HPLC determination of chlorate metabolism in bovine ruminal fluid

Abstract

Salmonella and Escherichia coli are two bacteria that are important causes of human and animal disease worldwide. Chlorate is converted in the cell to chlorite, which is lethal to these bacteria. An HPLC procedure was developed for the rapid analysis of chlorate (ClO(3)(-)), nitrate (NO(3)(-)), and nitrite (NO(2)(-)) ions in bovine ruminal fluid samples. Standard curves for chlorite, nitrite, nitrate, and chlorate were well defined linear curves with R(2) values of 0.99846, 0.99106, 0.99854, and 0.99138, respectively. Concentrations of chlorite could not be accurately determined in bovine ruminal fluid because chlorite reacts with or binds a component(s) or is reduced to chloride in bovine ruminal fluid resulting in low chlorite measurements. A standard curve ranging from 25 to 150 ppm ClO(3)(-) ion was used to measure chlorate fortified into ruminal fluid. The concentration of chlorate was more rapidly lowered (P < 0.01) under anaerobic compared to aerobic incubation conditions. Chlorate alone or chlorate supplemented with the reductants sodium lactate or glycerol were bactericidal in anaerobic incubations. In anaerobic culture, the addition of sodium formate to chlorate-fortified ruminal fluid appeared to decrease chlorate concentrations; however, formate also appeared to moderate the bactericidal effect of chlorate against E. coli. Addition of the reductants, glycerol or lactate, to chlorate-fortified ruminal fluid did not increase the killing of E. coli at 24 h, but may be useful when the reducing equivalents are limiting as in waste holding reservoirs or composting systems required for intense animal production.

Effect of dietary supplementation of rutin on lactation performance, ruminal fermentation and metabolism in dairy cows

“…Four millilitres rumen fluid and duodenal fluid were filtered with four layers of gauze and centrifuged (3824 g , 10 min). One millilitre ethyl acetate was added to 2 ml supernatant and plasma with sufficient mixing and centrifuged (3824 g , 5 min) to obtain the organic phase (Lodge‐Ivey et al., ; Beier et al., ). The organic phase was dried with nitrogen and dissolved to the mobile phase.…”

Chlorate Analyses in Matrices of Animal Origin

“…Ion chromatography with conductivity detection has been successfully used to measure parts per billion (ppb) concentrations of chlorate in drinking water 25 and, more recently, it has been used to measure the disappearance of chlorate salts from ruminal fluid cultures. 26 In the latter study, accurate measurements of chlorate were limited when chlorate concentrations were <25 ppm. For example, recoveries of analyte at 0.5 and 5 ppm were 718 and 145%, respectively.…”

“…For lamb urine, chlorate was eluted from these columns using sodium hydroxide mobile phases without interference (data not shown), but when urine from mature animals was analyzed, interferences prevented quantification of chlorate. Beier et al 26 utilized a Dionex AS9 column to quantify chlorate in diluted (1/10) and filtered, but otherwise unprocessed, ruminal fluid samples. Beier et al 26 reported recoveries of 718 and 142% at sample concentrations of 0.5 and 5 ppm, respectively.…”

“…Ion Chromatography. Because matrix components of urine completely interfered with the rapid colorimetric chlorate assay and because previous studies had indicated that very high concentrations of chlorate are excreted in urine, the ion chromatographic method of Beier et al 26 was modified for the detection of chlorate in urine. Briefly, urine samples (50-250 μL depending upon sodium chlorate content) were loaded onto Bond Elut Certify (200 mg; Varian, Lake Forest, CA) SPE cartridges conditioned with 2 mL of methanol followed by 4 mL of water.…”

Chlorate and Other Oxychlorine Contaminants Within the Dairy Supply Chain

“…Modern analytical developments have given rise to liquid chromatography with coupled mass spectral detection (LC‐MS) (Barron & Paull, ; Charles & Pepin, ), but although these methods have been developed for detection in water, their application to complex biological matrices has been explored to a lesser degree (Beier et al., ). Liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) has been developed as a multi‐residue analytical method for polar pesticides (Anastassiades et al., ; Hepperle et al., ) and has been employed by EFSA for the testing of fruits and vegetables for pesticide residues at levels of μg/kg (EFSA, ).…”