Author's personal copy Extraction and analysis of lipophilic brevetoxins from the red tide dinoflagellate Karenia brevis Michael J. Twiner a, * , Marie-Yasmine Bottein Dechraoui a , Zhihong Wang a , Christina M. Mikulski a , Michael S. Henry b , Richard H. Pierce b , Gregory J. Doucette a a Marine Biotoxins Program, Center for Coastal Environmental Health and Biomolecular Research, NOAA/National Ocean Service, Charleston, SC 29412, USA b Mote Marine Laboratory, Sarasota, FL 34236, USA Received 5 April 2007 Available online 22 June 2007 Abstract Efficient extraction and accurate analysis of lipophilic brevetoxins (PbTxs), produced by the harmful algal bloom (HAB) species Kare- nia brevis, are essential when assessing the toxicological potential of this dinoflagellate. One of the most commonly used brevetoxin extraction methodologies employs C18 solid-phase extraction (SPE). In this study, C18 SPEC discs were tested for extraction of spiked PbTx-3 in seawater and naturally produced brevetoxins from K. brevis. Quantification of brevetoxin in the extracts was determined using four independent methods: receptor binding assay (RBA), radioimmunoassay (RIA), neuroblastoma (N2A) cytotoxicity assay, and liquid chromatography/mass spectrometry (LC/MS). In addition to quantification of the brevetoxin concentration, LC/MS analysis pro- vided identification of individual congeners and each of their hydrolyzed products. SPEC disc extractions prepared from sonicated cul- tures of non-brevetoxin-producing Karenia mikimotoi cultures spiked with PbTx-3 yielded extraction efficiencies of 108, 99, and 125% as determined by the RBA, RIA, and N2A cytotoxicity assay, respectively. In SPEC disc extracts of brevetoxin-producing K. brevis (isolate SP3) cultures, LC/MS analysis yielded the highest total concentrations, possibly due to the concurrent detection of hydrolytic brevetoxin congeners that accounted for up to 20% of the congener profile. Relative to the brevetoxin concentration as determined by LC/MS, the RBA, RIA, and N2A cytotoxicity assay detected 73, 83, and 51% of the total brevetoxin concentration. Stability experiments demon- strated that brevetoxins remain stable on the SPEC discs for at least 30 days, making this extraction method suitable for shipboard collections. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Brevetoxin; Extraction; Harmful algal bloom; LC/MS; N2A cytotoxicity assay; Radioimmunoassay; Receptor binding assay Harmful algal blooms of the toxic dinoflagellate Karenia brevis (formerly Gymnodinium breve and Ptychodiscus bre- vis) are prominent in the U.S. Gulf of Mexico along the west Florida shelf [1] and along the coast of New Zealand [2]. Economic losses in the United States attributed to this toxic algal species exceed U.S. $38 million annually, due primarily to contamination and mortality of fishery resources as well as to lost tourism revenues [3]. The harm- ful effects of K. brevis result from production of potent neu- rotoxins called brevetoxins (PbTxs) 1 that bind with high affinity to site 5 of voltage-gated sodium channels (VGSCs) [4]. On binding, brevetoxins cause a shift in the threshold potential for channel activation concurrent with inhibition 0003-2697/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.06.031 * Corresponding author. Fax: +1 843 762 8700. E-mail address: Mike.twiner@noaa.gov (M.J. Twiner). 1 Abbreviations used: PbTx, brevetoxin; VGSC, voltage-gated sodium channel; NSP, neurotoxic shellfish poisoning; SPE, solid-phase extraction; RBA, receptor binding assay; RIA, radioimmunoassay; ELISA, enzyme- linked immunosorbent assay; N2A, neuroblastoma cytotoxicity assay; LC/MS, liquid chromatography/mass spectrometry; BSA, bovine serum albumin; PBS, phosphate-buffered saline; EDTA, ethylenediaminetetra- acetic acid; SIM, selected ion monitoring; ANOVA, analysis of variance; amu, atomic mass units. www.elsevier.com/locate/yabio Analytical Biochemistry 369 (2007) 128–135 ANALYTICAL BIOCHEMISTRY