The field of metabolomics seeks to characterize the suite of small molecules that comprise the endproducts of cellular regulation. Metabolomics has been used in biomedical applications as well as environmental studies that explore ecological and biogeochemical questions. We have developed a targeted metabolomics method using electrospray ionization-liquid chromatography tandem mass spectrometry to analyze metabolites dissolved in seawater. Preparation of samples from the marine environment presents challenges because dilute metabolites must be concentrated and desalted. We present the extraction efficiencies of 89 metabolites in our targeted method using solid phase extraction (SPE). In addition, we calculate the limits of detection and quantification for the metabolites in the method and compare the instrument response factors in five different matrices ranging from deionized water to spent medium from cultured marine microbes. High background organic matter content reduces the instrument response factor for only a small group of metabolites, yet enhances the extraction efficiency for other metabolites on the SPE cartridge used here, a modified styrene-divinylbenzene polymer called PPL. Aromatic or larger uncharged compounds, in particular, are reproducibly well retained on the PPL polymer. This method is suitable for the detection of dissolved metabolites in marine samples, with limits of detection ranging from < 1 pM to 2 nM dependent on the dual impacts of seawater matrix on extraction efficiency and on instrument response factors.Metabolomics is an "omics" technique that seeks to measure the small organic biomolecules produced by cells (Oliver et al. 1998;Fiehn 2002). Because these small molecules are the end-products of multiple levels of metabolic regulation, their concentrations provide a temporal snapshot of the metabolic state or phenotype of an organism. In particular, metabolites produced by nonenzymatic reactions, such as those formed by reaction with a radical oxygen species, or whose production is regulated by other small molecules, must be monitored directly because their production cannot be inferred from genomic or proteomic information. Metabolomics can be used as a diagnostic tool, identifying biomarkers of disease within the human metabolome, such as cancers (Armitage and Barbas 2014) and Crohn's Disease (Jansson et al. 2009). Metabolomics has also been applied in a wide range of organisms and environments, examining how metabolite abundances respond to environmental factors. In the oceans, marine metabolites have been a valuable source of new natural products, while other metabolomics applications are still rare but growing. For example, recent marine culture experiments have revealed metabolite production not predicted by genomic information (Baran et al. 2010;Fiore et al. 2015), metabolic shifts in response to a specific metabolite (Johnson et al. 2016), and changes in the quantity and composition of metabolite production during coculturing (Paul et al. 2012). Complementary field ...