Determination of ellagic acid in oak leaves and in sheep ruminal fluid by ion-pair RP-HPLC.

An isocratic ion-pair high-performance liquid chromatography (IP-RP-HPLC) method with UV detection was developed to identify and quantify ellagic acid (EA). This phenolic compound is widely distributed in the plants and is often present in the diet of ruminants. The method was validated and validation parameters were: linearity range 5-100 mg/L; correlation coefficient, 0.9995; mean recoveries (99.94 and 101.07%) and detection limit 1.4 mg/L. Method was applied for the determination of ellagic acid in oak leaves and in ruminal fluid from to a vitro ruminal system. The proposed method proved to be rapid and accurate and can be successfully used in ruminant nutrition studies.

Rapid and simple method for the determination of urinary benzoic and phenylacetic acids and their glycine conjugates in ruminants by reversed-phase high-performance liquid chromatography.

A simple, rapid and reproducible reversed-phase high-performance liquid chromatographic method for the simultaneous determination of benzoic acid (BA), phenylacetic acid (PAA) and their respective glycine conjugates hippuric acid (HA) and phenaceturic acid (PA) in sheep urine is described. The procedure involves only direct injection of a diluted urine sample, thus obviating the need for an extraction step or an internal standard. The compounds were separated on a Nova-Pak C18 column with isocratic elution with acetate buffer (25 mM, pH 4.5)-methanol (95:5). A flow-rate of 1.0 ml/min, a column temperature of 35 degrees C and detection at 230 nm were employed. These conditions were optimized by investigating the effects of pH, molarity, methanol concentration in the mobile phase and column temperature on the resolution of the metabolites. The total analysis time was less than 15 min per sample. At a signal-to-noise ratio of 3 the detection limits for ten-fold diluted urine were 1.0 microgram/ml for BA and HA and 5.0 micrograms/ml for PAA and PA with a 20-microliters injection.

Determination of creatinine and purine derivatives in ruminants' urine by reversed-phase high-performance liquid chromatography.

A procedure is described for the rapid and simultaneous determination of allantoin, creatinine, uric acid, hypoxanthine and xanthine in sheep urine. Separation was achieved on a Novapak C18 column under isocratic conditions. The mobile phase was potassium phosphate buffer (10 mM, pH 4.0). A flow-rate of 0.5 ml/min, detection at 218 nm and a column temperature of 25 degrees C were employed with a total analysis time of less than 15 min. Detection limits for allantoin, creatinine, uric acid, hypoxanthine and xanthine were 1.0, 0.5, 0.5, 0.5 and 0.2 micrograms/ml, respectively, at a signal-to-noise ratio of 3 in a 20-microliters injection volume of tenfold-diluted urine. This sensitivity permits the precise determination of these compounds in ruminants' urine.