Determination of Underivatized Fumonisin B₁ and Related Compounds by HPLC

Abstract

A method is presented for determining the purity of the mycotoxin fumonisin B₁ (FB₁) by high performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD). The ELSD is a universal HPLC detector that exhibits a non-linear relationship between analyte amount and the resulting response. A log-log plot of ELSD response with the mass of FB₁ injected was used as a calibration curve for determining the quantities of both FB₁ and also individual impurities present in samples. Assumptions related to the uniformity of ELSD response for different but related compounds and other issues implied in this use of ELSD data were examined.

One potential error produced by use of this method for purity analysis comes from the ELSD’s decreased sensitivity for low-concentration analytes. Because analytes become more dilute the longer they remain on a Chromatographic column, this sensitivity discrimination can be related to the retention times at which they appear. The ELSD response for FB₁ at retention time 15.5 minutes was used to construct a general purpose calibration curve. Whenever a peak appeared at any time other than 15.5 minutes, the discrimination effect was corrected using a an empirically determined weighting factor and a proportion calculated from the retention time difference compared to 15.5 minutes.

Purities for two fumonisin samples were calculated using both the ELSD method described above and an electrospray/mass spectrometric method. The quantitative assumptions underlying each method were discussed in order to understand and reconcile differences between the two sets of purity values obtained.