Gas chromatographic techniques are described for the separation and identification of fluoroacetate and fluorocitrate from fluoride-injured plant tissue and organs of animals suffering from severe fluorosis. Tissue was extracted with 9 5 z ethanol in a Soxhlet apparatus. The methyl esters of organic acids were separated on columns packed with 10% polyethylene glycol 6000 or 10% Reoplex 400 on Chromosorb W solid phase using a flame ionization detector. Evidence was presented for the occurrence of fluoroacetate and fluorocitrate in forage crops collected near a phosphate plant. The results obtained support the earlier findings suggesting that atmospheric HF may be incorporated into fluoroacetate and fluorocitrate by crested wheatgrass [Agropyron cristatum (L.) Gaerth]. luoroacetate and fluorocitrate have been shown to F occur in soybeans (Glycine max, Merr.) treated with atmospheric HF (Cheng, Yu, et al., 1968). More recently, forage crops collected near a phosphate plant were shown to contain these two compounds (Lovelace, Miller, et ai., 1968). Although these observations suggest that the biosynthesis of fluoroacetate and fluorocitrate may be a general property of plants, this has not yet been established. These analyses of the fluoroorganic compounds were conducted using paper chromatography, ashing, and fluoride determination. The concentrations reported for these compounds may be too high because of possible contamination with inorganic fluoride.Horses and cattle grazing near the above-mentioned industrial plant showed severe fluorosis. It was suspected that part of the injury might have been induced by the toxic fluoroorganic acids contained in the hay. A further investigation of the presence of fluoroorganic compounds in the organs of fluorotic animals was thus deemed of interest. Organs of these animals, control animals, crested wheatgrass growing near the phosphate plant, and control plants grown in a greenhouse were obtained and analyzed.
ProceduresMethylfluoroacetate was prepared from monofluoroacetate (Na salt, Sigma Chemical Co.) by dissolving the latter in water, acidifying with 10% H2S04, extracting with sulfuric ether, drying the ethereal extract over anhydrous sodium sulfate, and methylating by the method of Colowick and Kaplan (1963).Trimethylfluorocitrate was prepared from monofluorocitrate (Ba salt, Calbiochem.) by dissolving the latter in 1 N HCl solution, and removing the barium by addition of NazSOl followed by centrifugation. The solution was then passed through a n ion-exchange column @owex 50-X, Hf form) and the eluate evaporated to dryness in a rotary evaporator at 40' C. The residue was taken up into methanol and esterified (Colowick and Kaplan, 1963). Treatment with ion-exchange column chromatography facilitated methylation of fluorocitrate and resulted in a higher yield than otherwise.Samples of crested wheatgrass [Agropyron cristatum (L.) Gaerth] were collected within 2 miles of a n industrial phosphate plant, air-dried, and ground in a mill. Plants grown in a greenhouse in the abse...