9-diazomethylanthracene and dinophysistoxin-1

Several derivatization reagents for the conversion of okadaic acid and related DSP toxins to fluorescent derivatives for analysis by liquid chromatography have been examined, viz: 9-anthryldiazomethane (ADAM), 1-pyrenyldiazomethane (PDAM), 4-diazomethyl-7-methoxycoumarin (DMMC), 4-bromomethyl-7-methoxycoumarin (BrMMC), 4-bromomethyl-7,8-benzcoumarin (BrMBC), 4-bromomethyl-7-acetoxycoumarin (BrMAC), and 4-bromomethyl-6,7-dimethoxycoumarin (BrDMC). The ADAM reagent provides the greatest selectivity and sensitivity, but its application on a routine basis has been limited by its instability and cost. Improvement of this method was achieved through the production of ADAM in situ from the stable 9-anthraldehyde hydrazone. A detection limit of 30 ng/g hepatopancreas (equivalent to 6 ng/g whole tissue) was achieved. The other aryldiazomethane reagents were found to have insufficient reactivity. Of the bromomethylcoumarin reagents, BrDMC was found to have the greatest promise. The reagent is inexpensive and has excellent stability and purity. Quantitative derivatization may be achieved in a 2 hour reaction at 45 degrees C with N,N-diisopropylethylamine as a catalyst. Unfortunately, the lower reaction selectivity of BrDMC compared to that of ADAM limits its application to isolated toxins, plankton samples, and shellfish tissues with high levels of DSP toxins. The use of BrDMC for the determination of how toxin levels in shellfish tissues will require development of a more extensive clean-up prior to derivatization. Successful application of the ADAM and coumarin derivatization methods to real-world samples has been demonstrated.

Topics: Animals; Anthracenes; Chromatography, High Pressure Liquid; Coumarins; Dinoflagellida; Ethers, Cyclic; Fluorescent Dyes; Marine Toxins; Okadaic Acid; Pyrans; Pyrenes; Shellfish; Spectrometry, Fluorescence; Temperature; Umbelliferones

Okadaic acid and dinophysistoxin-1, the principal toxic components in diarrhetic shellfish poisoning, may be detected by high-performance liquid chromatography and fluorometric measurement as 9-anthrylmethyl esters. However, "greasy" samples may occur and the fluorescent reagent 9-anthryldiazomethane may decompose during storage, resulting in impurities that may seriously interfere with quantitative determination. Ultrasonic treatment of the samples during derivatization with 9-anthryldiazomethane was found to improve reproducibility. This may result from increased access to reactive sites on toxins by 9-anthryldiazomethane due to disruption of micelles formed by toxins and other partly hydrophobic compounds. A procedure for cleaning the derivatized samples, using a 0.1 g silica cartridge column and different eluent compositions from that reported by LEE et al. (1987), was found to facilitate chromatogram interpretation. Deoxycholic acid, a commercial available bile acid, was found to be an acceptable internal standard. The 9-anthrylmethyl esters of okadaic acid, dinophysistoxin-1 and deoxycholic acid, were stable at 4 degrees C for at least seven days when stored dry or in methanol.

Topics: Animals; Anthracenes; Bivalvia; Chromatography, High Pressure Liquid; Esters; Ethers, Cyclic; Fluorescent Dyes; Marine Toxins; Okadaic Acid; Pyrans; Reproducibility of Results; Ultrasonics