Study On On-line Concentration Techniques And Its Application In Biopharmaceutical Analysis

High-performance capillary electrophoresis(HPCE) and liquid chromatography-mass spectrometry(LC-MS) are both widely applied and actively studied in the field of separation science.However,due to the insufficient concentration sensitivity or the low sample throughput,their application in biopharmaceutical analysis is rather limited.On-line sample concentration techniques,with the advantages of simple operation,low cost,highly automation and good feasibility,is now getting more and more popularity as an approach to improve sensitivity.Study on on-line concentration techniques based on CE and LC-MS is of crucial importance to further explore the application in the area of in vivo pharmaceutical research and clinical practice. In this thesis,on-line sample preconcentration based on CE and on-line solid phase extraction coupled to LC-MS were studied in terms of application in biopharmaceutical analysis.On-line concentration methods were developed, optimized and validated according to the model drugs,which provided reliable,robust and economic quantification approach for pharmacokinetic study and therapeutic drug monitoring(TDM) in clinic.The main contents are summarized as follows:1.A micellar electrokinetic capillary chromatographic method was established for the simultaneous concentration,separation and detection of itraconazole and its main active metabolite hydroxyitraconazole in human plasma utilizing field-enhanced sample injection with reverse migrating micelles(FESI-RMM) as on-line enrichment approach.Plasma samples were prepared by liquid-liquid extraction to remove most of the proteins and salts as well as to off-line concentrate the target analytes.Under optimal concentration conditions,the sensitivity of the method can be improved by 2 magnitudes and the lower limit of detection was 0.05μg/mL.The described method boasted robust enrichment ability, no special requirement of instrument,lower cost as well as satisfactory feasibility, reproducibility and sensitivity.It has been adopted to the quantification of ITZ and HITZ in plasma samples from cryptococcal-meningitis-infected patients with the purpose to provide reference in clinical medication. 2.An automated on-line SPE LC-MS method was established to purify,enrich, separate and detect allylestrenol in human serum.The original LC system was minimal modified to form an on-line SPE configuration and the analyte was eluted in a normal-flush mode.All the serum samples were preliminarily extracted with ethyl acetate.Under the optimal conditions,the lower limit of quantification was 0.01μg/mL.The described method,boasting rapidity,simplicity,sensitivity as well as reliability,not only simplify the pretreatment procedure,but also improve the sample throughput.It can be applied to clinical TDM and pharmacokinetic study in vivo.