液相色谱串联质谱测定大鼠血浆中多黏菌素E甲磺酸钠及多黏菌素E方法的建立及应用

Establishment and application of LC-MS/MS method for determination of colistin methanesulfonate and polymyxin E in rat plasma

目的建立液相色谱串联质谱(LC-MS/MS)法测定大鼠血浆中多黏菌素E甲磺酸钠(CMS)及其活性成分多黏菌素E并进行应用。方法通过多黏菌素E主成分E1及E2浓度之和间接得到多黏菌素E的浓度。在大鼠血浆样品中加入硫酸对CMS进行水解,通过测定水解后与水解前血浆样品中多黏菌素E的浓度差间接测定CMS浓度。采用固相萃取法对样品进行预处理。色谱柱选择Phonomenex Kinetex XB-C18柱,质谱采用电喷雾电离源(ESI源)及多反应监测(multi-reaction monitoring,MRM)模式。其中待测物质多黏菌素E1、多黏菌素E2、内标多黏菌素B1的离子通道分别为m/z390.7→101.3、m/z 386.0→101.2、m/z 402.3→101.2。结果多黏菌素E1及E2的测定不受大鼠血浆中内源物质的影响,且分别在0.187~6.24 mg/L、0.0370~1.23 mg/L内线性良好,批内与批间精密度良好,变异系数均<15%。多黏菌素E1、E2准确度范围分别为100.1%~113.3%和94.9%~107.3%。多黏菌素E1、E2的提取回收率分别为21.8%、22.0%。CMS血浆样品室温下放置100 min稳定,多黏菌素E血浆样品室温下放置120 min稳定,以上样品均可在-20℃放置7 d,-70℃放置28 d,预处理后样品在自动进样器中放置24 h,在-20℃及-70℃下反复冻融3次后均稳定。对CMS样品进行1∶10(v/v)的稀释不影响该方法的检测。该方法对于CMS的水解稳定性良好,批内及批间平均水解率分别为73.9%、71.9%。结论该研究建立了一种灵敏、准确、采样量小的LC-MS/MS方法,通过测定水解后的多黏菌素E1及E2,间接测定大鼠血浆中CMS及其多黏菌素E的浓度。在降低采样量的同时也提高了检测灵敏度,该方法适用于大鼠CMS的PK研究。

Objective To establish and apply a liquid chromatography tandem mass spectrometry(LC-MS/MS)method suitable for determination of colistin methanesulfonate(CMS)and its active ingredient,polymyxin E,in rat plasma.Methods The concentration of polymyxin E was obtained indirectly by the sum of the concentrations of polymyxin E1 and E2,which are the main ingredients of polymyxin E.Sulfuric acid was added to the rat plasma samples to hydrolyze CMS.The concentration of CMS was measured indirectly by measuring the concentration difference of polymyxin E in plasma samples after and before hydrolysis.The concentration of CMS in the plasma sample was determined indirectly by measuring the concentration of polymyxin E after hydrolysis.The samples were pretreated by solid phase extraction.Phonomenex Kinetex XB-C18 column was selected for the chromatogram.Electrospray ionization(ESI)source and multi-reaction monitoring(MRM)mode were used for the mass spectrometry.The ion channels of polymyxin E1,polymyxin E2 and internal standard polymyxin B1 were m/z 390.7→101.3,m/z 386.0→101.2,m/z 402.3→101.2,respectively.Results The determination of polymyxin E1 and E2 were not affected by the endogenous substances in rat plasma.The linearity was good within 0.187-6.24 mg/L and 0.0370-1.23 mg/L,respectively(R2=1.00,0.991).The concentration of polymyxin E1/E2 in quality control samples were 0.187/0.0370,0.562/0.111,2.34/0.461,and 4.99/0.984 mg/L,respectively.Polymyxin E1 and E2 had good intra-day and inter-day precision,with coefficients of variation<15%.The accuracy range of polymyxin E1 and E2 was 100.1%-113.3%and 94.9%-107.3%,respectively.The matrix effect of polymyxin E1 and E2 was satisfactory.The extraction recoveries were 21.8%and 22.0%.CMS plasma samples can be stored at room temperature for 100 min,at-20°C for 7 d,and at-70°C for 28 d,and can be stored in an autosampler for 24 h after pretreatment.Polymyxin E samples can be placed at room temperature for 120 min,-20℃for 7 d,-70℃for 28 d,and also placed in the autosampler for 24 h.Dilution of CMS sample 1:10(v/v)did not affect the detection capability of this method.The hydrolysis stability of CMS was good.The average intra-day and inter-day hydrolysis rates were 73.9%and 71.9%,respectively.Conclusions In this study,a sensitive,accurate and small sampling LC-MS/MS method was established to indirectly determine the concentration of CMS in rat plasma by measuring hydrolyzed polymyxin E1 and B.The detection sensitivity was improved while the required sample volume was reduced,which makes this method more suitable for PK study of CMS in rats.