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Mar Drugs
2017 Aug 31;159:. doi: 10.3390/md15090278.
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HPLC-HRMS Quantification of the Ichthyotoxin Karmitoxin from Karlodinium armiger.
Andersen AJC
,
de Medeiros LS
,
Binzer SB
,
Rasmussen SA
,
Hansen PJ
,
Nielsen KF
,
Jørgensen K
,
Larsen TO
.
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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.
Figure 1. Performance of sample preparation procedures. Integrated peak areas of karmitoxin (peak area of the monoisotopic isotopomer from the most abundant adduct, [M + H]+), from HPLC-QTOF-MS analysis of different sample preparation methods and relative standard deviations of the five replicates (three replicates for solid phase extraction methods, indicated collectively with green bars) from each sample preparation procedures.
Figure 2. Combined integrated peak areas of monitored adducts for each of the external standards: chloramphenicol ([M + H]+, [M + Na]+, [3M + Fe(II)]2+, [2M − 2H + Fe(III)]+) and valinomycin ([M + H]+, [M + NH4]+, [M + Na]+), monitored during the HPLC-QTOF-MS analysis of samples prepared by lyophilisation, and by solid phase extraction Strata-X.
Figure 4. K. armiger culture grown in ammonium-substituted f/2 media: a comparison of culture cell concentration (cells·mL−1) to the concentration of karmitoxin (μg·mL−1). The concentration of karmitoxin correlated with cell concentration, a linear regression of karmitoxin per cell had an R2 value of 0.84 across all growth phases (Figure S2). The displayed standard deviation for each sample day is the standard deviation across all of the biological replicates. The average relative standard deviation across all sample points was 20%.
Figure 5. K. armiger culture grown in f/2 media in the presence of Rhodomonas salina as a food source: a comparison of cell concentration (cells·mL−1) to the concentration of karmitoxin (μg·mL−1). As with the cultures grown on ammonium-substituted f/2 media, the concentration of karmitoxin correlated with cell concentration, a linear regression of karmitoxin per cell had an R2 value of 0.93 across all growth phases (Figure S3). The stationary growth phase appeared to begin at day 8, two days later than that of K. armiger grown in ammonium-substituted f/2 media. The displayed standard deviation for each sample day is the standard deviation across all of the biological replicates. The average relative standard deviation across all biological replicates was 10%.
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