Quantitative Analysis In Plasma And Fragmentation Mechanism In Electrospray Ionization Mass Spectrometry For Colistin B Methanesulfonate

Colistin is a polypeptide antibiotic,which has attracted renewed attention recently because of its strong effect on Gram-negative bacteria and pan-drug-resistant infection.The launched colistin is usually the mixture of colistin A and colistin B,and these two components show similar antibiotic activity.However,it is reported that colistin A causes much more damage to kidney than colistin B.In another way,colistin methanesulfonate is an inactive prodrug of colistin,which is metabolized to colistin in vivo to kill bacteria.Colistin B methanesulfonate is a single component of colistin methanesulfonate,and it is more active and less toxic compared with the mixture.In this study,we established a quantitative method to study the pharmacokinetics of colistin B methanesulfonate and colistin B,and studied the chromatographic retention and mass spectrometry fragmentation of colistin B methanesulfonate.Firstly,this study established an LC-MS/MS method to determine the concentration of colistin B methanesulfonate and its metabolite colistin B in plasma of SD rats.Chromatography was carried out with a Shimadzu LC 30AD system,and selected a Luna Omega Polar C18?1.6?m,50×2.1 mm?column.The mobile phase consisted of 5 mM ammonium acetate with 0.2%formic acid and acetonitrile:methanol?1:1,v/v??Containing 0.1%formic acid?,the flow rate was 0.3 m L/min,gradient elution was selected,running time was 4.5 min.The mass spectrometry system was a TSQ Quantum Vantage triple quadrupole tandem mass spectrometer with HESI source,and positive ion detection and SRM scanning were selected.The precursor-product ion pairs were m/z 578??226.9+100.9?for colistin B and m/z602?100.8 for Polymyxin B₁?IS?.For determination of colistin B free concentration,we chose WCX cartridge solid phase extraction.The linear range of the method was 0.02-40?g/m L.The precision of RE was less than 4.4%,the accuracy was 89.2%-100.7%,and the recovery was 68.8%-69.5%.For determination of colistin B total concentration,we chose acetonitrile?containing0.2%formic acid?to precipitate the protein.The linear range of the method was0.05-100?g/m L,the precision was less than 3.8%,the accuracy was 93.9%-99.6%,and the recovery was 98.2%-103%.Secondly,a new method was developeded to study chromatographic retention or mass spectrometry for colistin B methanesulfonate.Ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry?UPLC-Q-TOF-MS?was used,and electrospray ionization anion?ESI^-?mode was selected.In addition,acquity UPLC HSS T3 Column?100 mm×2.1 mm,1.8?m?and gradient elution was selected for better separation of colistin B with different numbers of methanesulfonic acid groups.In order to analyse the possible fragmentation pathways of colistin B with different numbers of methanesulfonic acid groups,a chromatographic separation and mass detection method was established.It is concluded that the retention time of colistin B with different numbers of methanesulfonic acid groups depends on the numbers of methanesulfonic acid groups.The numbers of methanesulfonic acid groups were more,the retention time was shorter.In addition,colistin B with different numbers of methanesulfonic acid groups can be separated well through gradient elution,but isomers of the same number of methanesulfonic acid groups cannot be separated well as the property of these isomer are similar to each other.Results also reveal that colistin methanesulfonate with different numbers of methanesulfonic acid groups all have double charge quasi-molecular ion peak[M-2H]^2-.CID research shows CH₂SO₃,H₂SO₃,SO₂ and CH₃CHO are the main neutral fragments,without any amide bonds cleavage.The Quantitative method is rapid,accurate,selective and sensitive.It was successfully used to determine the concentration of colistin B methanesulfonate and colistin B in plasma samples of SD rats,and to further study the pharmacokinetic properties of both in rats.At the same time,we analyzed the fragmentation mechanism of colistin B methanesulfonate in ESI^-mode for the first time.This study provides a reference for the further study of the mass spectrometry of colistin methanesulfonate and derivatives,and also has a reference for the quantitative analysis,pharmacokinetic study and quality control of colistin methanesulfonate.