全自动二维液相色谱法测定甲氨蝶呤血药浓度及其临床应用  

Determination of methotrexate in the plasma by automatic two-dimensional liquid chromatography and its clinical application

作　　者：刘加涛[1] 孙小珊 宋帅 苏涌[1] 夏泉[1] LIU Jia-tao;SUN Xiao-shan;SONG Shuai;SU Yong;XIA Quan(Department of Pharmacy,the First Affiliated Hospital of Anhui Medical University,Hefei 230022)

机构地区：[1]安徽医科大学第一附属医院药剂科,合肥230032

出　　处：《中南药学》2022年第2期366-370,共5页Central South Pharmacy

基　　金：安徽省“十三五”临床重点专科建设项目(No.卫科教秘[2017]529号);吴阶平医学基金会临床科研专项资助基金(No.320.6750.2020-03-6)。

摘　　要：目的采用全自动二维液相色谱(2D-LC-UV)法快速测定甲氨蝶呤的血药浓度。方法采用10%三氟乙酸去蛋白剂对样本进行前处理后取上清液直接进样,待测物在一维柱Aston SC2(C18,4.6 mm×25 mm,5μm)上初步分离,通过中间柱Aston SN(苯基柱,4.6 mm×10 mm,5μm)截取保留,最后转移到二维色谱柱Aston SX1(氰基柱,4.6 mm×75 mm,5μm)上进一步分离、测定。一维流动相为甲醇-乙腈-10 mmol·L^(-1)磷酸铵水溶液=1∶1∶10(V/V/V),流速0.6 mL·min-1;二维流动相为水(含1.0 mmol·L^(-1)磷酸氢二铵水溶液,磷酸调pH至7.40)-水(含1.0 mmol·L^(-1)磷酸氢二铵水溶液,磷酸调pH至3.00)-甲醇-乙腈(16∶60∶10∶14)的混合体系,流速1.0 mL·min^(-1),柱温40℃;紫外检测波长304 nm。使用本方法监测2019年10月—2020年12月期间使用甲氨蝶呤治疗的48份样本的甲氨蝶呤血药浓度。结果甲氨蝶呤可在10 min内完整出峰且无杂质干扰,在0.05~12.8μmol·L^(-1)与峰面积线性关系良好(r=0.9997),方法学考察均符合要求。检测的48份甲氨蝶呤血药浓度样本中72.9%(35/48)的样本在标准曲线范围内,10.4%(5/48)的样本因使用剂量低未检出,另有16.7%(8/48)的样本使用大剂量甲氨蝶呤后血药浓度超出正常值上限。结论本方法操作简单,灵敏度、专属性高,稳定性好,能够满足甲氨蝶呤用药后药物浓度监测及解救的需要。Objective To determine the concentration of methotrexate in the plasma by automatic two-dimensional liquid chromatography(2D-LC-UV).Methods The sample was injected for determination after being deproteinized with 10%trifluoroacetic acid agent.The sample was initially separated with a one-dimensional Aston SC2 column(C18,4.6 mm×25 mm,5μm).Then methotrexate was intercepted and retained on an intermediate Aston SN column(phenyl column,4.6 mm×10 mm,5μm).Finally,it was transferred to the two-dimensional Aston SX1(cyano column,4.6 mm×75 mm,5μm)column for further separation and determination.The one-dimensional mobile phase was methyl alcohol-acetonitrile-10 mmol·L^(-1) ammonium phosphate aqueous solution(1∶1∶10,V/V/V)with a flow rate of 0.6 mL·min^(-1).The two-dimensional mobile phase was a mixed system of water(1.0 mmol·L^(-1) diammonium phosphate water solution,with pH adjusted to 7.40 with phosphoric acid)-water(1.0 mmol·L^(-1) diammonium phosphate water solution,with pH adjusted to 3.00)-methyl alcohol-acetonitrile(16∶60∶10∶14).The flow rate was 1.0 mL·min^(-1).The column temperature was 40℃and the wavelength was 304 nm.The method was used for 48 samples treated with methotrexate from October 2019 to December 2020.Results Methotrexate had a complete chromatographic peak within 10 minutes without impurity interference.The linearity was good at 0.05 to 12.8μmol·L^(-1)(r=0.9997).The methodology met all the requirements.Totally 48 samples were detected,35(72.9%)of which were within the range of the standard curve,5(10.4%)of which were not detected due to low dose,and 8(16.7%)of which exceeded the upper limit of normal value due to the use of high-dose methotrexate.Conclusion The two-dimensional liquid chromatography method is simple,sensitive,specific and good stable,which meets the needs of drug concentration monitoring and rescue after methotrexate administration.

关 键 词：二维液相色谱 甲氨蝶呤 血药浓度 治疗药物监测 

分 类 号：R96[医药卫生—药理学]