J. A. Resines

Simultaneous determination of allantoin and creatinine in urine by a rapid reversed-phase liquid-chromatographic method

Abstract A simple, rapid, reproducible RP-HFLC method for a simultaneous analysis of allantoin and creatinine in sheep urine, is described. Separation was achieved on a Novapak-C18 (3.9×300 mm) column by isocratic elution. Quantitation and detection limits were determined. Detection was effected at 218 nm. Retention times of allantoin and creatinine were 4.4 and 5. 8 min, respectively. The proposed RP-HPLC method was verified for linearity, accuracy, precision and applicability.

Quantitative estimation of urinary metabolites in ruminants by high-performance liquid chromatography

Abstract A reversed-phase high-performance liquid chromatographyc method for a simultaneous determination of cretinine (C); Uric Acid (A); Hipoxanthine (HX); Xanthine (X); Hippuric Acid (HA); Benzoic Acid (BA) and Phenylacetic Acid (PAA) in urine of ruminants is described. Separation was achieved on a Nova-PaK column by-gradient elution. Quantitation and detection limits were determined Detection is effected by UV absorption at 254 nm with a total analysis time of less than 30 min. An aliquot of diluted urine is injected directly into the liquid chromatographic column. The proposed HPLC method was verified for linearity, accuracy, precision and applicability.

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.

High-performance liquid chromatographic determination of phenolic acids isolated from plants

Abstract A simple and rapid method is described for the estimation by high-performance liquid chromatography of cis and trans isomers of the substituted cinnamic acids, p-coumaric (p-CA) and ferulic (FA), using p-anisic acid (p-AA) as internal standard. Chromatographic separation and quantification were performed on a reversed-phase Nova-Pak C18 column with isocratic elution water-n-butanol-acetic acid (98:1.5:0.5). A flow-rate of 1.5 ml/min, a column temperature of 35°C and detection at 270 nm were employed. As little as 175ng/ml for p-CA and 63 ng/ml for FA can be estimated by this procedure with a 20 μl injection. Application of this method are illustrated by estimation of these acids in extracts, prepared with 1 M sodium hydroxide of barley straw, mixed grass hay and alfalfa hay.

Determination of phenolic compounds derived from hydrolysable tannins in biological matrices by RP-HPLC.

An RP-HPLC method for the determination of four phenolic compounds: gallic acid (GA), pyrogallol (PY), resorcinol (RE) and ellagic acid (EA), derived from hydrolysable tannins is reported. Separation was achieved on a SunFire C18 (250 x 4.6 mm id, 5 microm) column at 40 degrees C with gradient elution. UV detection at 280 nm was applied. The developed method was validated in terms of linearity, accuracy and precision. Satisfactory repeatability and between day precision were noticed with RSD values lower than 3%. Recoveries from different biological samples ranged from 91.50 to 105.25%. The LODs were estimated as 1.70 mg/L for PY, 1.68 mg/L for GA, 1.52 mg/L for RE and 0.98 mg/L for EA with a 20 microL injection volume. The method was applied for the determination of these compounds in oak leaves and in ruminal fluid and urine samples taken from beef cattle fed with oak leaves. The proposed method could be used in ruminant nutrition studies to verify the effect that a diet rich in tannins have on ruminal fermentation and to determine the toxicity of these compounds.

ION-PAIR REVERSED-PHASE HPLC DETERMINATION OF CREATININE IN URINE

high performance liquid chromatography (HPLC) with isocratic ion-pair-reversed-phase separation and UV detection at 230 nm is proposed for the determination of creatinine in urine. The analysis is achieved on a C18-Symmetry column (4 μm, 150mm × 3.9mm ID) using a mixture of 10 mM phosphate buffer pH=4, containing 3mM 1-octane-sulfonic acid, sodium salt and methanol (97:3). A flow-rate of 0.5mL/min and a column temperature of 30°C were employed. We validated the method for linearity, precision, and accuracy and the results were found to be excellent. The procedure proposed here is an alternative to less specific colorimetric methods.

Statistical Evaluation of Agreement Between HPLC and Colorimetric Methods for Analysis of Allantoin in Ruminants' Urine

Abstract In this study we compare an HPLC method for the determination of allantoin developed by us [1] with the traditional colorimetric method according to the Rimini-Schryver reaction modified by Fujihara [2]. Results were compared by using product-moment correlation (r = 0-962) as a measure, and by using the interclass correlation as the correct statistic for assesing agreement between both of them (r1 = 0.957, lower limit = 0.916) for a 95% of confidence interval.