Quantitative profiling of lipid mediators in sperm cells through on-line dilution on-line polymer matrix-based solid-phase extraction liquid chromatography with mass spectrometric detection

An automated on-line dilution on-line solid-phase extraction liquid chromatography-mass spectrometry method was developed for the straightforward and versatile quantitative profiling of lipid mediators such as various eicosanoids, endocannabinoids, and arachidonic acid in human sperm. The LC-MS/MS system utilized a polymer matrix-based trap column (TurboFlow Cyclone™) for sample pre-treatment, followed by chromatographic separation using an ODS 3 μm, 100 × 2 mm column, and detection via a triple-quadrupole mass spectrometer equipped with an electrospray interface. The sample preparation process employed a simple “dilute and shoot” approach, with a centrifugation step to remove proteins. This method has significantly improved sensitivity and selectivity while minimising matrix effects, with limits of quantification in the low picogram range for most analytes. Linearity, accuracy, precision, and recovery all met the required criteria for bioanalytical method validation. This LC-MS/MS approach was successfully applied to determine basal levels of selected eicosanoids, endocannabinoids, and arachidonic acid in human spermatozoa.