Determination Of Insulin Lispro In Rat Plasma By LC-MS/MS And Its Application In A Pharmacokinetics Study

There is only one protein hormone in the human body that can help regulate blood sugar,insulin,which plays a pivotal role in human health.Insulin Lispro is the main research object in this study.It is the representative research result of the third-generation insulin-insulin analogs.Compared with human insulin,it absorbs faster after subcutaneous injection and lowers blood sugar.The effect is faster and the peak blood concentration is higher,which can better control the hyperglycemia caused by diabetes after meals.So far,the quantitative analysis of insulin and its analogs has been widely studied at home and abroad,and most of them are investigated by means of liquid chromatography tandem mass spectrometry（LC-MS/MS）.However,there are still many problems in the analysis methods,such as poor chromatographic separation.,low recovery rates,etc.,there is an urgent need to establish a method that is cost-effective and suitable for routine laboratory analysis.Section one:In this study,we took insulin lispro as the research object,and established a ultra-high liquid chromatography-tandem mass spectrometry（UPLC-MS/MS）method to analyze insulin lispro.Using the electrospray ion source in positive mode and the multiple reaction detection mode,the precursor ions were determined by a full scan in the range of 50 to 1500 Da of the mass spectrometer,and then the product ions with the highest response were obtained by collision-induced dissociation with different energies to determine the desired ions.Yes,the final ion pairs used are insulin lispro m/z（1162.5→217.2）and bovine insulin m/z（1157.5→136.0）.Using bovine insulin as the internal standard,it was found that the non-precipitation recovery rate was the highest after the sample pretreatment conditions were optimized,and the electrostatic adsorption problem that insulin was easily generated was solved by adding0.1%blank rat plasma to the preparation solvent.After solid-phase extraction,plasma samples were separated into a chromatographic system.The chromatographic column was UPLC Peptide CSH C18 Column（2.1 mm×50 mm,1.7μm）produced by Waters,and the total running time of each sample was 5 min..Section two:Method validation with reference to FDA and Chinese Pharmacopoeia,the results show that the standard curve range of 0.1～100 ng·m L^-1is set as expected;The intra-day and inter-day accuracies and precision of the developed method were also within the acceptable range of±20%,and the recoveries were in the range of 63.1%～68.1%.The stability results under different conditions also met the expected standards and can be used as a routine laboratory assay.Compared with previous methods reported in literature,the developed method has excellent chromatographic separation and doubled the recovery rate.Section three:Taking rats as the animal experimental object,the developed and validated UPLC-MS/MS analytical method for analyzing insulin lispro was applied to its single-dose subcutaneous injection pharmacokinetics study.90μg·kg^-1insulin lispro was subcutaneously injected into 18 SD rats by the cross-bleed method.According to the calculation of pharmacokinetic parameters,the drug reached its peak time in 15minutes,and its peak concentration was 12.46 ng·m L^-1.The half-life is 36.49 min,the average residence time is 52.07 min,and the area under the drug-time curve is 776.47min·ng·m L^-1.All the pharmacokinetic data results are in line with the literature reports,providing the pharmacokinetic results of insulin lispro.