High-performance liquid chromatography bioassay using

Other kinds of bloassays have been used to detect the presence of specific allelochemical effects (8), effects on N2 fIxatlon (9), the presence of volatile compounds (10) and of Inhibitory substances produced by marine microalgae (11). Putnam and Duke (12) have summarized the extraction techniques and bioassay methods used In allelopathy research. Recent developments In high performance liquid chromatography (HPLC) separation of allelochemlcals from plant extracts dictates the need for bloassays with sensitivity to low concentrations of compounds contained In small volumes of eluent. Einhellig at al. (13) described a bloassay using Lemna minor L. growing In tissue culture cluster dish wells that maximizes sensitivity and minimizes sample requirements. 

Currently, all five toxic components from concavum are being subjected to column and high performance liquid chromatography. Products of these treatments will be evaluated and characterized using the mouse bioassay and isolated nerve-muscle preparations. 

Unfortunately, the dose-survival times for the DSP toxins in the mouse assay fluctuate considerably and fatty acids interfere with the assay, giving false-positive results consequently, a suckling mouse assay that has been developed and used for control of DSP measures fluid accumulation after injection of the shellfish extract. Considerable effort has been applied recently to development of chemical assays to replace these bioassays. As a result a good high performance liquid chromatography (HPLC) procedure has been developed to identify individual PSP toxins (detection limit for saxitoxin = 20 fg per 100 g of meats 0.2 ppm), an excellent HPLC procedure (detection limit for okadaic acid = 400 ngg 0.4 ppm), a commercially available immunoassay (detection limit for okadaic acid=lfg per 100 g of meats 0.01 ppm) for DSP, and a totally satisfactory HPLC procedure for ASP (detection limit for domoic acid = 750 ngg 0.75 ppm). 

High performance liquid chromatography has been used as a criterion of specificity when coupled with a bioassay or radioimmunoassay. With regard to the opioid peptides, brain extracts were chromatographed on an HPLC column that had previously been calibrated with known opioid peptides (Lewis et ai, 1979), and the collected fractions were assayed for activity (Stern et ai, 1980) (Fig. 12). The bioassay provides the quantitation and the HPLC provides the identity of each component. It would be interesting to use HPLC coupled with radioimmunoassay to confirm the identity of the many peptides that have been found in brain by immunohistochemical techniques. 

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