反相高效液相色谱法测定人血浆中咪达唑仑浓度

Determination of Plasma Concentration of Midazolam by RP-HPLC

目的建立反相高效液相色谱法测定人血浆中咪达唑仑浓度的方法。方法采用Hypersil-C18反相色谱柱(250mm×4.6mm,5μm),水(pH=7.3,内含三乙胺0.15%,磷酸0.05%)-乙腈(46∶54)为流动相;流速为1.0mL.min-1;柱温为30℃;样品经碱化提取分离后用流动相溶解;采用紫外检测,检测波长为223nm。结果本方法测定咪达唑仑血药浓度在50～2000ng.mL-1范围内线性关系良好(r=0.9996),咪达唑仑的保留时间为9.60min,其低、中、高(200、500、2000ng.mL-1)浓度的提取回收率分别为92.11%、90.25%、93.27%,方法回收率分别为96.81%、101.97%、101.58%,日内、日间RSD均<5.0%。咪达唑仑血浆样品预处理室温放置10h及咪达唑仑标准贮备液于4℃下15d稳定。结论本法简单,准确,快速,稳定,灵敏度高,可满足咪达唑仑血药浓度的检测及临床药代动力学研究要求。

OBJECTIVE To establish a method for the determination of midazolam in human plasma.METHODS The separation was carried out by a reversed-phase C18 column（250mm×4.6mm,5um） with a mobile phase of acetonitrile-H2O（pH=7.3,containing 0.15% mol·L-1 TEA and 0.05%mol·L-1 phosphoric acid（46∶54）.The flow rate was 1.0mL·min-1,the column temperature was 30℃.Midazolam was extracted from ackalinizing plasma and soluted in the mobile phase then detected at 223 nm.RESULTS The calibration curve showed good linearity in a concentration range of 50～2000ng·mL-1（r=0.9996）.The retention times of midazolam was 9.60 min.The average recovery was 92.11% for 200ng·mL-1,90.25% for 600ng·mL-1,93.27% for 2000ng·mL-1,the relative recovery was 96.81% for 200ng·mL-1,101.97% for 500ng·mL-1,101.58% for 2000ng·mL-1,The intra-and inter-day assay variability values were less than 5.0%,respectively.midazolam in human plasma was stable after storage for 10h under temperature or midazolam mobile phase after freeze-storing at 4℃ for 15 days.CONCLUSION The method is simple,accurate,specific,stable and highly sensitive and may be applied to the clinical pharmacokinetic study of midazolam.