Tata P N, Fu C H, Bramer S L
Otsuka America Pharmaceutical, Inc., Development Department, Rockville, MD 20850, USA.
J Pharm Biomed Anal. 2001 Jan;24(3):381-9. doi: 10.1016/s0731-7085(00)00426-x.
A high performance liquid chromatography (HPLC) method with ultraviolet detection for the simultaneous quantification of cilostazol, and its known metabolites in human urine was developed and validated. Cilostazol, its metabolites and the internal standard OPC-3930 (structural analogue of cilostazol) were extracted from human urine using liquid-liquid extraction with chloroform. The organic extract was then evaporated and the residue was reconstituted in 8% acetonitrile in ammonium acetate buffer (pH 6.5). The reconstituted solution was injected onto an HPLC system and was subjected to reverse-phase HPLC on a 5-microm ODS column. A gradient mobile phase with different percentages of acetonitrile in acetate buffer (pH 6.5) was used for the resolution of analytes. Cilostazol, its metabolites and the internal standard were well resolved at baseline with adequate resolution from constituents of human urine. The lower limit of quantification was 100 ng/ml for cilostazol and all metabolites. The method was validated for a linear range of 100-3000 ng/ml for all the metabolites and cilostazol. The overall accuracy (% relative recovery) of this method ranged from 86.1 to 116.8% for all the analytes with overall precision (%CV) being 0.8-19.7%. The long-term stability of clinical urine samples was established for at least 3 months at -20 degrees C in a storage freezer. During validation, calibration curves had correlation coefficients greater than or equal to 0.995 for cilostazol and the seven tested metabolites. The method was successfully used for the analysis of cilostazol and its metabolites in urine samples from clinical studies, demonstrating the reliability and robustness of the method.
建立并验证了一种采用紫外检测的高效液相色谱（HPLC）方法，用于同时定量测定人尿中西洛他唑及其已知代谢物。采用氯仿液 - 液萃取法从人尿中提取西洛他唑、其代谢物和内标OPC - 3930（西洛他唑的结构类似物）。然后将有机萃取液蒸发，残渣用含8%乙腈的醋酸铵缓冲液（pH 6.5）复溶。将复溶后的溶液注入HPLC系统，在5微米的ODS柱上进行反相HPLC分析。使用在醋酸盐缓冲液（pH 6.5）中不同乙腈百分比的梯度流动相来分离分析物。西洛他唑、其代谢物和内标在基线处得到良好分离，与人类尿液成分有足够的分离度。西洛他唑及其所有代谢物的定量下限为100 ng/ml。该方法在100 - 3000 ng/ml的线性范围内对所有代谢物和西洛他唑进行了验证。该方法对所有分析物的总体准确度（相对回收率%）范围为86.1%至116.8%，总体精密度（%CV）为0.8%至19.7%。临床尿液样本在 - 20℃的储存冰箱中至少可稳定保存3个月。在验证过程中，西洛他唑和七种测试代谢物的校准曲线相关系数大于或等于0.995。该方法成功用于临床研究尿液样本中西洛他唑及其代谢物的分析，证明了该方法的可靠性和稳健性。
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