固相萃取荧光检测高效液相色谱法测定人血浆中罗格列酮浓度

Determination of Rosiglitazone in Human Plasma by Solid Phase Extraction and EDPLC with Fluorescence Detection

目的:建立适用于人体动力学研究的高灵敏度和高选择性的人血浆中罗格列酮的HPIC测定法。方法:以罗格列酮的同系物SB-204882为内标,采用C2固相萃取荧光检测的HPIC分析法,C18分析柱(5μm,150mm×4.6 mm),流动相:乙酸铵(0.01 mol·L-1,用10%氨水调节pH 8.0)-乙腈(64:36),流速:1.0 mL·min-1,激发波长247 nm,发射波长367 nm;取血样0.2 mL,加入内标和0.05 mol·L-1盐酸0.5 mL,涡旋1 min后上固相萃取柱,真空抽干,并用正己烷-异丙醇(80:20)0.5 mL洗涤弃去,继续真空抽干,接收氯仿-异丙醇(80:20)1 mL的洗脱液,通氮挥干,残渣用流动相100μL充分溶解,进样20μL。结果:最低定量限为2.00 ng·mL-1,线性范围为2.00-500.0 ng·mL-1。血浆内源物质不干扰测定。结论:本法的准确度和精密度符合要求,方法可靠、灵敏、高选择性,可用于药物动力学研究。

Objective:To develop a highly sensitive and selective HPLC method for the determination of rosiglita zone in human plasma. Methods: The method involves the use of solid - phase extraction (C2) followed by HPLC with fluorescence detection, using a chemical analogue, SB - 204882, as the internal standard. The assay was performed on C18 analytical column with mobile phase(0. 01 mol·L-1 ,pH 8. 0 ammonium acetate - acetonitrile,64: 36 ) flow rate was 1. 0 mL ?·min -1. The fluorescence detector set at excitation wavelength of 247 nm, emission wavelength of 367 nm. To 200μL of plasma sample was added 25 μL of internal standard solution ,0. 5 mL of hydrochloric acid(0. 05 mol·L-1) and vortexed for 1 minute. The mixture solution loaded onto the solid - phase extraction cartridge( Bond - Elut C2,100 mg, 1 mL) under reduced pressure, and washed with hexane - 2 - propanol(80= 20, 0. 5 mL). The compounds of interest were eluted with chloroform - 2 - propanol(80:20,1 mL) under low vacuum and collected in a 4. 5 mL polypropylene tube. The extract was evaporated to dryness under a stream of nitrogen, reconstituted with 100 μL mobile phase,transferred to an autosampler vial with plastic insert,and injected 20 μL onto the HPLC system. Results: Using 200 μL of plasma the limit lower of quantification for rosiglitazone was 2. 00 ng·mL-1 . Linear responses in analyte/internal standard peak area ratios were observed for analyte concentrations ranging from 2. 00 ng·mL -1 to 500 ng·mL-1. Chromatograms of drug - free plasma showed no interfering peaks with retention times similar to those for rosiglitazone or internal standard. Conclusion: The assay demonstrated appropriate sensitivity and robustness to determine pharmacokinetics parameters.