On-line immunoaffinity capture, coupled with HPLC and electrospray ionization mass spectrometry, for automated determination of fumonisins

An automated on-line method for the quantitative detection of fumonisins B-1, B-2, and B-3 and hydrolyzed fumonisin B-1 in corn-based feed was developed using a combination of immunoaffinity capture (IAC)/cleanup, coupled with reversed phase liquid chromatography and electrospray ionization mass spectrometry. Blocking the C2 amine of fumonisin B-1 with 9-fluorenylmethylchloroformate allowed coupling to keyhole limpet hemocyanin through the tricarballylic end, leaving the amino terminus exposed on the immunogenic conjugate after removal of the blocking group. Antiserum had specificity for the C1-C10 fumonisin domain and was used for ELISA and to prepare immunoaffinity columns. The quantitation limit (s/n = 10) for fumonisin B-1 standard was 250 pg using the protonated molecule signal (m/z 722). Similar sensitivity was observed for fumonisins B-2 and B-3 (m/z 706) and hydrolyzed fumonisin B-1 (m/z 406). The on-line IAC/LC/ESI-MS method provided a linear response from the detection limit to 5 ng for fumonisin B-1 and has the capability to analyze low-level contamination of rodent feed samples for fumonisins. The dynamic range can be adjusted as necessary by varying the volume of the sample injected for IAC capture.