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

Abstract

Abstract. 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.

Analysis and measurement of serotonin

“…A validated LC/MS/MS method was used to determine serotonin concentrations in lamb plasma with a sensitivity that reached the low ng/mL range (Szeitz, Nguyen, Riggs, & Rurak, ). Similar sensitivity was reported with a validated LC/MS/MS method that was used to measure serotonin levels in platelet‐poor equine plasma from healthy horses and horses suffering from small intestine colic (Torfs, Maes, Delesalle, Deprez, & Croubels, ).…”

Comparison of serum serotonin and serum 5-HIAA LC-MS/MS assays in the diagnosis of serotonin producing neuroendocrine neoplasms: A pilot study

“…The LOQ for our new serotonin LC-MS/MS assay was 10 nmol/l and the linear range is 10-10000 nmol/l.A better sensitivity can be achieved by using 200 µl sample volume instead of 100 µl as we used for PPP samples with <10 nmol/l serotonin. Previously described serotonin assays have LOQ of 1-50 nmol/l and linear range of 3.5-5000 nmol/l[9,10,[16][17][18]. Our assay compares favorably with these with inter-and intra-assay CVs of <10 %.Methods based on immunoassays and HPLC paired with fluorescence, UV or electrochemical detection may be laborious and suffer from interference from related compounds present in samples.…”

Clinical Aspects Related to Plasma Serotonin in the Horse

“…5-HT plasma concentrations in horses are subject to physiological variations such as age [17,18], exercise [7][8][9], stress [19], seasonal, circadian and nycthemeral rhythms [15,20], altitude [21], reproductive status [22], and type of anticoagulant and laboratory technique [23][24][25]. In addition, these factors also influence the analytical results of this neurotransmitter.…”

“…It is believed that 5-HT in PPP reflects the amount of 5-HT synthesized and recently secreted in EC cells. Although ELISA [23] and HPLC [18,21] have been used, the tandem chromatographic mass spectrometry (LC-MS/MS) method is suitable for determining the plasma reference values of 5-HT and analyzing changes in 5-HT associated with pathological conditions.…”