oven temperature was optimized at 35 °C and the flow rate was 0.8 mL/min. The detection wavelength was fixed at 230 nm for entire analysis. The calibration curve was found to be linear over the concentration range of 50-2000 ng/mL (=0.9992). The limit of quantification (LOQ) of the method was 50 ng/mL. The % RSD values of accuracy and precision values for intra and inter days were <15% at quality control (QC) concentrations. Recovery, stability and robustness were studied within the acceptable range according to ICH guidelines. The method was efficiently applied to a pharmacokinetic study in healthy Wistar rats.
建立了一种采用光电二极管阵列（PDA）检测的新型等度反相高效液相色谱（RP-HPLC）法，用于测定大鼠血浆中的多巴酚丁胺（DBT）。在优化了各种色谱条件和其他实验参数后，对该方法进行了验证。使用高香草胺作为内标。甲醇用作制备血浆样品的萃取溶剂。样品在Symmetry C18（250 mm×4.6 mm内径，5μm）分析柱上分离。使用乙腈和15 mM磷酸二氢钾（用0.3%三乙胺调至pH 5.0）（20:80，v/v）。柱温箱温度优化为35℃，流速为0.8 mL/min。整个分析过程的检测波长固定为230 nm。校准曲线在50-2000 ng/mL浓度范围内呈线性（r = 0.9992）。该方法的定量限（LOQ）为50 ng/mL。在质量控制（QC）浓度下，日内和日间准确度和精密度的相对标准偏差（%RSD）值均<15%。根据ICH指南，在可接受范围内研究了回收率、稳定性和耐用性。该方法有效地应用于健康Wistar大鼠的药代动力学研究。
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