Quantitative analysis of anandamide and related acylethanolamides in human seminal plasma by ultra performance liquid chromatography tandem mass spectrometry

Abstract

The endocannabinoids anandamide, palmitoylethanolamide and oleoylethanolamide have been detected in human seminal plasma and are bioactive lipids implicated in regulation of sperm motility, capacitation and acrosome reaction. Several methods exist for endocannabinoid quantification but none have been validated for measurement in human seminal plasma. We describe sensitive, robust, reproducible solid phase and isotope-dilution UHPLC-ESI-MS/MS methods for the extraction and quantification of anandamide, palmitoylethanolamide and oleoylethanolamide in human seminal plasma. Precision and accuracy were evaluated using pooled seminal plasma over a 4 day period. For all analytes, the inter- and intraday precision (CV%) was between 6.6–17.7% and 6.3–12.5%, respectively. Analyses were linear over the range 0.237–19 nM for anandamide and oleoylethanolamide and 0.9–76 nM for PEA. Limits of detection (signal-to-noise >3) were 50, 100 and 100 fmol/mL and limits of quantification (signal-to-noise >10) were 100, 200 and 200 fmol/mL, respectively for anandamide, palmitoylethanolamide and oleoylethanolamide. Anandamide and oleoylethanolamide were stable at −80 °C for up to 4 weeks, but palmitoylethanolamide declined significantly. We assessed seminal plasma from 40 human donors with normozoospermia and found mean (inter-quartile range) concentrations of 0.21 nM (0.09–0.27), 1.785 nM (0.48–2.32) and 15.54 nM (7.05–16.31) for anandamide, oleoylethanolamide and palmitoylethanolamide, respectively. Consequently, this UHPLC-ESI-MS/MS method represents a rapid, reliable and reproducible technique for the analysis of these endocannabinoids in fresh seminal plasma.