Development Of Immunochromatography Assay For Several PDE-5 Inhibitors

The Phosphodiesterase type 5(PDE5)inhibitors are prescription drugs commonly used to treat erectile dysfunction.Therefore,they are often illegally added to anti-fatigue and immune-enhancing health products,which cause potential threat to people's health.At present,the main analytical methods of PDE5 inhibitors are liquid chromatography-tandem mass spectrometry.However,these methods are unable to achieve high-throughput on-site screening because time-consuming,expensive and professional.Gold immunochromatography assay(GICA)has the advantages of simple pretreatment,short time-consuming,low cost and sensitive,which is very suitable for on-site screening of actual samples.Up to now,there are few reports and products on the detection of PDE5inhibitors by GICA.Thus,in this study,we taken Tadalafil and Sildenafil as well as their analogues as research objects.The multi-residue GICA were developed based on the prepared monoclonal antibodies and polyclonal antibodies.And the research contents and experimental results are as follows:(1)Two haptens were designed for Tadalafil and Sildenafil.And the relative artificial antigen synthesized with carrier protein by mixed anhydride method and active ester method were used to immunize the female Balb/c mice and New Zealand rabbits.Effective polyclonal antibody and monoclonal antibodies by cell fusion were prepared.The inhibition rates of Tadalafil and Sildenafil at 1 ug/m L were 80.5%,84.0%,respectively.(2)The microwell lateral flow immunochromatographic assay for detecting Tadalafil drugs was established based on the polyclonal antibody.The limit of detection of this method(IC₂₀)was 0.05 ng/m L,the IC₅₀ranges were 0.64-4.02 ng/m L,the detection ranges were 1.56-76.92 ng/m L,and cut-off value in buffer solution were 50-200 ng/m L.The total time of detection was 10 minutes.The cross-reactivities with other Tadalafil analogues were less than 0.1%.The average recovery of spiked samples ranged from 72.29%to117.80%with a relative standard deviation less than 15%,which had good correlation with the method of HPLC-MS/MS(R²=0.96).(3)The GICA for detecting Sildenafil and its analogues was established based on the monoclonal antibody.This method can be used for qualitative and quantitative analysis of other sildenafil drugs besides qualitative analysis of vardenafil(cut-off value is 0.625ng/m L).The limit of detection(IC₂₀)of this method was 0.12-2.55 ng/m L,the IC₅₀ranges were 0.31-8.45 ng/m L,the detection ranges were 0.12-28.05 ng/m L,and cut-off value in buffer solution were 5.0-80.0 ng/m L.The total time of detection was 6 minutes.The cross-reactivities with Avanafil and Tadalafil as well as their analogues were less than 0.1%.The average recovery of spiked samples ranged from 70.53%to 118.87%with a relative standard deviation less than 15%,which is suitable for preliminary screening for practical samples.(4)Microwell lateral flow immunochromatographic kit for detection of Sildenafil and drugs was developed based on monoclonal antibodies.The result of the experiment was judged by contrast method.Under the optimum conditions,the visual detection limit of Sildenafil and its analogues was less than 50 ng/m L,which met the national detection requirements.In the detection of actual samples,the experimental results were confirmed by the method of HPLC-MS/MS,and there were no false positive or false negative phenomena.The kit can be stored at 37°C for more than 6 months through stability test.The product has high sensitivity and good stability,and is suitable for the detection of actual samples.