Satoshi Kawai

Determination of glyphosate and its metabolite, (aminomethyl)phosphonic acid, in serum using capillary electrophoresis

Capillary electrophoresis has been used to separate and quantitate glyphosate and its major metabolite, (aminomethyl)phosphonic acid (AMPA), in serum. The two compounds, after derivatization with p-toluenesulphonyl chloride, were clearly separated with 0.1 M boric acid-sodium hydroxide buffer (pH 9.6) containing 10% methanol. The separation was completed within 15 min at an applied potential of 30 kV. Calibration curves for the assay were linear over both the lower (0.5-10 micrograms/ml) and the higher (10-100 micrograms/ml) concentration ranges. The within-run and day-to-day coefficients of variation of peak area were 1.4-4.4 and 4.4-8.5%, respectively, for glyphosate and 1.8-2.9 and 1.8-2.9%, respectively, for AMPA. The within-run and day-to-day precisions of the migration time for both compounds were less than 1.8% and less than 2.5%, respectively. The detection limit of both derivatives was 0.1 microgram/ml in spiked sera, and the recoveries of glyphosate and AMPA were 87.9-88.8 and 78.4-86.9%, respectively. In this study, the reproducibility and the effect of pH changes on the electropherograms were especially examined.

Determination of malonaldehyde in oxidized biological materials by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method was used to determine the level of malonaldehyde (MA) in materials containing unsaturated fatty acids and rat liver microsomes peroxidized in vitro. The detection limit was 8.3 pmol for fatty acid samples and 25 pmol for microsomal samples. The method was specific to MA and the relative standard deviation was 4.34-5.14%. The recovery of MA was about 100%. In general, the MA values in oxidized materials obtained by the proposed HPLC method were lower than those obtained by the thiobarbituric acid method, although similar results were obtained with both methods for microsomal samples oxidized by NADPH. The effect of temperature on the HPLC results was investigated and it was found that the MA values obtained by derivatization at 25 degrees C, followed by separation using HPLC, reflected the situation of the peroxidation more accurately.

High-performance liquid chromatographic determination of glyphosate and (aminomethyl)phosphonic acid in human serum after conversion into p-toluenesulphonyl derivatives

We have developed a simple, highly sensitive and fast assay method for determining glyphosate and its major metabolite, (aminomethyl)phosphonic acid (AMPA), in serum by high-performance liquid chromatography with ultraviolet detection. Both compounds were successfully extracted with an anion-exchange resin column and allowed to react with p-toluenesulphonyl chloride. The detection limits were 0.3 microgram/ml for glyphosate and 0.2 microgram/ml for AMPA. Recoveries of glyphosate and AMPA spiked to serum were ca. 75% and ca. 88%, respectively. We are convinced that this procedure, in practice, allows medical examiners to analyse both compounds in the serum of poisoned patients within a short time.

Vibrational and NMR spectra of phenylpyruvic acid and its salts in aqueous solution

Abstract The IR, Raman and NMR spectra of phenylpyruvic acid and its sodium and lithium salts were recorded in aqueous solution. The spectral data indicate that the acid exists mostly in the hydrated keto form (gem-diol form) in acidic solution and that the sodium and lithium salt hydrates take predominantly the normal keto form in aqueous solution in contrast with those in the solid state. Also the solution of the acid and the salts contain a small amount of the enol form. A solvent isotope effect, i.e. the enol content differs between the H2O and D2O solutions of the sodium salt, was found.

Free Malondialdehyde Levels in the Urine of Rats Intoxicated with Paraquat.

We examined the excretion of free malondialdehyde (MDA) in the urine of rats to which a herbicide, Gramoxone, had been orally administered. The herbicide was administered for 2 days at a dose of 60 mg paraquat/kg body weight/day. As a result, the concentration of free MDA decreased following the intake of Gramoxone. The total amount of free MDA increased temporarily, but then it decreased significantly to below normal values. Rats that died during this experimental period did not excrete any free MDA. In the surviving animals, the MDA concentration in serum and lung microsomes decreased, while that in liver microsomes increased slightly after intake of the poison. Although the cause of the decrease in the urinary free MDA level remains unclear, the marked changes may provide valuable information regarding a toxic mechanism of paraquat intake.

