Assessing melatonin and its oxidative metabolites amounts in biological fluid and culture medium by liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS)

Abstract

The introduction of precise analytical technologies in the biological area is needed to increase the knowledge of the fundamental processes occurring in the animal and human domain. The objective of this study was to develop a highly sensitive and selective method based on liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) using electrospray ionization for the simultaneous detection and quantification of melatonin (MEL) and its metabolites, 6-hydroxymelatonin (6-HMEL), N²-acetyl-N¹-formyl-5-methoxykynuramine (AFMK) and N¹-acetyl-5-metoxykynuramine (AMK). Using this methodology, we have studied the role of melatonin and its metabolites in the mammalian embryo in vitro production system, which is fundamental due to the antioxidant and signaling roles of these compounds. Samples comprised of bovine follicular fluid (FF) and tissue culture medium (TCM 199). The optimized procedure uses liquid–liquid extraction with MS monitoring via selective reaction monitoring (SRM). Low limits of detection/quantification were obtained, 3/10 pg mL⁻¹ for MEL, AFMK and AMK and 30/100 pg mL⁻¹ for 6-HMEL, respectively using deuterated melatonin (MEL-d₄) as the internal standard (IS). Validation and correlation coefficients were higher than 0.999 and recoveries were 80–108%. Precision was evaluated as repeatability and intermediate precision with relative standard deviation values <3.55%. The method has been successfully applied to the analysis of pooled FF and TCM 199 samples and results suggest that MEL and its metabolites are involved in oocyte maturation and that their proper quantitation is essential to monitor and study their role in such a process.