Study On New PDE5 Inhibitors Adulterated In Health Food And Establishment Of Surface Enhanced Raman Spectrum Database Including 21 PDE5 Inhibitors

Purpose:Analysis and verification of high-performance liquid phase,mass spectrometryand nuclear magnetic resonance proton spectroscopy of new phosphodiesterase type 5?PDE5?inhibitors found in health foods,and establish related inspection standards to provide future food and drug supervision effective support.A fingerprint database of 21 PDE5 inhibitors based on surface-enhanced Raman spectroscopy was established to enable rapid screening of PDE5 inhibitors.Methods:1.The current standard method was used for the initial screening,and 180 batches of market samples were tested.Two unknown compounds were found.These two compounds were extracted and purified by preparative liquid phase,and the structure of the compound was analyzed by NMR.2.High performance liquid chromatography-tandem mass spectrometry was used,with 0.1% formic acid acetonitrile-0.1%formic acid aqueous solution as mobile phase,gradient elution,column temperature 35?,flow rate:300?L·min^-1,injection volume:2?L,electrical Spray ion source,scanning method:positive ion scanning,multi-reactive ion monitoring,establishment of demethyl carbadenafil,propoxy adenafil,hydroxypropyl desmethyl tadalafil and N-ethyl tadalafil investigate the test standards for four compounds of lafite and study their methodology.3.High-performance liquid chromatography with acetonitrile-0.1%trifluoroacetic acid as mobile phase,column temperature of 35?,detection wavelength:230nm,flow rate:1.0 mL·min^-1,injection volume:10?L,and desmethylstata were established.Lafida,2-hydroxypropyl desmethyl tadalafil,2-hydroxyethyl desmethyl tadalafil and tadalafil impurity 37 test standards for four compounds,methodological research;and use of liquid Mass chromatography,using 0.02moL·L^-1ammonium acetate solution?0.1%acetic acid?-acetonitrile-methanol?45:25:30?as mobile phase,flow rate:0.2mL·min^-1;column temperature:35?;electricity Spray ionization source?ESI+?,positive ion detection,adopting full-level scanning,second-level and third-level scanning,mass range 50-600,establish the qualitative confirmation method of the above four compounds.4.Using surface-enhanced Raman spectroscopy technology,taking demethylcarbadinafil as the research object,through orthogonal experiments,determine four instrument parameters of integration time,integration times,smoothing times,power,and concentration ratio of the enhancing reagent,and apply this Methods A database of surface enhanced Raman spectroscopy fingerprints of 21 illegal additives of sildenafil,tadalafil and vardenafil was established.Results:1.The new compounds found in the samples of the health food market were desmethyl carbodenafil and tadalafil impurity 37.The molecular formula of desmethylcarbadinafil was C₂₃H₃₀O₃N₆ and the chemical name was 5-?5-?4-Methylpiperazine-1-carbonyl?-2-ethoxyphenyl?-1-methyl-3-propyl-1H-pyrazole[4,3-d]pyrimidin-7?6H?-one;tada Raphi impurity 37 molecular formula is C₂₄H₂₃O₅N₃,chemical name is 6-?benzo[d][1,3]dioxo-5-yl?-2-?3-hydroxypropyl?-2,3,6,7,12,12a-hexahydropyrazino[1'2':1,6]pyrido[3,4-b]indole-1,4-dione.2.Using high performance liquid chromatography-mass spectrometry and methodological nvestigations,desmethyl carbodenafil,propoxy adenafil,2-hydroxypropyl desmethyl tadalafil and N-ethyl tadalafil in the corresponding concentration range,the linear relationship was greater than0.99,the detection limits were between 0.4111 and 41.3520 ng·mL^-1,the spiked recoveries were71.2%to 109.7%,and the RSD was between 0.6%and 9.1%.It shows that the method is stable and feasible,with high repeatability and high sensitivity.3.Quantitative determination by HPLC and methodological investigations,desmethyl tadalafil,2-hydroxypropyl desmethyl tadalafil,2-hydroxyethyl desmethyl tadalafil and tadalafil impurity 37 in the corresponding concentration range,the linear relationship was greater than 0.99,and the etection limits were between 1.243 and 75.912 ng·mL^-1.The spiked recoveries were 96.62%to104.76%,and the RSD were all 0.1%to 3.9%,indicating that the method is easy to operate and stable and feasible.Using qualitative liquid chromatography-mass spectrometry?LC-MS?qualitative detection,the secondary and tertiary fragment ions of four tadalafil compounds were identified.4.Using surface-enhanced Raman spectroscopy technology,through orthogonal experiments,determine the integration time is 30s,the number of integrations is 3 times,the number of smoothing times is 3 times,the power is 300mW and the ratio of the enhanced reagent gold gel OTR 202 reagent to OTR103 reagent is 600:100.A database of 21 types of PDE5 inhibitor surface enhanced Raman spectroscopy fingerprints was established by this method,and the detection limit of the above21 compounds was determined to be 15?g·mL^-1.This method is simple and easy to operate,and is suitable for rapid detection in the field.Conclusion:After analysis and confirmation,the new illegal additives found in health foods are desmethylcarbadinafil and tadalafil impurity 37.The above two compounds are reported for the first time in China and should attract the attention of inspection workers.Established LC-MS methods for four compounds such as demethylcarbadinafil,which are suitable for rapid and accurate quantitative detection in different complex matrices,four tadalafil derivatives such as tadalafil impurity 37 established the test method can be applied to qualitative and quantitative detection under different complex matrices.The established database of PDE5 inhibitor surface enhanced Raman spectroscopy fingerprint database is suitable for rapid screening and quasi-determinism of illegally added PDE5 inhibitors.