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

Chemistry Central Journal

Syed Ghulam Musharraf²  Qamar ul Arfeen²  Wardah Mazhar¹  Nayab Kanwal² 

[1] Department of Chemistry, University of Karachi, Karachi 75270, Pakistan

[2] H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan

关键词: Stress degradation;    TLC-densitometry;    Stanozolol;   

Others  :  787851
DOI  :  10.1186/1752-153X-7-142

 received in 2013-05-17, accepted in 2013-08-15,  发布年份 2013

【 摘 要 】

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 λ_max 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 R_f 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 R_f 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.

【 授权许可】

   
2013 Musharraf et al.; licensee Chemistry Central Ltd.

【 图 表 】

Figure 1.

Scheme 1

【 参考文献 】

• [1]Clinton RO, Manson AJ, Stonner FW, Beyler AL, Potts GO, Arnold A: Steroidal [3,2-C]pyrazoles. J Am Chem Soc 1959, 81(6):1513-1514.
• [2]Massé R, Ayotte C, Bi HG, Dugal R: Studies on anabolic steroids. III. Detection and characterization of stanozolol urinary metabolites in humans by gas chromatography–mass spectrometry. J Chromatogr 1989, 497:17-37.
• [3]Chesnut CH 3rd, Ivey JL, Gruber HE, Matthews M, Nelp WB, Sisom K, Baylink DJ: Stanozolol in postmenopausal osteoporosis: therapeutic efficacy and possible mechanisms of action. Metabolism 1983, 32(6):571-80.
• [4]Beringer TR, Ardill J, Taggart HM: Effect of calcium and stanozolol on calcitonin secretion in patients with femoral neck fracture. Bone Miner 1986, 1(4):289-95.
• [5]Bates PC, Chew LF, Millward DJ: Effects of the anabolic steroid stanozolol on growth and protein metabolism in the rat. J Endocrinol 1987, 114:373-381.
• [6]Van De Wiele M, De Wasch K, Vercammen J, Courtheyn D, De Brabander H, Impens S: Determination of 16beta-hydroxystanozolol in urine and faeces by liquid chromatography-multiple mass spectrometry. J Chromatogr A 2000, 904(2):203-209.
• [7]Yuan A, Liu C, Huang X: Treatment of 34 cases of chronic aplastic anemia using prepared Rehmannia polysaccharide associated with stanozolol. Zhongguo Zhong Xi Yi Jie He Za Zhi 1998, 18(6):351-353.
• [8]ICH, Q1A: Stability Testing of New Drug Substances and Products, Proceedings of the International Conference on Harmonization. Geneva: IFPMA; 2000.
• [9]ICH Q1A (R2): Stability testing of new drug substances and products, proceedings of the International Conference on Harmonization. 2003. http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q1A_R2/Step4/Q1A_R2__Guideline.pdf webcite
• [10]Guidance for Industry: Drug Stability Guidelines. http://www.fda.gov/downloads/AnimalVeterinary/GuidanceComplianceEnforcement/GuidanceforIndustry/ucm051556.pdf webcite
• [11]Yao S, Zhao Y, Li X, Chen X, Jin M: Determination of 11 anabolic hormones in fish tissue by multi-function impurity adsorption solid-phase extraction-ultrafast liquid chromatography-tandem mass spectrometry. Se Pu 2012, 30(6):572-577.
• [12]Zeng Z, Liu R, Zhang J, Yu J, He L, Shen X: Determination of seven free anabolic steroid residues in eggs by high-performance liquid chromatography-tandem mass spectrometry. J Chromatogr Sci 2013, 51(3):229-236.
• [13]De Brabander HF, De Wasch K, van Ginkel LA, Sterk SS, Blokland MH, Delahaut P, Taillieu X, Dubois M, Arts CJM, van Baak MJ, Gramberg LG, Witkamp RF, Schilt R, van Bennekom EO, Courtheyn D, Vercammen J, Witkamp RF: Multi-laboratory study of the analysis and kinetics of stanozolol and its metabolites in treated calves. Analyst 1998, 123:2599-2604.
• [14]Guan F, Uboh CE, Soma LR, You Y, Liu Y, Li X: High-throughput UHPLC-MS/MS method for the detection, quantification and identification of fifty-five anabolic and androgenic steroids in equine plasma. J Mass Spectrom 2010, 45(11):1270-1279.
• [15]Srivastava MM (Ed): High-Performance Thin-Layer Chromatography (HPTLC). Berlin Heidelberg: Springer Verlag; 2011.
• [16]Musharraf SG, Ul Arfeen Q, Shoaib M: Development and validation of TLC-densitometric method for the quantification of a steroidal drug, danazol in its pharmaceutical formulations. J Planar Chromatogr - Mod TLC 2012, 25:331-337.
• [17]Musharraf SG, Fatima U, Sultana R: Stress degradation studies and development of stability-indicating TLC-densitometry method for determination of prednisolone acetate and chloramphenicol in their individual and combined pharmaceutical formulations. Chem Cent J 2012, 6:7. BioMed Central Full Text
• [18]Musharraf SG, Gulzar U: Effective separation and simultaneous analysis of anabolic androgenic steroids (AAS) in their pharmaceutical formulations by a validated TLC-densitometry method. Chem Cent J 2012, 6:54. BioMed Central Full Text
• [19]Musharraf SG, Kanwal N, Arfeen Q: Stress degradation studies and stability-indicating TLC-densitometric method of glycyrrhetic acid. Chem Cent J 2013, 7(1):9. BioMed Central Full Text
• [20]Bi H, Massé R, Just G: Studies on anabolic steroids. 9. Tertiary sulfates of anabolic 17 alpha-methyl steroids: synthesis and rearrangement. Steroids 1992, 57(7):306-312.