Dual liquid-liquid extraction followed by LC-MS/MS method for the simultaneous quantification of melatonin, cortisol, triiodothyronine, thyroxine and testosterone levels in serum: Applications to a photoperiod study in rats.

Hypothalamic Pituitary (PH) axes directly affects the functionality of the thyroid gland, the adrenal gland, and the gonads and their alteration has been related to several pathologies. Therefore, the global analysis of representative hormones from each axis, together with melatonin, would be a very good strategy for therapeutic diagnosis. Hence, an accurate, economic and effective analytical method has been developed and validated for the simultaneous measurement of the melatonin, cortisol, triiodothyronine (T3), thyroxine (T4) and testosterone levels in serum. The protocol consists in two liquid-liquid extractions followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis with electrospray ionization in positive mode. The isotopically labelled internal standards melatonin-D4 , cortisol-D4 , l-thyroxine-13 C6 and testosterone-13 C3 were added to serum samples. Multiple reaction monitoring (MRM) mode was performed to target fragment ions for the hormones and internal standards. Excellent linearity (r2  ≥ 0.993) of this method was observed within the concentration range of 0.004-0.5 ng/mL for melatonin and 0.4-50 ng/mL for cortisol, T3, T4 and testosterone in rat sera. The mean recovery of all compounds ranged from 51.3% to 76.7%. The relative standard deviations (RSDs) of intra-day and inter-day precision were within the acceptable limits of ±15% at all of the concentrations tested. The method developed here has been successfully applied to study the changes of these hormones induced by the duration of light exposure in rat serum, as a physiological model of HP axes modulation. The results obtained from rat sera showed the suitability of this analytical strategy.