中国临床药理学杂志2024，Vol.40Issue(1)：117-120,4.DOI:10.13699/j.cnki.1001-6821.2024.01.024
高效液相色谱荧光法测定人红细胞内甲氨蝶呤及其谷氨酸聚合物浓度
Determination concentration of methotrexate and its polyglutamates in human erythrocyte by high-performance liquid chromatographic fluorescence method
摘要
Abstract
Objective To establish a sensitive,accurate and simple method for the determination of methotrexate and methotrexate polyglutamates(MTXPG2 and MTXPG3)in human erythrocytes.Methods A dual three element gradient liquid chromatograph with a fluorescence detector was used,the C18-WP column(20 mm ×4 mm,5μm)was used as the online SPE column,and the Athena C18-WP column(150 mm x4.6 mm,3 μm)was used as the analytical column.Erythrocyte lysate was precipitated with zinc sulphate-10％formic acid methanol(100:90,v/v),and postcolumn photo-oxidation of MTXPGs to fluorescent analytes using H2O2.The fluorescence excitation wavelength was 274 nm,the emission wavelength was 470 nm,the column temperature was 40 ℃,and the injection volume was 100 μL.The specificity,standard curve,lower limit of quantitation,precision,recovery and stability of the method were investigated.Results MTX,MTXPG2 and MTXPG3 had good linearity in the range of 12.5-400.0 nmol·L-1.The standard curve of MTX was y=763.8x-2 961.1(R2=0.999 5),and the extraction recovery rate was 60.7％-66.1％;the standard curve of MTXPG2 was y=1 017.8x-239.8(R2=0.998 4),and the extraction recovery rate was 67.2％-67.3％;the standard curve of MTXPG3 was y=1 069.1x-819.6(R2=0.999 4),the extraction recovery rate was 62.9％-70.1％.Intra-day precision RSD＜8.8％,inter-day precision RSD＜10.8％.Conclusion This method is accurate and reproducibility,and the online solid-phose extraction enrichment and separation of target compounds simplify the sample pretreatment steps,improve the analysis efficiency,and is suitable for detecting the concentration of MTX,MTXPG2 and MTXPG3 in erythrocytes of patients with rheumatoid arthritis.关键词
甲氨蝶呤/多聚谷氨酸甲氨蝶呤/在线固相萃取/高效液相色谱/类风湿性关节炎Key words
methotrexate/methotrexate polyglutamates/online solid phase extraction/high performance liquid chromatography/rheumatoid arthritis分类
医药卫生引用本文复制引用
张桂杰,廖婷,接红宇,陈文瑛,李强..高效液相色谱荧光法测定人红细胞内甲氨蝶呤及其谷氨酸聚合物浓度[J].中国临床药理学杂志,2024,40(1):117-120,4.基金项目
广东省药学会临床用药研究基金资助项目(2023JZ15) （2023JZ15）
