Development And Application Of Quantitative Method For Protein Polypeptide Drug Liraglutide Based On LC-MS/MS

Background:Liraglutide,a structural analogue of glucagon-like peptide-1（GLP-1）,an endogenous substance in humans,can bind and activate GLP-1 receptors,promote insulin secretion in a glucose-dependent manner,inhibit glucagon release,and in addition slow gastric emptying and reduce energy intake to play a weight-loss role.Although liraglutide is widely used in clinical practice,the development of quantitative detection methods for this substance is relatively lagging behind.Traditional immunological methods have been widely used since their development because they are simple and rapid to perform and can be operated in general laboratories.However,when the molecular weight of the analyte is relatively small,the specificity of immunological methods will become worse due to cross-reaction and other reasons.Immunological methods can only detect one compound in one experiment,and if the drug is used in combination,multiple experiments are required for one sample.At present,more and more laboratories at home and abroad use LC-MS/MS method instead of immunological method to carry out plasma concentration detection.Objectives:To establish a selective,sensitive and reproducible LC-MS/MS method for the determination of liraglutide in K₂EDTA plasma of SD rats and its pharmacokinetics.Methods:The processed samples were separated by C18 column using liraglutide-Valine-D8 as internal standard and 100%absolute ethanol containing 2%ammonia as protein precipitant.The ionization mode was positive ion mode,electrospray ionization（ESI）,and the scanning mode was multiple reaction monitoring（MRM）,and the monitored ion pairs were liraglutide（m/z 938.8→1064.3）and liraglutide-Valine-D8（m/z 940.8→527.6）,respectively.The mobile phase was 0.1%FA in water and 0.1%FA in acetonitrile using a gradient elution program with a flow rate of 0.6 m L/min and an analysis time of 3 min.Results:1.The interference of blank matrix to liraglutide and internal standard was 5.3%and 0.2%,respectively,the interference of analyte to internal standard was 7.5%and the interference of internal standard to analyte was 4.3%;the range of calibration standard curve was 1.00～1000 ng/m L;the intra-batch precision（%CV）was less than8.2%,and the accuracy deviation（%RE）was-14%～13%;the inter-batch precision（%CV）was less than 6.2%,and the accuracy deviation（%RE）was-3.7%～1.1%;the extraction recoveries of low,medium and high quality control samples were 45.6%,50.4%and 52.5%,respectively,the coefficients of variation were 7.4%,5.1%and 7.3%,respectively,the extraction recovery of internal standard was 48.7%,and the coefficient of variation was 7.5%;the samples beyond the upper limit of concentration could be diluted by 5-fold concentration;the stability of three freeze-thaw cycles,the stability of stock solution at-20°C for 30 days,the stability of working solution at room temperature for 24 hours,the stability of plasma samples at-80°C for 30 days and the stability of autosampler for 24 hours were within the acceptable range.This method has been successfully applied to the analysis of plasma samples from SD rats.2.The results of LC-MS/MS and ELISA in subcutaneous and tail vein plasma samples were statistically analyzed,and there were no significant differences.3.The main pharmacokinetic parameters C_max、AUC_0-t、AUC_0-∞、T_max、t_1/2、MRT、CL and V_d measured by LC-MS/MS were 203±31.6 ng/m L,2672±278h·ng/m L,2685±279 h·ng/m L,6 h,4.24±0.133 h,9.18±0.273 h,0.0375±0.00377m L/h/kg,0.230±0.0251 m L/kg,respectively,for SD rats given 100μg/ml liraglutide subcutaneously.The main pharmacokinetic parameters C_max、AUC_0-t、AUC_0-∞、T_max、t_1/2、MRT、CL and V_d by ELISA were 201±32.047 ng/m L,2676.4±265.9 h·ng/m L,2693.8±266.4 h·ng/m L,5.33±1.155 h,4.50±0.091 h,9.30±0.287 h,0.0374±0.00359 m L/h/kg,and 0.243±0.0258 m L/kg,respectively.Except for MRT,there was no significant difference in other PK parameters,and the overall results were basically the same.4.The main pharmacokinetic parameters C_max、AUC_0-t、AUC_0-∞、T_max、t_1/2、MRT、CL and V_d measured by LC-MS/MS were 1749.333±402.394 ng/m L,9918.954±321.413 h·ng/m L,9906.765±320.964 h·ng/m L,0.83±0h,3.624±0.143 h,5.75±0.345 h,0.0101±0.000349 m L/h/kg and 0.0528±0.00205 m L/kg,respectively.The main pharmacokinetic parameters C_max、AUC_0-t、AUC_0-∞、T_max、t_1/2、MRT、CL and V_d measured by ELISA were 1777±358 ng/m L,9905±288 h·ng/m L,9920±292h·ng/m L,0.83±0h,3.77±0.509 h,5.72±0.334 h,0.0101±0.000284 m L/h/kg and0.0548±0.00670 m L/kg,respectively.There was no significant difference in PK parameters,and the overall results were basically the same.Conclusions:1.A rapid and simple LC-MS/MS method was established and successfully applied to the determination of liraglutide concentration in K₂EDTA plasma of SD rats after systematic validation.2.The results of LC-MS/MS and ELISA were in good agreement,which verified the applicability of LC-MS/MS in the study of liraglutide kinetics.In contrast,LC-MS/MS analysis provides a good alternative to immunoassays with its advantages of good reproducibility and high selectivity,which can shorten method development time and improve specificity.