A validated stability-indicating TLC-densitometric method for the determination of stanozolol in pharmaceutical formulations.

Background: 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.

Results: 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.

Conclusion: 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.