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

We report a highly sensitive and rapid strategy for characterizing biological toxins based on capillary electrokinetic chromatography with multiphoton-excited fluorescence. In this approach, aflatoxins B₁, B₂, and G₁ and the cholera toxin A-subunit are fractionated in ~80 s in a narrow- bore electrophoretic channel using the negatively charged pseudostationary phase, carboxymethyl-β-cyclodextrin. The aflatoxins-highly mutagenic multiple-ringed heterocycles produced by Aspergillus fungi-are excited at the capillary outlet through the simultaneous absorption of two to three 750-nm photons to yield characteristic blue fluorescence; cholera toxin A-subunit, the catalytic domain of the bacterial protein toxin from Vibrio cholera, is excited through an unidentified multiphoton pathway that apparently includes photochemical transformation of an aromatic residue in the polypeptide. The anionic carboxymethyl-β-cyclodextrin, used to chromatographicarly resolve the uncharged aflatoxins, enhances emission from these compounds without contributing substantially to the background. Detection limits for these toxins separated in 2.1-μm-i.d. capillaries range from 4.4 zmol (~2700 molecules) for aflatoxin B₂ to 3.4 amol for the cholera toxin A-subunit. Larger (16-μm-i.d.) separation capillaries provide concentration detection limits for aflatoxins in the 0.2-0.4 nM range, severalfold lower than achieved in 2.1-μm capillaries. These results represent an improvement of > 10⁴ in mass detectability compared to previously published capillary separations of aflatoxins and demonstrate new possibilities for the analysis of proteins and peptides.