Comparative analysis of serotonin in equine plasma with liquid chromatography--tandem mass spectrometry and enzyme-linked immunosorbent assay.

Serotonin is regularly measured in equine platelet-poor plasma in research settings. However, reported reference values vary between studies, partially because plasma serotonin concentrations are very low and a reliable and affordable detection method is lacking. A simple, rapid, and sensitive method for serotonin determination in equine platelet-poor plasma using liquid chromatography--tandem mass spectrometry (LC-MS/MS) was developed and validated. Results of a commercially available enzyme-linked immunosorbent assay (ELISA) were compared to the LC-MS/MS results, in order to validate a test more suitable for use in a clinical situation. For LC-MS/MS, 500 µl of plasma was required, and deuterated serotonin was used as an internal standard. The sample preparation was based upon a simple liquid extraction into ethyl acetate. Chromatographic separation was performed with an acetic acid--acetonitrile mobile phase gradient elution. Linearity was demonstrated between 3 ng/ml and 100 ng/ml. A limit of quantification of 3 ng/ml was achieved, corresponding to a limit of detection of 0.10 ng/ml. Comparison of LC-MS/MS and ELISA with Passing-Bablok regression and Bland--Altman plotting showed a poor agreement between the 2 methods, with an increasing difference within the higher range of measurements. Caution is needed when extrapolating results from sources using different analytical techniques.