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Mar. Drugs 2017, 15(9), 278; doi:10.3390/md15090278

HPLC-HRMS Quantification of the Ichthyotoxin Karmitoxin from Karlodinium armiger

1
National Food Institute, Technical University of Denmark, Kemitorvet Building 202, 2800 Lyngby, Denmark
2
Department of Biotechnology and Biomedicine, Technical University of Denmark, Søtofts Plads, Building 221, 2800 Lyngby, Denmark
3
Departamento de Química, Universidade Federal de São Paulo (UNIFESP), Rua São Nicolau, 210, CEP 09913-030 Diadema-SP, Brazil
4
Marine Biological Section, Department of Biology, University of Copenhagen, Strandpromenaden 5, 3000 Helsingør, Denmark
*
Author to whom correspondence should be addressed.
Received: 18 May 2017 / Revised: 21 August 2017 / Accepted: 27 August 2017 / Published: 31 August 2017
(This article belongs to the Special Issue Harmful Marine Phytoplankton)
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Abstract

Being able to quantify ichthyotoxic metabolites from microalgae allows for the determination of ecologically-relevant concentrations that can be simulated in laboratory experiments, as well as to investigate bioaccumulation and degradation. Here, the ichthyotoxin karmitoxin, produced by Karlodinium armiger, was quantified in laboratory-grown cultures using high-performance liquid chromatography (HPLC) coupled to electrospray ionisation high-resolution time-of-flight mass spectrometry (HRMS). Prior to the quantification of karmitoxin, a standard of karmitoxin was purified from K. armiger cultures (80 L). The standard was quantified by fluorescent derivatisation using Waters AccQ-Fluor reagent and derivatised fumonisin B1 and fumonisin B2 as standards, as each contain a primary amine. Various sample preparation methods for whole culture samples were assessed, including six different solid phase extraction substrates. During analysis of culture samples, MS source conditions were monitored with chloramphenicol and valinomycin as external standards over prolonged injection sequences (>12 h) and karmitoxin concentrations were determined using the response factor of a closely eluting iturin A2 internal standard. Using this method the limit of quantification was 0.11 μg·mL−1, and the limit of detection was found to be 0.03 μg·mL−1. Matrix effects were determined with the use of K. armiger cultures grown with 13C-labelled bicarbonate as the primary carbon source. View Full-Text
Keywords: harmful algal bloom; ichthyotoxic; karlotoxin; amphidinol; polyether; polyketide; quantify; quantitation harmful algal bloom; ichthyotoxic; karlotoxin; amphidinol; polyether; polyketide; quantify; quantitation
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MDPI and ACS Style

Andersen, A.J.C.; de Medeiros, L.S.; Binzer, S.B.; Rasmussen, S.A.; Hansen, P.J.; Nielsen, K.F.; Jørgensen, K.; Larsen, T.O. HPLC-HRMS Quantification of the Ichthyotoxin Karmitoxin from Karlodinium armiger. Mar. Drugs 2017, 15, 278.

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