| Glucocorticoids (GC) are a class of steroid hormones that bind to the glucocorticoid receptor (GR), which is present in almost every vertebrate animal cell. Hydrocortisone, prednisone, prednisolone, dexamethasone, methylprednisolone, triamcinolone, betamethasone and so on are popular GC drugs. GCs are part of the feedback mechanism in the immune system that turns immune activity down. They are therefore used in medicine to treat diseases caused by an overactive immune system, such as allergies, asthma, autoimmune diseases and sepsis. GCs have many diverse effects, including potentially harmful side effects, and as a result are rarely sold over the counter. Therefore, to understand and know the drug concentration in vivo during the treatment process using GC drugs will be very helpful to reduce side effects. It is necessary and important to establish a sensitive and rapid method for GC drugs in biopharmaceutical analysis.In this thesis, we studied on development of methods for determination of halometasone, dexamethasone and prednisone in biopharmaceutical analysis. Liquid chromatography coupled with tandem mass spectrometer (LC-MS/MS) was chosen for the determination because of its high sensitive, separation efficiency and specificity. A LC-MS/MS method was established for determination of halometasone in human plasma and for determination of halometasone, dexamethasone and prednisone in mouse plasma and tissue simultaneously. Sample preparation method, chromatographic conditions and mass spectrometry conditions were validated and optimized during the experiment.For the sample preparation method, Micro solid phase extraction (MSPE) was used for pre-processing of the sample. Compared with traditional extraction and concentration methods such as protein precipitation (PPT), liquid-liquid extraction (LLE) and solid phase extraction (SPE), the MSPE method, using of the Oasis μElution plate (Waters, US), resulted in a higher level of analyte recovery, less volume of plasma required and simple to use. That improved time economy and laboratory productivity observablyFor the chromatographic conditions, chromatographic column and mobile phase were optimized. We use a micro-diameter column (Agilent ZORBAX SB-C18) to determination of halometasone in human plasma. It provided a higher sensitive, less injection volumn and better matrixeffects. In order to maintain the stability and reproducibility of the adducted molecules, CH3COONH4 and HCOONH4 were added to the mobile phase. The chemical reagents 0.2%o acetic acid,0.2 mM ammonium formate and 0.2 mM ammonium acetate were investigated.0.2 mM ammonium acetate was deemed the most appropriate agent to use, where the method was better in sensitivity and reproducibilityFor the mass spectrometry conditions, Electrospray ionization (ESI) was used as the ionization mode for the MS detector and the analytes were detected by multiple reaction monitoring (MRM).In contradiction to the common perception about LC-MS/MS based methods, the selectivity obtained with selected reaction monitoring (SRM) acquisition modes has been questioned. The presence of unknown and undetectable (by SRM) components in biological matrices could induce an alteration of the analyte response that could limit and compromise the reliability of the results. This alteration of the analyte response is known as matrix effects (ME) and may be reflected in an increased (ion enhancement) or a decreased (ion suppression) signal.Therefore, we paid more attention onAssessing ME to optimize the method’s sensibility, stability and reproducibility.Finally, a reliable and validated LC-MS/MS method was established for determination of halometasone in human plasma and for determination of halometasone, dexamethasone and prednisone in mouse plasma and tissues. The LC-MS/MS conditions has been investigated and optimized. The method showed significant improvement in specificity, linearity, precision and sensitivity, and it has been successfully applied in clinical research. |
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