Standardization Study On Evaluation Of Bioequivalence Of Salbutamol Sulfate Inhalation Aerosol

Objectives:（1）The liquid chromatography-mass spectrometry method has the characteristics of strong specificity and high sensitivity,and has a wide range of applications in the field of biopharmaceutical analysis.The liquid chromatography-tandem mass spectrometry method to determine the concentration of salbutamol in human and rat plasma was established and validated in this study.（2）Salbutamol sulfate inhalation aerosol is a kind of smetered dose inhalers.In this study,the pharmacokinetic equivalence evaluation technology and process of salbutamol sulfate inhalation aerosol were established.（3）Medicinal charcoal is an adsorbent which is clinically used for the treatment of food and alkaloid poisoning,diarrhea,flatulence and other diseases.This study would verify the applicability of medicinal charcoal suspension in the evaluation of the bioequivalence of salbutamol sulfate inhalation aerosol.Methods:（1）For the LC-MS/MS method to determine concentration of salbutamol in human plasma,the internal standard was albuterol-d9,and the protein in plasma was precipitated by methanol.The supernatant was dried and reconstituted.The Welch Ultimate XS-C18 column（3.0×100 mm,3μm）was applied with the Welch Ultimate XB-C18 guard column（2.1×5mm,5μm）being as the guard column.Gradient elution was conducted with the mobile phase 5%acetonitrile containing 0.1%formic acid 5mmol·L^-1ammonium acetate and pure acetonitrile.Electrospray ionization and multi-reaction monitoring was applied to determine the salbutamol concentration in positive ion mode.For the LC-MS/MS method to determine concentration of salbutamol in rat plasma,the internal standard was albuterol-d9,and the protein in plasma was precipitated by acetonitrile.The supernatant was dried and reconstituted.The Welch Ultimate XB-C18 column（2.1×50mm,5μm）was applied.Gradient elution was conducted with the mobile phase water containing 0.05%formic acid 5mmol·L^-1ammonium acetate and pure acetonitrile.Electrospray ionization and multi-reaction monitoring was applied to determine the salbutamol concentration in positive ion mode.（2）Six healthy subjects orally inhaled 200μg（2 puffs）of Salbutamol Sulfate Inhalation Aerosol under fast condition in two study periods,and the washout period between two study periods was 7 days.The concentration of salbutamol in human plasma was determined by ultra performance liquid chromatography-mass spectrometry.The plasma drug concentration-time data were statistically analyzed by the DAS 3.2.8 software.Calculate the pharmacokinetic parameters C_max,AUC_0-20min,AUC_0-tand AUC_0-∞and their within-subject coefficient of variations.（3）Rats in the salbutamol group,low-dose medicinal charcoal group,and high-dose medicinal charcoal group were administrated respectively,drawn whole blood at the time of blood sampling and the plasma was separated.The concentration of salbutamol in rat plasma was determined by ultra performance liquid chromatography-mass spectrometry.The plasma drug concentration-time data was analyzed by DAS 3.2.8 software.The pharmacokinetic parameters C_max,AUC_0-twere calculated.The pharmacokinetic parameter T_maxwas Observed.Results:（1）As for the LC/MS method for detecting salbutamol in human plasma,the linear range of salbutamol is 5-2000pg·m L^-1,the selectivity,residue,linear range,accuracy and precision,extraction recovery rate,matrix effect,Hemolysis effect,high-fat effect,dilution reliability,stability and sample volume of this method were verified.As for the LC/MS method for detecting salbutamol in rat plasma,the linear range of salbutamol is 0.2-20ng·m L^-1,the selectivity,residue,linear range,accuracy and precision,extraction recovery,matrix effect,hemolysis effect and high-fat effect of the method were verified.（2）The main pharmacokinetic parameters of Salbutamol Sulfate Inhalation Aerosol in the first period and the second period were C_max,（285.33±158.83）pg·m L^-1and（222.12±108.02）pg·m L^-1,respectively;T_max,（0.72±0.52）h and（1.03±1.03）h,respectively;t_1/2z,（6.39±1.75）h and（5.91±1.22）h,respectively;AUC_0-20min,（63.77±42.27）pg·h·m L^-1and（47.56±33.54）pg·h·m L^-1,respectively;AUC_0-t,（1469.79±701.96）pg·h·m L^-1and（1 292.24±498.59）pg·h·m L^-1,respectively;AUC_0-∞,（1 596.30±772.95）pg·h·m L^-1and（1 383.21±488.82）pg·h·m L^-1,respectively.The within-subject coefficient of variations of the pharmacokinetic parameters C_max,AUC_0-20min,AUC_0-tand AUC_0-∞were 29.04%,17.64%,27.12%and 27.63%,respectively.（3）Compared with the salbutamol group,the C_maxof the low-dose medicinal charcoal group significantly decreased by 58.44%（p=0.010<0.05）,the AUC_0-tsignificantly decreased by 60.48%（p=0.004<0.05）,and there was no significant difference in the T_maxbetween the two groups（p=0.934>0.05）.Compared with the salbutamol group,the C_maxof the high-dose medicinal charcoal group was significantly reduced by56.37%（p=0.037<0.05）,AUC_0-twas significantly reduced by 81.23%（p=0.004<0.05）,and the Significant difference in T_maxwas not found between the two groups（p=0.288>0.05）.Conclusions:（1）The LC-MS method established in this study for the determination of salbutamol in human plasma is simple,accurate,sensitive,robust,economical,and is suitable for clinical pharmacokinetic studies and bioequivalence evaluation of salbutamol sulfate inhalation aerosol;The LC/MS method for the determination of salbutamol in rat plasma is simple,economical,accurate,and is suitable for the study of the blocking effect of medicinal charcoal suspension on the absorption of salbutamol in the gastrointestinal tract of rats.（2）This study established a simple,economical and accurate pharmacokinetic equivalence evaluation technology and process of salbutamol sulfate inhalation aerosol,and adopted simple and feasible protection methods to enable subjects and pharmacists to avoid drug contamination,which saved the economic cost of establishing a negative pressure administration room,providing a reference for the bioequivalence evaluation of salbutamol sulfate inhalation aerosol and other metered dose inhalers.（3）This study proved that both low-dose medicinal charcoal suspension and high-dose medicinal charcoal suspension can significantly block the absorption of salbutamol in the gastrointestinal tract of rats.The blocking effect of the two on Cmax are similar while the blocking effect of high-dose medicinal charcoal suspension on AUC0-t may be better,providing a reference for the applicability of the medicinal charcoal suspension and the optimization of the medicinal charcoal suspension dosing regimen in the bioequivalence study of salbutamol sulfate inhalation aerosol.