Development Of Immunoassay For Tadalafil And Its Analogues In Health-care Food

Tadalafil, the second generation of PDE-5 enzyme inhibitor, is a kind of prescription drugs for treatment of male erectile dysfunction. It often be illegally added to health-care food which claims having functions of resisting fatigue, enhancing immunity and improving sexual ability and vitality. However clinically significant side effects have been reported and the PDE-5 enzyme inhibitors have potentially serious drug-drug interaction with nitrates, that lead severe hypotension or even death. Thus it's very important to strengthen monitoring of the PDE-5 enzyme inhibitors illegally usage in health-care food. At present, the detection methods of the PDE-5 enzyme inhibitors mainly include liquid chromatography(LC), gas chromatography(GC), LC-MS and GC-MS. But these analysis methods have some disadvantages, such as high cost of equipment usage,inconvenience of on-site usage and professional operator requirement. Thus it's difficult to popularize, especially in some economically underdeveloped city. Immunoassay is a rapid and sensitive detection technology which combined antigen-antibody reaction with enzymatic reaction. For its low cost and convenient to on-site test, it can supplement the disadvantages of instrumental analysis methods. To our best knowledge, no reports on the immunoassay of tadalafil and its analogues had surfaced. Therefore, in this research, a series of haptens and antigens were designed and synthesized. Furthermore, the broad specific antibodies against tadalafil were achieved by immunizing New Zealand white rabbit. Then colloidal gold immunochromatographic test strip(GICA) and indirect competitive enzyme-linked immunosorbent assay(ic-ELISA) were developed. The detailed results as follows:(1) Five haptens(H1~H5) were designed, synthesized and then coupled to carrier protein to form antigens. Two specific polyclonal antibodies PAb-H5-Am TD-MA and PAb-H5-Am TD-MA-KLH were obtained by immunizing the New Zealand white rabbit.The inhibition rates of the antibodies were 89% and 95% at 1 ?g/m L tadalafil respectively.(2) The ic-ELISA method was established based on the PAb-H5-Am TD-MA against tadalafil and its analogues. The optimal assay conditions as follows: the concentration of coating antigen was 31.25 ng/m L; the concentration of antibody was diluted 8k folds; the diluent buffer of tadalafil was PBST(p H 8.4, 0.01 mol/L, containing 0.2% Tween-20); the reaction time was 40 min and the concentration of CH3 OH was lower than 5%. The results showed that the LOD for tadalafil of the developed ic-ELISA was 0.027 ng/m L; the IC50 was 0.90 ng/m L and the linear range was 0.099~8.22 ng/m L. The average recoveries of tadalafil in spiked samples ranged from 84.92% to 116.19%, and the coefficient of variation below 14.28%. The test results of the ic-ELISA are very compatible with that of HPLC-MS/MS(R2=0.9993).(3) The immunochromatographic test strip was established based on the PAb-H5-Am TD-MA-KLH against tadalafil and its analogues. The optimal assay conditions as follows: the p H value of colloidal gold solution was 8.5; the concentration of antibody for labeling colloidal gold was 0.75 ?g/m L; the colloidal gold-antibody conjugate and coating antigen was original concentration; the material of sample pad was GF-06; the buffer of immersing sample pad was Tris-HCl(0.2 mol/L, p H 7.5); the diluent buffer of tadalafil was PBST(0.01mol/L, p H 7.4); the concentration of goat anti rabbit Ig G was 0.27mg/m L and the concentration of CH3 OH was lower than 5%. Under the above conditions,the developed GICA strip gave the IC50 of 10.04 ng/m L and the linear range of 0.71~141.99ng/m L for tadalafil. The average recoveries of tadalafil in spiked samples ranged from93.34% to 121.50%, and the coefficient of variation below 13.33%. The test results of the GICA are very compatible with that of HPLC-MS/MS(R2=0.9598).(4) The results of blind samples analysis by the developed methods exhibited that three among nine health wine samples were positive and four among thirteen solid samples were positive and the concentrations were above the ?g/m L. The test results of the methods are very compatible with that of HPLC-MS/MS(R2=0.9758 for ic-ELISA and R2=0.9287 for GICA strip).