CHAPTER 17 • Characterization of Marine Toxins with LC-ESI-MS
329
widely used to detect them. Recently, Cole and co-workers developed an LC-ESI-MS
method that proved to be 3 to 100 times more sensitive than LC-UV, depending on the
specific toxin to be determined (Hua et al.I995 1996). They used a microbore column,
100 x 1 mm i.d., with an eluent flow rate of 4-8 III min -I. The eluent was split into a
311 volume ratio and the two portions were conveyed respectively into a UV detector
and a mass spectrometer with electrospray ionization, so that the corresponding chromatograms could be compared to one another. When the mass analyser was scanned,
the total ion chromatogram profile was similar to that arising from UV detection, even
if some peak resolution was lost in the longer and more complex ESI interface.
Information of higher quality is gained from single ion chromatograms and ESI
mass spectra, examples of which are provided in Fig. 17.6, relative to a "red tide" seawater extract. On the right side, a series of ESI mass spectra are reported, corresponding to seven different brevetoxins. The main peak in each spectrum is provided by the
adduct ion formed between the brevetoxin and one sodium ion, present as ubiquitary
contaminant even after sample cleanup. This finding confirms the large affinity of
brevetoxins toward alkali metal ions. Other mass peaks typical of ESI correspond to
the sodiated brevetoxin dimer [2 M + Nat and a doubly-charged adduct formed by
three brevetoxin molecules and two sodium ions [3 M + 2 Na]2+. Obviously, these doubly charged ions, frequently encountered in ESI, appear at an mlz value equal to one
half of the adduct ion total mass. On the left side of Fig. 17.6, the single ion chromatograms relative to the most abundant ion of the brevetoxin spectra are plotted. The peaks
are very neat and unaffected by interferences. Their quantitation, by external calibration with authentic standards, proved accurate. In contrast, some quantitative results
determined by LC-UV were significantly higher (for example, 65 ng Ilrl instead of
18 ng Ilrl) than expected (Hua et al. 1996). These higher concentrations were attributed to interfering substances coeluting with brevetoxins. In LC-ESI-MS analysis not
only the component peaks are separated in different diagrams, but also these interferences were removed.
17.4
Saxitoxins
Saxitoxins are potent neurotoxins causing paralytic shellfish poisoning (PSP), a syndrome of variable gravity, with occasional cases of human death. They are also related
to toxic red algal blooms, from a particular species of dinoflagellates. The family of
saxitoxins comprises more than thirty closely related compounds, and new structures
are continuously identified by the use of NMR and ESI -MS techniques (Arakawa et al.
1995; Onodera et al.1997). Most important from an analytical point of view is that, while
the basic structure of saxitoxin carries two positive charges, most analogs carry one
or more easily-ionizable hydroxysulfate groups, which compensate for the positive
charge (see Fig. 17.7). As the ESI efficiency strongly depends on the ionic state of
desovated analyte ions, setting up experimental conditions (both chromatographic and
mass spectrometric) suitable for all saxitoxin analogs is extremely difficult. Traditionally, the analytical problem was solved by LC with post-column oxidation of saxitoxins and fluorescence detection. More recently, Pleasance and co-workers compared
several mass spectrometric approaches (Pleasance et al. 1992a), finding out that FIAESI-MS and FIA-ESI-MS-MS were particularly suited for the structural identification
329
widely used to detect them. Recently, Cole and co-workers developed an LC-ESI-MS
method that proved to be 3 to 100 times more sensitive than LC-UV, depending on the
specific toxin to be determined (Hua et al.I995 1996). They used a microbore column,
100 x 1 mm i.d., with an eluent flow rate of 4-8 III min -I. The eluent was split into a
311 volume ratio and the two portions were conveyed respectively into a UV detector
and a mass spectrometer with electrospray ionization, so that the corresponding chromatograms could be compared to one another. When the mass analyser was scanned,
the total ion chromatogram profile was similar to that arising from UV detection, even
if some peak resolution was lost in the longer and more complex ESI interface.
Information of higher quality is gained from single ion chromatograms and ESI
mass spectra, examples of which are provided in Fig. 17.6, relative to a "red tide" seawater extract. On the right side, a series of ESI mass spectra are reported, corresponding to seven different brevetoxins. The main peak in each spectrum is provided by the
adduct ion formed between the brevetoxin and one sodium ion, present as ubiquitary
contaminant even after sample cleanup. This finding confirms the large affinity of
brevetoxins toward alkali metal ions. Other mass peaks typical of ESI correspond to
the sodiated brevetoxin dimer [2 M + Nat and a doubly-charged adduct formed by
three brevetoxin molecules and two sodium ions [3 M + 2 Na]2+. Obviously, these doubly charged ions, frequently encountered in ESI, appear at an mlz value equal to one
half of the adduct ion total mass. On the left side of Fig. 17.6, the single ion chromatograms relative to the most abundant ion of the brevetoxin spectra are plotted. The peaks
are very neat and unaffected by interferences. Their quantitation, by external calibration with authentic standards, proved accurate. In contrast, some quantitative results
determined by LC-UV were significantly higher (for example, 65 ng Ilrl instead of
18 ng Ilrl) than expected (Hua et al. 1996). These higher concentrations were attributed to interfering substances coeluting with brevetoxins. In LC-ESI-MS analysis not
only the component peaks are separated in different diagrams, but also these interferences were removed.
17.4
Saxitoxins
Saxitoxins are potent neurotoxins causing paralytic shellfish poisoning (PSP), a syndrome of variable gravity, with occasional cases of human death. They are also related
to toxic red algal blooms, from a particular species of dinoflagellates. The family of
saxitoxins comprises more than thirty closely related compounds, and new structures
are continuously identified by the use of NMR and ESI -MS techniques (Arakawa et al.
1995; Onodera et al.1997). Most important from an analytical point of view is that, while
the basic structure of saxitoxin carries two positive charges, most analogs carry one
or more easily-ionizable hydroxysulfate groups, which compensate for the positive
charge (see Fig. 17.7). As the ESI efficiency strongly depends on the ionic state of
desovated analyte ions, setting up experimental conditions (both chromatographic and
mass spectrometric) suitable for all saxitoxin analogs is extremely difficult. Traditionally, the analytical problem was solved by LC with post-column oxidation of saxitoxins and fluorescence detection. More recently, Pleasance and co-workers compared
several mass spectrometric approaches (Pleasance et al. 1992a), finding out that FIAESI-MS and FIA-ESI-MS-MS were particularly suited for the structural identification