LC-MS/MS Detection of Karlotoxins Reveals New Variants in Strains of the Marine Dinoflagellate Karlodinium veneficum from the Ebro Delta (NW Mediterranean)

Abstract

Abstract:A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the detection and quantitation of karlotoxins in the selected reaction monitoring (SRM) mode. This novel method was based upon the analysis of purified karlotoxins (KcTx-1, KmTx-2, 44-oxo-KmTx-2, KmTx-5), one amphidinol (AM-18), and unpurified extracts of bulk cultures of the marine dinoflagellate Karlodinium veneficum strain CCMP2936 from Delaware (Eastern USA), which produces KmTx-1 and KmTx-3. The limit of detection of the SRM method for KmTx-2 was determined as 2.5 ng on-column. Collision induced dissociation (CID) spectra of all putative karlotoxins were recorded to present fragmentation patterns of each compound for their unambiguous identification. Bulk cultures of K. veneficum strain K10 isolated from an embayment of the Ebro Delta, NW Mediterranean, yielded five previously unreported putative karlotoxins with molecular masses 1280, 1298, 1332, 1356, and 1400 Da, and similar fragments to KmTx-5. Analysis of several isolates of K. veneficum from the Ebro Delta revealed small-scale diversity in the karlotoxin spectrum in that one isolate from Fangar Bay produced KmTx-5, whereas the five putative novel karlotoxins were found among several isolates from nearby, but hydrographically distinct Alfacs Bay. Application of this LC-MS/MS method represents an incremental advance in the determination of putative karlotoxins, particularly in the absence of a complete spectrum of purified analytical standards of known specific potency.

LC-MS/MS Method Development for the Discovery and Identification of Amphidinols Produced by Amphidinium

“…The AM profiles of these strains (ACRN02, ACRN03, CCMP1314, and DN241EHU) were dominated by one or two AM as the main components and the other AM variants only occurred in small amounts. A similar pattern of karlotoxins that are structurally very similar to AM was found in different Karlodinium strains [ 22 ]. However, the quantitative expression of AM in Amphidinium resulting in varying AM cell quotas seems to be variable as ACRN02 in this study showed a higher number and higher AM cell quota than ACRN03 ( Table 5 ), which contrast with a previous study that showed that the latter produce large amounts of LP-D and LS-A [ 11 ].…”

“…This should preferably be the adduct form that occurs at the highest abundance and thus allows a high sensitivity. In the case of LP-D and the structurally closely-related karlotoxins [ 22 ], this applies to the sodium adduct ( Figure 1 ). The stabilizing effect of the sodium ion [ 24 ] is the reason for the need for relatively high collision energies of up to 85 eV for the fragmentation of AM in the collision cell of the tandem mass spectrometer.…”

“…This work aimed to develop a standardized and sensitive analytical method for the quantification of AM as well as detection of novel AM. Similar methods have been successfully established for many shellfish toxins and, more recently, for karlotoxins [ 22 ]. To achieve the methodological development for AM, we used a purified standard of luteophanol D (LP-D) [ 11 ] to establish the method for the LC-MS/MS identification and quantification and then applied this method to other known members of the AM family.…”

Biochemical characteristics support the recently described species Karlodinium zhouanum (Gymnodiniales, Dinophyceae)

“…() and Krock et al . () were adopted with modifications. Briefly, 600 ml of each algal culture was used for toxin extraction.…”

Mass Spectrometry-Based Characterization of New Spirolides from Alexandrium ostenfeldii (Dinophyceae)

“…Tandem mass spectroscopy coupled to liquid chromatography (LC-MS/MS), as a complementary technique, can provide partial structural information and requires only several orders of magnitude lower amounts of samples due to its lower limit of detection relative to NMR. As the requirement of relatively high amounts of pure substances poses a severe limitation in many cases, much of our understanding on the structural variability of phycotoxins is based on mass spectral data [ 12 , 21 , 32 , 33 ]. LC-tandem high-resolution mass spectrometry (LC-HRMS/MS) has successfully complemented low resolution MS in the screening for unknown phycotoxins as well as in the characterization of structural information when the toxin concentration is too low for NMR analysis [ 34 ].…”