2-4-dinitrophenylhydrazine and glyoxylic-acid

A method is reported for determination of allantoin in urine and plasma based on high-performance liquid chromatography (HPLC) and pre-column derivatization. In the derivatization procedure, allantoin is converted to glyoxylic acid which forms a hydrazone with 2,4-dinitrophenylhydrazine. The hydrazone appears as syn and anti isomers at a constant ratio. These derivatives are separated by HPLC using a reversed-phase C18 column from hydrazones of other keto acids possibly present in urine and plasma and then monitored at 360 nm. All components were completely resolved in 15 min. Both the reagents and derivatization products are stable. Recovery of allantoin added to urine and plasma was 95 +/- 3.7% (n = 45) and 100 +/- 7.5% (n = 64), respectively. The lowest allantoin concentration that gave a reproducible integration was 5 mumol/l. The between-assay and within-day coefficients of variation were 2.8 and 0.6%, respectively.

Topics: Allantoin; Animals; Chromatography, High Pressure Liquid; Glyoxylates; Hydrazones; Keto Acids; Phenylhydrazines; Reproducibility of Results

A high-performance liquid chromatographic method was developed for the determination of glyoxylate in the liver. Alpha-keto acids in charcoal-treated acid-extract of the liver were converted to the corresponding 2,4-dinitrophenylhydrazones and purified as the derivatives by successive extractions with ethyl acetate and sodium bicarbonate solution. The dinitrophenylhydrazones were then quantitatively converted to the corresponding substituted 2-hydroxyquinoxalines by reaction with o-phenylenediamine, followed by analysis by high-performance liquid chromatography with fluorescence detection. As a control to correct the recovery of tissue glyoxylate, an acid-extract of the liver prepared with the addition of standard glyoxylate (25-50 nmol/g wet weight of tissue) was simultaneously subjected to the analytical procedure. The maximum sensitivity of the glyoxylate measurement as 2-hydroxyquinoxaline (the quinoxaline derivative corresponding to glyoxylate) was defined as the peak area reading five times as high as the blank value obtained without sample and was approximately 10 pmol per injection. Glyoxylate in the addition compound with tris(hydroxymethyl)aminomethane was quantitatively recovered as 2-hydroxyquinoxaline. The addition compounds of glyoxylate with bisulfite and cysteine did not react with 2,4-dinitrophenylhydrazine under the conditions employed and were not detectable as glyoxylate by this method, while the adduct-forming substances added to the acid-extract of the liver did not interfere with the glyoxylate determination. No glyoxylate was detected when the liver extract had been incubated at neutral pH with a large excess of cysteine, indicating that little artificial production of glyoxylate occurred during the analytical procedure. Among 64 compounds tested for possible artificial production of glyoxylate or possible interference with the chromatographic determination of 2-hydroxyquinoxaline, p-hydroxyphenylpyruvate was the only compound which was converted to glyoxylate during the procedure. However, p-hydroxyphenylpyruvate was easily removed from the acid-extract of the tissue by charcoal treatment. The amount of glyoxylate in the liver of fasted rat was measured by the present method to be approximately 5 nmol per g of wet weight.

Topics: Animals; Chromatography, High Pressure Liquid; Glyoxylates; Liver; Male; Phenylenediamines; Phenylhydrazines; Rats; Rats, Inbred Strains