Separation and identification of microcystins in cyanobacteria by frit-fast atom bombardment liquid chromatography/mass spectrometry

Abstract

In order to separate and identify microcystins, a new analytical method was developed using a frit probe as an interface for fast atom bombardment mass spectral analysis of high performance liquid chromatographic (HPLC) effluents. Two types of HPLC conditions were designed for separation of standard microcystins RR, YR and LR. The HPLC conditions, for example, methanol:0.01% trifluoroacetic acid = 61:39 (containing 0.8% glycerol) as a mobile phase and 0.5 ml/min as a flow rate, provided a base line separation of standard microcystins RR, YR and LR. The HPLC conditions were also effective for separation of the nontoxic geometrical isomers of microcystins RR and LR. The total ion chromatogram of a mixture of standard microcystins showed excellent correlation with the HPLC separation using a u.v. detector. The method was subsequently applied to analysis of microcystins contained in both a culture strain and a field sample, and the procedure from toxin extraction to identification of microcystins was performed within 1 day. The mass chromatogram monitored at m/z 135 that is always observed with abundance in the FAB mass spectra of the purified microcystins, differentiated between microcystins and other types of compounds. This technique allowed the rapid identification of unknown microcystins without standard samples. Additionally, compounds other than microcystins were also found, which would not be seen by u.v. detection at 238 nm.