Domoic acid HPLC analysis

FIGURE 19.2 HPLC-fluorescence analysis of domoic acid (DA). (1) Standard DA (50 ng/mL). (2) The extract of Nitzschia navis-varingica (VSP 974-1) isolated from Vietnam, (a) Detected by UV monitor (242 nm). (b) Detected by fluorescence monitor (Ex. 264 nm, Em. 313 nm). 

FIGURE 19.6 HPLC-UV analysis of the extract of N. navis-varingica isolated from Bulacan estuary (a) and the Canadian standard (DACS-l-D) (h). 1 unknown peak, 2 same retention time as lA, 3 same retention time as tryptophan. Analytical conditions Column Develosil ODS-5 (Nomura, 4.6 mm x 250 mm), mobile phase 10% acetonitrile in 20 mM phosphate buffer (pH 2.5), column temperature 35°C, Flow rate 0.8 mL/min, UV detection 242 nm. a Elution order of peaks 1 and 2 in this analysis was reverse of the HPLC-fluorescenee analysis in which DA and isomers are derivatized with FMOC-Cl reagent. b Lack of domoic acid peak. 

To 1-2 g samples of seaweeds an equal volume of 0.1 N HCl was added, and the mixture heated in a water bath for 5 min, then filtered through filter paper. The filtrate was applied to a Sep-pak Cl 8-cartridge (Waters, Milford, MA, USA). Ten pi of eluate was submitted to HPLC analysis for domoic acid on a Tosoh HPLC equipped with a Finepack SIL 18-T-5 column (4.6 x 250 mm), using a mixture of acetonitrile, heptafluorobutyric acid, water (12 0.43 87.57) as the eluant at a rate of 1.0 ml per min. The domoic acid in the eluate was monitored at 242 nm with a Tosoh UV 8011 monitor. The quantity of domoic acid was calculated using a calibration curve of amount of authentic domoic acid (Sigma, St. Louis, MO, USA) vs absorbance at 242 nm, as follows ... 

Cells and culture broths of 25 microorganisms isolated by Dr. K. Ohwada, Ocean Research Institute, University of Tokyo, from C. armata collected in January 1993 at Yakushima were submitted to HPLC analysis for domoic acid. 

Figure 1. HPLC analysis for domoic acid of red algae (1) Chondria armata (994 ppm), (2) Coelothrix irregularis (6 ppm), (3) Digenea simplex (<1 ppm), (4) standard of domoic acid.

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