sample preparation and liquid chromatography-tandem mass spectrometry for multiple steroids in mammalian and avian circulation样品制备和液体chromatography-tandem质谱为多个类固醇在哺乳动物和鸟类的血液循环.pdfVIP

Sample Preparation and Liquid Chromatography- Tandem Mass Spectrometry for Multiple Steroids in Mammalian and Avian Circulation 1. 1. 2 1 Lee Koren , Ella S. M. Ng , Kiran K. Soma , Katherine E. Wynne-Edwards * 1 Department of Comparative Biology Experimental Medicine, Faculty of Veterinary Medicine, and Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada, 2 Department of Psychology, University of British Columbia, Vancouver, British Columbia, Canada Abstract Blood samples from wild mammals and birds are often limited in volume, allowing researchers to quantify only one or two steroids from a single sample by immunoassays. In addition, wildlife serum or plasma samples are often lipemic, necessitating stringent sample preparation. Here, we validated sample preparation for simultaneous liquid chromatography – tandem mass spectrometry (LC-MS/MS) quantitation of cortisol, corticosterone, 11-deoxycortisol, dehydroepiandrosterone (DHEA), 17b-estradiol, progesterone, 17a-hydroxyprogesterone and testosterone from diverse mammalian (7 species) and avian (5 species) samples. Using 100 mL of serum or plasma, we quantified (signal-to-noise (S/N) ratio $10) 4–7 steroids depending on the species and sample, without derivatization. Steroids were extracted from serum or plasma using automated solid-phase extraction where samples were loaded onto C18 columns, washed with water and hexane, and then eluted with ethyl acetate. Quantitation by LC-MS/MS was done in positive ion, multiple reaction-monitoring (MRM) mode with an atmospheric pressure chemical ionization (APCI) source and heated nebulizer (500uC). Deuterated steroids served as internal standards