High‐performance liquid chromatographic separation of microcystins derivatized with a highly fluorescent dienophile

Abstract

AbstractMicrocystins are potent hepatotoxins produced by cyanobacteria, and are also tumor promoters as well as potent inhibitors of the catalytic subunits of protein phosphatases 1 and 2A. In order to establish a physicochemical method for individual detection and determination of trace amounts of microcystins, we developed a derivatization method for fluorescence (FL) and chemiluminescence (CL) detection, in which a highly fluorescent dienophile, DMEQ‐TAD (4‐[2‐(6,7‐dimethoxy‐4‐methyl‐3‐oxo‐3,4‐dihydroquinoxalinyl) ethyl]‐1,2,4‐triazoline‐3,5‐dione), was used as the labeling reagent. DMEQ‐TAD reacted smoothly with the conjugated diene of the Adda moiety to give 2 stereoisomers of the adducts. As a result of the extensive experiments, the following reaction conditions were optimized for the labeling: sample amount, 10 μg; reaction solvent, DMF:acetonitrile (1:1); reaction time, 15 minutes; reaction temperature, 70°C; amount of DMEQ‐TAD used relative to that of microcystin, 80 equivalent. The resulting 6 adducts from microcystins‐LR, ‐YR, and ‐RR can be separated from one another using the following reversed phase HPLC conditions in combination with a clean‐up using ODS silica gel: column, Cosmosil 5C18‐AR (150 × 4.6 I.D. mm); mobile phase, methanol:0.05M phosphate buffer (pH 3) (1:1); flow rate, 1.0 ml/min; detection, FL λex 370 nm, λem 440 nm. The detection limits of the DMEQ‐TAD derivatives were estimated to be 100 and 500 pg for LR, and 65 and 2,500 pg for RR using FL and CL detections, respectively; and the detection behavior was different from that of the Dns‐Cys derivatives, which were more sensitive to CL than FL. Nat. Toxins 5:201–207, 1997. © 1998 Wiley‐Liss, Inc.