Fast and Reproducible 96-Well Plate-Based Method for the Evaluation of the Antigerminative Potential of Plant Extracts and Phytotoxic Compounds.

With the aim of evaluating the antigerminative activity of plant extracts, a miniaturized assay using 96-well plates (WP assay) was developed and compared to the long-established assay using Petri dishes (PD assay). The WP assay yielded results comparable to those of the PD assay using an ethanolic extract of the Himalayan balsam and lawsone as a standard. It also allowed the needed volume of the test solution to be cut by half and the number of required cress seeds to be cut by more than 1.5. The WP assay was then successfully applied to various extracts of Himalayan balsam, molecules (2,4-dichlorophenoxyacetic acid (2,4-D), glyphosate, and 2-methoxy-1,4-naphthoquinone (2-MNQ)) and target seeds (radish, lettuce, and wheat). By being adapted to a 96-well plate format, the antigerminative WP assay is a promising alternative to the PD assay. Besides, its convenience and low resource consumption make it ready for accelerated and high-throughput screening, as well as automation.

Alpha-ribazole, a fluorescent marker for the liquid chromatographic determination of vitamin B12 in foodstuffs.

A method to determine the contents of free vitamin B12 in various foods by reversed phase liquid chromatography-fluorimetry is reported. It includes a purification of the samples by passage through an immunoaffinity column and a pre-column conversion of vitamin B12 into the fluorescent alpha-ribazole (successive treatments of the extract with 2.5 M sodium hydroxide (at 100 degrees C for 15 min) and alkaline phosphatase (7.5 U) at 37 degrees C and pH 8 for 16 h). An enzymatic hydrolysis prior to the purification step (pepsin at 37 degrees C and pH 4 for 3 h) made it possible to release the vitamin B12 bound to proteins and thus to obtain the total vitamin B12 contents of these foodstuffs. The method proposed for the determination of free and bound vitamin B12 gives a good recovery rate (95-100%) and a satisfactory repeatability (R.S.D.r between 1.0 and 5.4%). Owing to its low quantification limit (3 ng g(-1)) and the good resolution of the alpha-ribazole peak, it could most probably be applied to the determination of this vitamin in any foodstuff.

Fluorimetric determination of pantothenic acid in foods by liquid chromatography with post-column derivatization.

A method to determine the content of free pantothenic acid in various foods by reverse phase liquid chromatography-fluorimetry is reported. It includes a purification of the samples by successive passages through anion and cation exchange cartridges and a post-column derivatization of pantothenic acid as the fluorescent 1-alkylthio-2-alkylisoindole (reaction of beta-alanin, formed by hot alkaline hydrolysis of pantothenic acid, with orthophthaldialdehyde in the presence of 3-mercaptopropionic acid). An enzymatic hydrolysis prior to the purification step (pepsin at 50 degrees C for 3 h, then pantetheinase and alkaline phosphatase at 20 degrees C for 18 h) made it possible to release the bound pantothenic acid and thus to obtain the total Vitamin B5 content of these foodstuffs. The method proposed for the determination of free and bound pantothenic acid gives a good recovery rate (96-101%) and a satisfactory repeatability (R.S.D.r less than 8%). Owing to its low detection limit (0.65 microg g(-1)) and the good resolution of the pantothenic acid peak, it could most probably be applied to the determination of this vitamin in any foodstuff.

Determination of folates in foods by high-performance liquid chromatography with fluorescence detection after precolumn conversion to 5-methyltetrahydrofolates.

A liquid chromatographic-fluorimetric determination of folates in foodstuffs including their extraction, without or with deconjugation, chemical conversion to 5-CH3-H4PteGlu(n) and purification of the extract by affinity chromatography is reported. The conversion enables the analysis of total folates and also of the contents of the different mono- and polyglutamate forms of the folates. The method has a satisfactory day-to-day repeatability (never,more than 10%) and a very low detection limit (0.02 pmol per injection). Depending on the folate studied, the recovery rates varied from 78% (10-CHO-PteGlu) to 98% (5-CHO-H4PteGlu). Furthermore it has been possible to show that the deconjugation of the folates by rat plasma conjugase was incomplete in foodstuffs whereas chicken pancreas conjugase effectively converted the different folate polyglutamates into folate diglutamates. It could not be demonstrated that prior hydrolysis with a protease and amylase was useful for the analysis of the different foodstuffs studied (yeast, spinach, beef liver, beef fillet and peas) when deconjugation was performed with the chicken pancreas conjugase.