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

The research article focuses on the development and validation of a new method for measuring serotonin levels in equine platelet-poor plasma using liquid chromatography–tandem mass spectrometry (LC-MS/MS). It also compares the results obtained using this method with a traditional enzyme-linked immunosorbent assay (ELISA) to confirm its efficacy and practicality for use in clinical scenarios.

Research Methodology

The researchers developed a method for determining serotonin in equine plasma using LC-MS/MS and compared its results to the ones from a commercially available ELISA test. Key highlights in the methodology include:

• For the LC-MS/MS test, 500 µl of plasma was required and deuterated serotonin was used as the internal standard.
• The sample preparation was based on a simple liquid extraction into ethyl acetate, and chromatographic separation was achieved with an acetic acid-acetonitrile mobile phase gradient elution.
• The developed method demonstrated linearity between 3 ng/ml and 100 ng/ml, achieving a limit of quantification of 3 ng/ml and a detection limit of 0.10 ng/ml.

Comparison of LC-MS/MS and ELISA methods

In order to validate the new method’s reliability and suitability for clinical use, the results it produced were compared with those of a conventional ELISA test using Passing-Bablok regression and Bland-Altman plotting techniques. This comparative analysis yielded the following results:

• The agreement between the LC-MS/MS method and the traditional ELISA was found to be poor. Moreover, the discrepancy increased within the higher range of measurements.
• This variation between the results obtained through the two different techniques highlights the need for caution when extrapolating results from sources that employ different analytical methods.

Significance of the study

This study is important because it not only creates a new, highly sensitive and simple method for quantifying equine plasma serotonin but also elucidates the discrepancies that can arise when comparing different measurement techniques. As such, the findings of this research contribute to the improvement of the reliability and accuracy of serotonin detection in equine plasma, which is crucial for diagnostic and research purposes.