Quantification of a sea lamprey (Petromyzon marinus) pheromone antagonist in river water using ion pairing solid phase extraction coupled with liquid chromatography-tandem mass spectrometry

Pheromones mediate species-wide communication for many aquatic organisms, and the measurement of pheromones in natural waters is essential to understanding the environmental context of their function. However, chemical measurement of environmental pheromones and their antagonists is technically demanding and remains underdeveloped relative to assays for characterizing biological functions and application efficacy. In this study, we developed and validated an accurate and sensitive method to quantify a sea lamprey (Petromyzon marinus) pheromone and its antagonists. In this species, males release a multi-component sex pheromone containing 3-keto petromyzonol sulfate (3kPZS) that attracts females, while related compounds petromyzonol sulfate (PZS) and petromyzonol tetrasulfate (3sPZS) antagonize and disrupt female attraction. Developing methods to quantify 3sPZS in river water that contains pheromone is essential for understanding concentration-dependent effects of antagonists on invasive sea lamprey spawning. The target compound 3sPZS was extracted using triethylamine as an ion-pairing reagent during solid phase extraction followed by quantification using liquid chromatography-tandem mass spectrometry. The method showed a limit of detection of 0.1 ng/mL and limit of quantification of 0.5 ng/mL with linearity in the range of 10-1000 ng/mL. The intra- and inter-day accuracy, precision, recovery, and matrix effect of this method were evaluated. The method was applied to quantify 3sPZS, PZS, and 3kPZS in water sampled during field application in a river with sea lamprey and further evaluated for robustness by quantifying 3sPZS in 16 rivers across diverse environmental matrices. Our approach may be adapted to inform management strategies for detecting and mitigating invasive or imperiled aquatic species.