Musharraf Syed Ghulam, Arfeen Qamar Ul, Mazhar Wardah, Kanwal Nayab
H,E,J, Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan.
Chem Cent J. 2013 Aug 27;7(1):142. doi: 10.1186/1752-153X-7-142.
Stanozolol is a synthetic derivative of dihydrotestosterone (DHT), and one of the frequently detected anabolic steroids in doping analysis. The current work describes the development and validation of the stability-indicating TLC-densitometric method for sensitive and specific estimation of stanozolol even its degradation product being there. Precoated silica gel TLC aluminium plates were utilized as the stationary phase and the eluent comprised of petroleum ether: acetone (6:4, v/v). Densitometric analysis of stanozolol was achieved at λmax 750 nm in the absorbance mode after staining with phosphomolybdic acid (PMA). Stress degradation of stanozolol was carried out under various reaction conditions including acid, base and neutral hydrolysis, wet and dry heating treatment, oxidation, and photo-degradation. Resulted stress samples and pharmaceutical products were analyzed with the developed TLC method.
This system showed a compact spot for stanozolol at Rf value of 0.46 ± 0.02. The data of linear regression analysis indicated a good linear relationship over the range of 200-1200 ng/spot concentrations. The method was validated for robustness, precision and recovery. The LOD and LOQ were 1.6 and 5.1 ng/spot, respectively. Under various stressed conditions, stanozolol showed degradation only under acidic hydrolysis. Peak of a degraded product was well resolved from the stanazolol with reasonably different Rf value and identified as 17, 17-dimethyl-l8-nor-5α-androst-13(14)-eno [3,2c] pyrazole through 1D- and 2D-NMR spectroscopic techniques and ESI-QqTOF-MS/MS analysis.
Result reflected that the stanozolol is majorly affected by the acidic condition. Statistical analysis indicated the application of the developed stability-indicating TLC-densitometric method for routine analysis of stanozolol in the presence of its degradation product.
司坦唑醇是二氢睾酮（DHT）的合成衍生物，是兴奋剂检测中经常检测到的合成代谢类固醇之一。目前的工作描述了一种稳定性指示薄层色谱 - 密度测定法的开发和验证，该方法用于灵敏且特异的测定司坦唑醇，即使存在其降解产物。采用预涂硅胶薄层色谱铝板作为固定相，洗脱剂由石油醚：丙酮（6:4，v/v）组成。用磷钼酸（PMA）染色后，在750nm波长下以吸光度模式对司坦唑醇进行密度测定分析。司坦唑醇在各种反应条件下进行强制降解，包括酸、碱和中性水解、湿热和干热治疗、氧化和光降解。用所开发的薄层色谱法分析所得的强制降解样品和药品。
该系统在Rf值为0.46±0.02处显示出司坦唑醇的致密斑点。线性回归分析数据表明，在200 - 1200 ng/斑点浓度范围内具有良好的线性关系。该方法在稳健性、精密度和回收率方面得到了验证。检测限和定量限分别为1.6和5.1 ng/斑点。在各种强制条件下，司坦唑醇仅在酸性水解下发生降解。降解产物的峰与司坦唑醇峰通过合理不同的Rf值得到良好分离，并通过一维和二维核磁共振光谱技术以及电喷雾四极杆 - 飞行时间串联质谱（ESI - QqTOF - MS/MS）分析鉴定为17,17 - 二甲基 - 18 - 降 - 5α - 雄甾 - 13(14) - 烯[3,2c]吡唑。
结果表明司坦唑醇主要受酸性条件影响。统计分析表明所开发的稳定性指示薄层色谱 - 密度测定法可用于在其降解产物存在的情况下对司坦唑醇进行常规分析。
