Quantitative Analysis Of Corticosterone And Testosterone In Wild Birds Plasma By Liquid Chromatography-tandem Mass Spectrometry

We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative analysis of corticosterone and testosterone in wild birds plasma. Compared with radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), it has characteristic of simple and fast sample preparation, excellent specific and accurate so as to apply in routine analysis of corticosterone and testosterone in wild birds (wild animals) plasma.The result shows that:1. A method was developed for the determination of corticosterone in wild birds plasma using liquid chromatography-tandem mass spectrometry. After liquid-liquid extraction with diethyl ether, liquid chromatography separation was achieved by using an Agilent Eclipse Plus-C18 column and acetonitrile-0.1% formic acid solution (containing 0.01mol/L ammonium formate) gradient as a mobile phase, then detected using MS/MS system with electrospray ionization (ESI) in multi-reaction monitoring (MRM) mode. The detection limits ranged from 1ng/mL to 100ng/ml. The recoveries ranged from 85.1% to 87.4%, and the inter-and intra-day precisions (measured as relative standard deviations) were less than 5.94% and 9.51%. With No derivatisation, simple and fast sample preparation, the method was excellent sensitive, specific and accurate for determination of corticosterone in wild birds plasma. The method has been successfully applied in wild birds study and is based procedures in routine analysis of endogenous steroids such as corticosterone in wild birds plasma.2. A method was developed for the determination of testosterone in wild birds plasma using liquid chromatography-tandem mass spectrometry. Plasma proteins were precipitated with acetonitrile and eliminated lipid Compounds by hexane. Testosterone were derivatized by reaction with hydroxylamine hydrochloride.then were extracted with tert-butyl methyl ether, liquid chromatography separation was achieved by using an Agilent Eclipse Plus-C18 column and acetonitrile-0.1% formic acid solution (containing 0.01mol/L ammonium formate) gradient as a mobile phase, then detected using MS/MS system with electrospray ionization in multi-reaction monitoring mode. The detection limits ranged from 0.1ng/mL to 100ng/mL. The recoveries ranged from 75.8% to 79.3%, and the inter-and intra-day precision(measured as relative standard deviations) were less than 6.97% and 9.81%. With simple and fast sample preparation, the method was excellent sensitive, specific, accurate and lower LOD for determination of testosterone in small volumes of wild birds plasma. 3. A method was developed for the simultaneous determination of corticosterone and testosterone in wild birds plasma using liquid chromatography-tandem mass spectrometry. After liquid-liquid extraction with diethyl ether/dichloromethane (V:V/6:4), liquid chromatography separation was achieved by using an Agilent Eclipse Plus-C18 column and acetonitrile-0.1% formic acid solution (containing 0.01mol/L ammonium formate) gradient as a mobile phase, then detected using MS/MS system with electrospray ionization in multi-reaction monitoring mode. The detection limits of corticosterone and testosterone ranged from 1ng/ml to 100ng/ml and 0.5ng/mL to 100ng/mL respectively. The recoveries of corticosterone and testosterone ranged from 81.7% to 82.9% and 84.2% to 87.2% respectively. The inter-and intra-day precisions (measured as relative standard deviations) were less than 7.82% and 9.60% respectively. With simple and fast sample preparation, the method was excellent sensitive, accurate and specific for simultaneous determination of corticosterone and testosterone in wild birds plasma.