Determination of biological toxins using capillary electrokinetic chromatography with multiphoton excited fluorescence

We report: a highly sensitive and rapid strategy for characterizing biologi cal toxins based on capillary electrokinetic chromatography with multiphoto n-excited fluorescence, In this approach, aflatoxins B-1, B-2, and G(1) and the cholera toxin A-subunit are fractionated in similar to 80 s in a narro w-bore electrophoretic channel using the negatively charged pseudostationar y phase, carboxymethyl-beta-cyclodextrin. The aflatoxins-highly mutagenic m ultiple-ringed heterocycles produced by Aspergillus fungi-are excited at th e capillary outlet through the simultaneous absorption of two to three 750- nm photons to yield characteristic blue fluorescence; cholera toxin A-subun it, the catalytic domain of the bacterial protein toxin from Vibrio cholera , is excited through an unidentified multiphoton pathway that apparently in cludes photochemical transformation of an aromatic residue in the polypepti de, The anionic carboxymethyl-beta-cyclodextrin, used to chromatographicall y resolve the uncharged aflatoxins, enhances emission hom these compounds w ithout contributing substantially to the background. Detection limits for t hese toxins separated in 2.1-mu m-i.d. capillaries range from. 4.4 zmol (si milar to 2700 molecules) for aflatoxin B-2 to 3.4 amol for the cholera toxi n A-subunit. Larger (16-mu m-i.d.) separation capillaries provide concentra tion detection limits for aflatoxins in the 0.2-0.4 nM range, severalfold l ower than achieved in 2.1-mu m capillaries, These results represent an impr ovement of > 10(4) in mass detectability compared to previously published c apillary separations of aflatoxins and demonstrate new possibilities for th e analysis of proteins and peptides.