Simultaneous Determination of Cortisol and Testosterone from Rat Serum by Liquid Chromatography-Tandem Mass Spectrometry

A liquid chromatography-electrospray ionization tandem mass spectrometry in a positive mode (LC-ESI-MS/MS) was developed to determine simultaneously the levels of cortisol and testosterone in rat serum. The MRM (Multiple Reaction Monitoring) was used to optimize ion precursor, fragmentor voltage, ion product, and collision energy. Analytical samples were treated by solid-phase extraction with Bond Elut C18 cartridge. The method was validated according to FDA guidance for bioanalytical method validation. The results indicated that this method showed a wide linearity range (0.1-100 ng/mL for cortisol and 0.025-100 ng/mL for testosterone), good linearity (r2 > 0.999), low limit of quantification (LLOQ values of cortisol and testosterone were 0.1 ng/mL and 0.025 ng/mL, respectively), good recovery (90.4% - 108.0%), suitable repeatability (CV values were in the range of 2.7% - 14.8%), good stability in different conditions. 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