HPLC法测定大鼠血浆中乙醛的含量

HPLC determination of acetaldehyde in rat plasma

目的:建立检测大鼠血浆中乙醛(ACH)浓度的HPLC方法。方法:选择正丁醛-2,4-二硝基苯肼(DNPH)为内标,采用ZORBAX Eclipse XDB-C18(250 mm×4.6 mm,5μm)色谱柱,以甲醇-乙腈-水(25∶35∶40,v/v/v)为流动相,流速1.0 mL.min-1,在紫外波长为360 nm处检测。血浆样品需经乙腈沉淀蛋白,离心后取上清与DNPH发生衍生化反应,正己烷-乙酸乙酯(5∶1,v/v)萃取衍生化产物ACH-DNPH,进行高效液相色谱测定。结果:乙醛在0.05～10.00μg.mL-1浓度范围内线性关系良好(r=0.9999),检测限为0.01μg.mL-1,定量限为0.05μg.mL-1;高、中、低3个浓度的相对回收率为102.7%～109.0%;日内RSD为1.6%～4.3%,日间RSD为3.3%～6.3%。结论:本法可用于大鼠体内乙醇代谢动力学研究。

Objective：To establish a method for determination of acetaldehyde （ACH） in rat plasma by HPLC Methods：With butyl aldehyde - 2,4 - dinitrophenylhydrazine （ BUH - DNPH） as the internal standard, ACH - DN- PH was detected by UV detector at the wavelength of 360 nm with the ZORBAX Eclipse XDB -C18 （250 mm ×4.6 mm, 5 μm） column. The mobile phase was methanol - acetonitrile - water （ 25： 35： 40, v/v/v） and the flow rate was 1.0 mL ·min^-1. The plasma samples were treated with acetonitrile to precipitate proteins. After centrifugation, the supernatant was reacted with DNPH reagent. The derived acetaldehyde was then isolated by n-hexane - acetidin （5 ： 1, v/v） and quantitated by high performance liquid chromatography. Results： The assay linearity of ACH was con- firmed over the range of 0.05 - 10. 00 μg·mL^-1 （r = 0. 9999）. The limit of detection was 0.01 μg·mL^-1and the limit of quantitation was 0.05 μg·mL^-1. The relative recoveries at high, medium and low concentrations were with- in 102.7% - 109.0% ,and the RSD of intra - day and inter - day assays were 1.6% -4.3% and 3.3% -6.3% , respectively. Conclusion：The method is suitable for the pharmacokinetic research of alcohol in rats.