Page 95 - Modern Derivatization Methods for Separation Sciences

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B peak was not possible due to the presence of substrate matrix interference. Disadvantages of the
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latter two fluorescence derivatizing reagents for HPLC are formation of dual peaks with fluorescamine
and instability of the derivatives with OPA/2-ME (half-life period 1-2 min). The dual peaks from
fluorescamine resulted from the formation of the acid alcohol and the lactone derivatives of the
fluorescent complex both exhibiting identical fluorescent characteristics. To solve these problems, the
use of derivatizing reagents such as 4-fluoro-7-nitrobenzofurazan (NBD-F) (λex460 nm, λem500 nm)
[214], 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2, 1,3-benzoxadiazole (DBD-F) (λex450 nm, λem590
nm) [215], naphthalene-2,3-dicarboxaldehyde (NDA) (λex420 nm, λem500 nm) [214,216], FMOC
(λex263 nm, λem313 nm) [217], AQC (λex395 nm, λem418 nm) [218] has been considered.
Determing the usefulness of each reagent is not easy, but reacting conditions with pre-column method
are: 1 min at 60° C for NBD-F; 60 min at 60 °C for DBD-F; 30s at room temperature for FMOC; and
10 min at 55 °C for AQC, and the reacting conditions for FMOC appear to be the best. The stability of
fumonisin derivatives at room temperature is reported as: 30 min for NBD-F; 48 hours for DBD-F; 24
hours for NDA; 72 hours for FMOC and 48 hours for AQC, the use of FMOC is also excellent for
stability. However, the derivatives with FMOC require removal of excess reagents after reaction and
extraction with pentane is carried out.

The use of the post-column method with OPA/NAC was developed [219]. A derivative with OPA/NAC
is more stable than that with OPA/2-ME and the reagent has been used for the analysis of amines with
the on-column method.

Thus, various derivatization methods for fumonisins have been investigated, extraction and cleanup
procedures for samples such as corn are almost the same; after extracted with 50% acetonitrile or 75%
methanol, samples are subject to cleanup with SPE (C18 and/or SAX). A clean-up using
immunoaffinity column (Fumonitest column) specific to fumonisin B after 80% methanol extraction
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was also reported [220].

1.2.6.2—
Marine Toxins

Paralytic Shellfish Poisoning Toxins

Poisoning by seafoods contaminated with marine toxins are posing serious problems to public health
and fishery industries worldwide. Paralytic shellfish poisoning (PSP) is a potentially deadly illness
caused by ingestion of shellfish that have accumulated potent neurotoxins produced by marine algae
such as those of the genus Alexandrium. The group of compounds known as PSP toxins is composed of
saxitoxin and its derivatives. For toxin detection, derivatization of the toxins to fluorescent compounds
by oxidation under alkaline condition yields methods with high sensitivity and fairy good specificity for
the toxins [221]. This chemical assay is 100 times more sensitive than the existing bioassay, and
precolumn and post-column fluorescent derivatization has been applied to HPLC. The representative
precolumn method employs peroxide or periodate as an oxidizing agent. PSP toxins in mussel were
thermally extracted with diluted hydrochloride followed by clean-up with SPE (C18 and SAX),
oxidization with peroxide or periodate to form PSP fluorescent derivatives, separation with
reversedphase gradient HPLC and detection (λex330 nm, λem400 nm) [222,223]. The main
disadvantage of the pre-column method is that some toxins (PSP analogues) cannot be separated (e.g.,
neosaxitoxin and B , GTX-1 and GTX-4, GTX-2 and GTX-3) on HPLC due to the similar chemical
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structures of PSP oxidized products.

On the other hand, available post-column methods employ periodate as a reaction reagent [224], and

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