HPLC-HRMS Quantification of the Ichthyotoxin Karmitoxin from Karlodinium armiger

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.

Development of an Indirect Quantitation Method to Assess Ichthyotoxic B-Type Prymnesins from Prymnesium parvum

“…Future improvements of the method should focus on enhancing the recovery of prymnesins during the reconstitution step. Still, the apparent recovery was comparable to that achieved for similar compounds, e.g., karmitoxin [18].…”

“…Quantitative analytical methods for algal toxins that are harmful to humans, like those accumulating in shellfish, are in general well established [15]. However, only few quantitative and semi-quantitative analytical methods have been developed for ichthyotoxic metabolites [16,17,18]. Themost limiting factor in developing analytical methods is the lack of commercially available standards [19] or reliable standards at all.…”

“…Themost limiting factor in developing analytical methods is the lack of commercially available standards [19] or reliable standards at all. The compounds have to first be isolated from cultures, which is often a huge challenge due to the low natural production, unknown stability and solubility characteristics [18]. Furthermore, some ichthyotoxic metabolites have been described as “sticky”, adhering to different kinds of surfaces (especially plastics), which further challenges purification yields and especially the recovery in analytical methods [16,18,20].…”

Deeper insight into Gambierdiscus polynesiensis toxin production relies on specific optimization of high-performance liquid chromatography-high resolution mass spectrometry

“…An alternative to confirm the presence of known compounds or to detect related, unknown compounds in complex extracts is high-resolution mass spectrometry (HRMS) [34,41]. With hybrid high resolution systems such as quadrupole-time-of-flight (Q-TOF) technology, both targeted and untargeted analyses can be performed allowing for retrospective data analysis [43,44]. Compared to MRM mode performed in tandem mass spectrometry (i.e.…”

The blue mussel Mytilus edulis is vulnerable to the toxic dinoflagellate Karlodinium armiger—Adult filtration is inhibited and several life stages killed

“…armiger and only few reached the larval stage ( S4 Fig ). At present it is unknown whether karmitoxins were involved or not, but it seems highly likely, since karmitoxin has been shown to impact copepods and rainbow trout gill cells [ 49 , 51 ].…”