M. Vincenti . A. Irico
Sometimes, only one compound is responsible for the toxicity, while in other cases
several molecules with distinct structures contribute to the overall poisoning effect,
making toxicological evaluations problematic. Therefore, the traditional bioassay tests
based on acute effects in mice and rats proved to be scarcely sensitive, unspecific and
occasionally unreliable. The analytical methods initially developed were based on the
derivatization of toxins by fluorescent markers followed by LC with fluorimetric detection (Quilliam 1995; Shen et al. 1997). The refinement and increased availability of
LC-ESI-MS made this intrumentation the analytical technique of choice for the identification and detection of DSP toxins, since it is more sensitive and specific than fluorimetric detection,does not require derivatization pretreatments and allows much
higher pre concentration factors (Pleasance et al. 1992b). Figure 17.2 provides a clear
picture of the superior MS selectivity, which is reflected in an improved SIN ratio and,
consequently, in extremely high sensitivity.
Most experimental work has been addressed to the detection of OA and its isomers,
homologues and derivatives collectively defined DTXs, since most of DSP cases proved
to be related to the presence of these toxins. The kind of samples considered were either mussels (Marr et al. 1992; Pleasance et al. 1992b; Draisci et al. 1995; Quilliam 1995;
James et al. 1997b; Draisci et al.1998b) or marine phytoplankton specimen (James et al.
1997a; Draisci et al. 1998a; Morton et al. 1998). Since the mussels are generally available in large amounts and tend to accumulate the toxins, the analysis of their hepatopancreas apparatus is relatively simple and does not require extensive cleanup, provided that LC separation is combined with a selective detection method, such as ESIMS (Draisci et al. 1995; Draisci et al. 1998b). In this case, a conventional 4.6 mm LC
column has been generally used. By means of a rather complex sample treatment (including up to five different chromatographic steps (Draisci et al. 1998a», the single
toxins were isolated and subsequently used as analytical standards, whenever not commercially available. Since the analysis of single compounds did not require LC separation, they were generally flow-injected into the ESI-MS apparatus in order to optimize the experimental parameters (solvent flow rate, nebulizer gas pressure, tip voltage, skimmer voltage) (Quilliam 1995; Draisci et al. 1995). In contrast to mussels, pure
phytoplankton specimen are generally available in limited amount, since various phytoplankton species are generally mixed together, especially when they can not be cultured in a laboratory. Draisci, James and co-workers were able to analyse single phytoplankton species by picking individual cells from a microscope slide (James et al.
1997a; Draisci et al. 1998a). The resulting extract, available in a very small amount, was
subsequently analysed by micro-LC-MS-MS, requiring only 0.2111 injection.
It should be noted that the decreased amount of sample loaded in micro-LC (approximately 100 times lower than in conventional LC) is compensated for by a largely
improved ionization and ion-trasmission efficiency associated with the low solvent
flow rate of micro-LC, resulting in comparable sensitivities, in terms of analyte concentration (Pleasance et al. 1992b; Quilliam 1995; James et al. 1997a).
The ESI positive ion mass spectra of OA and DTXs are characterized by an abundant protonated molecular ion and a series of fragments corresponding to consecutive losses of water molecules, due to the presence of several labile hydroxyl groups in
the toxin structures. The relative abundance of such fragment ions strongly depends
on the experimental parameters adopted, particularly the skimmer voltage, which can
induce the molecular ion dissociation (Marr et al.1992). The selective detection of these
Sometimes, only one compound is responsible for the toxicity, while in other cases
several molecules with distinct structures contribute to the overall poisoning effect,
making toxicological evaluations problematic. Therefore, the traditional bioassay tests
based on acute effects in mice and rats proved to be scarcely sensitive, unspecific and
occasionally unreliable. The analytical methods initially developed were based on the
derivatization of toxins by fluorescent markers followed by LC with fluorimetric detection (Quilliam 1995; Shen et al. 1997). The refinement and increased availability of
LC-ESI-MS made this intrumentation the analytical technique of choice for the identification and detection of DSP toxins, since it is more sensitive and specific than fluorimetric detection,does not require derivatization pretreatments and allows much
higher pre concentration factors (Pleasance et al. 1992b). Figure 17.2 provides a clear
picture of the superior MS selectivity, which is reflected in an improved SIN ratio and,
consequently, in extremely high sensitivity.
Most experimental work has been addressed to the detection of OA and its isomers,
homologues and derivatives collectively defined DTXs, since most of DSP cases proved
to be related to the presence of these toxins. The kind of samples considered were either mussels (Marr et al. 1992; Pleasance et al. 1992b; Draisci et al. 1995; Quilliam 1995;
James et al. 1997b; Draisci et al.1998b) or marine phytoplankton specimen (James et al.
1997a; Draisci et al. 1998a; Morton et al. 1998). Since the mussels are generally available in large amounts and tend to accumulate the toxins, the analysis of their hepatopancreas apparatus is relatively simple and does not require extensive cleanup, provided that LC separation is combined with a selective detection method, such as ESIMS (Draisci et al. 1995; Draisci et al. 1998b). In this case, a conventional 4.6 mm LC
column has been generally used. By means of a rather complex sample treatment (including up to five different chromatographic steps (Draisci et al. 1998a», the single
toxins were isolated and subsequently used as analytical standards, whenever not commercially available. Since the analysis of single compounds did not require LC separation, they were generally flow-injected into the ESI-MS apparatus in order to optimize the experimental parameters (solvent flow rate, nebulizer gas pressure, tip voltage, skimmer voltage) (Quilliam 1995; Draisci et al. 1995). In contrast to mussels, pure
phytoplankton specimen are generally available in limited amount, since various phytoplankton species are generally mixed together, especially when they can not be cultured in a laboratory. Draisci, James and co-workers were able to analyse single phytoplankton species by picking individual cells from a microscope slide (James et al.
1997a; Draisci et al. 1998a). The resulting extract, available in a very small amount, was
subsequently analysed by micro-LC-MS-MS, requiring only 0.2111 injection.
It should be noted that the decreased amount of sample loaded in micro-LC (approximately 100 times lower than in conventional LC) is compensated for by a largely
improved ionization and ion-trasmission efficiency associated with the low solvent
flow rate of micro-LC, resulting in comparable sensitivities, in terms of analyte concentration (Pleasance et al. 1992b; Quilliam 1995; James et al. 1997a).
The ESI positive ion mass spectra of OA and DTXs are characterized by an abundant protonated molecular ion and a series of fragments corresponding to consecutive losses of water molecules, due to the presence of several labile hydroxyl groups in
the toxin structures. The relative abundance of such fragment ions strongly depends
on the experimental parameters adopted, particularly the skimmer voltage, which can
induce the molecular ion dissociation (Marr et al.1992). The selective detection of these