Application of a metal capillary column in gas chromatographic determination of catechol-O-methyltransferase activity

The utility of a deactivated metal capillary column, Rascot, in the measurement of an enzymatic reaction, in this case measurement of rat catechol-O-methyltransferase activity, was examined. 3,4-Dihydroxybenzaldehyde, 3,4-dihydroxybenzylalcohol and 3,4-dihydroxybenzoic acid were used as substrates and the m- and p-O-methylated products were separated by using Rascot after derivatization. The peaks on the chromatograms were symmetrical. The data obtained were compared with those reported in previously published papers. Good agreement with previous results proved that Rascot is able to withstand practical use in biological materials.

High-performance liquid chromatographic determination of malonaldehyde using p-nitrophenylhydrazine as a derivatizing reagent

Abstract A high-performance liquid chromatographic procedure for the specific determination of free malonaldehyde (MA) is described. MA solution was mixed with 1 M acetate buffer (pH 3.76) and was reacted with p -nitrophenylhydrazine (NPH) hydrochloride solution in ethanol. An aliquot of the reaction mixture was injected into a C 18 -5 column with a mobile phase consisting of acetonitrile-isopropanol-0.01 M sodium dihydrogenphosphate (30:10:60, v/v/v) and detection at 315 nm. MA reacted readily with NPH in a weakly acidic medium at room temperature, giving 1-( p -nitrophenyl)pyrazole. The method is specific for free MA. The response was linear in the range 36–720 ng/ml of MA and the detection limit was 6 ng/ml with a 20-μl injection.

Improved direct injection method and extractive methylation method for determination of valproic acid in serum by gas chromatography

Two simple methods for the determination of valproic acid in serum by gas chromatography are described. One is a direct injection method. To the serum sample are added an acetonitrile solution including an internal standard and hydrochloric acid. The sample is deproteinized and after centrifugation valproic acid in the supernatant is measured in the free form by direct injection into the gas chromatograph. The other is an extractive methylation method. To the serum sample are added an internal standard solution and a counter-ion solution, and the mixture is extracted into methylene chloride containing methyl iodide. Valproic acid is extracted into methylene chloride and simultaneously the methylation reaction proceeds. After centrifugation, an aliquot of the lower layer without evaporation is injected into the gas chromatograph. Both methods have been applied successfully to monitoring routine serum levels of valproic acid.

Combined action of paraquat and superoxide on the peroxidation of detergent-dispersed linolenic acid

We investigated the peroxidative effect of paraquat and active oxygens on detergent-dispersed linolenic acid in phosphate buffer (pH 7.5) from the malondialdehyde (MDA) level. Our complete system and further inclusion of catalase were effective in stimulating MDA formation. On the other hand, xanthine oxidase (XOD) or paraquat omission, superoxide dismutase (SOD) inclusion or anaerobic incubation inhibited the formation of MDA. Ferrous ion was weakly associated with phosphate of the buffer, forming a complex, and the release of ferrous ion from the complex intensified the MDA levels with the complete and catalase inclusion systems. The electron paramagnetic resonance (EPR) spectra using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) showed that superoxide, produced immediately after the addition of XOD, played a crucial role. We could obtain a DMPO-OOH signal at the starting stage whenever MDA stimulation was observed. The omission of paraquat, however, produced no increase in MDA level in spite of an appearance of DMPO-OOH signal, indicating that paraquat also plays an important role. On the other hand, Desferal, a ferric chelator, showed a concentration-dependent inhibition effect. There was an immediate strong intensity of DMPO-OOH and paraquat signals. We did not, however, observe MDA stimulation at 250 microM Desferal, which confirms that ferrous ion plays an essential role in the lipid peroxidation. These results indicate a combined action of paraquat (or its radical) and superoxide on the accessibility of ferrous ion, including its release from the complex with phosphate, which may be an endogenous chelator. The possibility of ternary complex participation is also discussed.

High-performance liquid chromatographic separation of p-hydroxyphenylpyruvic acid

Abstract This paper reports the development of a reliable method for the separation and determination of p -hydroxyphenylpyruvic acid by high-performance liquid chromatography. An aqueous solution of p -hydroxyphenylpyruvic acid was injected into a Cosmosil C 18 -5 column with a mobile phase consisting of acetonitrile-acetic acid-water (18:1:81, v/v/v) with detection at 283 nm. Three peaks were observed on the chromatogram, and were identified as the keto-enol tautomers and the decomposition product p -hydroxybenzaldehyde. The enol form and p -hydroxybenzaldehyde were determined without derivatization and the keto form as its oxime from their peak heights by comparison with those of known amounts of the enol form, p -hydroxybenzaldehyde and the oxime standard of the keto form.