Microassay for GM1 ganglioside beta-galactosidase activity using high-performance liquid chromatography

A simple and sensitive assay for GM1 ganglioside (GM1) beta-galactosidase activity was devised by direct measurement of released D-galactose using high-performance liquid chromatography (HPLC). GM1 beta-galactosidase activity in crude samples such as brain homogenates could be measured by this method. After incubation of brain homogenate for 1 h with GM1 at 37 degrees C and pH 4.4 in the presence of sodium taurodeoxycholate, the reaction was terminated by heating at 100 degrees C for 2 min and the supernatant from the centrifuged sample was analysed directly by HPLC. D-Galactose isolated by HPLC was converted into a fluorescent compound by a post-column reaction with arginine at 150 degrees C and the fluorescence intensity at 430 nm was measured with excitation at 320 nm. By this method 10 pmol of D-galactose could be measured and the fluorescence intensity was linear up to 1 mmol of D-galactose. Using this method, the optimal conditions for the activity of this enzyme were re-examined. As an application, the enzyme activity in the brain of a patient with GM1 gangliosidosis was examined. This method can be applied to any natural substrates, glycolipids or glycoproteins, the terminal galactose of which is hydrolysed by this enzyme.     

Highly sensitive assay for gamma-glutamyltranspeptidase activity by high-performance liquid chromatography with electrochemical detection

A highly sensitive assay for gamma-glutamyltranspeptidase activity involving high-performance liquid chromatography (HPLC) with electrochemical detection was devised. gamma-Glutamyl-DOPA, a new synthetic dipeptide, which consists of naturally occurring amino acids, was found to be a good substrate for gamma-glutamyltranspeptidase purified from Proteus mirabilis. Enzymatically formed DOPA was adsorbed on an aluminium oxide column, eluted with 0.5 M hydrochloric acid and determined by HPLC with electrochemical detection. The sensitivity limit of this method was 0.5 pmol of DOPA formed. Some properties of gamma-glutamyltranspeptidase purified from P. mirabilis were investigated using gamma-glutamyl-DOPA as a substrate. In the presence of 0.15 M glycylglycine, the KM value of the enzyme for gamma-glutamyl-DOPA was 0.013 mM, and the maximum velocity was 247 nmol/min per mg protein. This method was applied to the assay of the enzymatic activity in human serum.     

Highly sensitive assay for choline acetyltransferase activity by high-performance liquid chromatography with electrochemical detection

A highly sensitive assay for choline acetyltransferase activity by high-performance liquid chromatography with electrochemical detection was devised. This assay method is based on the separation of acetylcholine and choline on a Develosil Ph-5 reversed-phase column (a phenyl column), followed by their enzymatic conversion to hydrogen peroxide through post-column reaction with acetylcholinesterase and choline oxidase. The sensitivity of the system is excellent and 5 pmol of acetylcholine enzymatically formed could be detected. The linearity between the peak height and the amount of acetylcholine was observed over the range of 5 pmol to 5 nmol. Some enzymatic properties were investigated by using a soluble fraction of bovine caudate nucleus as enzyme. The Michaelis constants of the enzyme for choline and acetyl coenzyme A were 0.3 mM and 0.03 mM, respectively. The enzyme exhibited the maximum activity over the pH range 7.4-9.5. The regional distribution of choline acetyltransferase activity in rat brain was examined. The order of the activity from the highest to the lowest agreed with the reported brain distribution of the enzyme: striatum, pons plus medulla oblongata, cerebral cortex, thalamus plus hypothalamus, olfactory bulb and cerebellum.     

Gunshot residue determination by high-performance liquid chromatography with electrochemical detection

A procedure for the detection of gunshot residue via the organic constituent diphenylamine is described. The method incorporates high-performance liquid chromatography with electrochemical detection.     

Determination of platelet monoamine oxidase activity by high-performance liquid chromatography with electrochemical detection.

A procedure for determining human platelet monoamine oxidase (MAO) with dopamine (DA) as substrate is described. High-performance liquid chromatography (HPLC) with electrochemical detection (ED) was used to separate and detect components of the reaction mixture. The method for platelet preparation was also improved and only 2 ml of blood were required. Following a 10-min incubation of the platelet preparation with DA in 0.1 M Tris buffer (pH 9.0), excess DA substrate was removed by adsorption on a cation-exchange resin. The reaction product, 3,4-dihydroxyphenylacetaldehyde, was adsorbed on acid-washed alumina, eluted with 0.1 M perchloric acid and analyzed by HPLC. Simple, clean chromatograms were obtained with good reproducibility using 3,4-dihydroxybenzylamine as an internal standard. The within-sample, between-samples and between-day relative standard deviations were 0.9, 3.7 and 6.1%, respectively. The apparent Michaelis constant and maximum velocity were 0.10 mM and 0.37 nmol/min.mg protein, respectively. This HPLC-ED method offers a good alternative to methods using radioactivity.     

Sensitive derivatization reagents for thiol compounds in high-performance liquid chromatography with electrochemical detection

Three N-substituted maleimides were tested as derivatizing reagents; N-(4-anilinophenyl) maleimide (APM) was the most favorable in terms of sensitivity and reactivity. N-Acetyl-l-cysrteine, glutathione, l-cysteine and d-penicillamine were readily converted into the adducts with APM. Picogram levels of these thiol compounds were separated and quantified.     

Analysis of vinca alkaloids in plasma and urine using high-performance liquid chromatography with electrochemical detection

The reversed-phase high-performance liquid chromatography with electrochemical detection was used to quantify plasma and urine levels of vinblastine, vincristine, vindesine and a metabolite of vinblastine, desacetylvinblastine. Sample clean-up consisted of solid-phase extraction with a Bond Elut CN column. The extracts were separated on a Hypersil ODS column. The mobile phase consisted of a mixture of methanol and 10 mM phosphate buffer (pH 7.0). The limit of sensitivity using electrochemical detection was 100 pg on-column for all compounds with a signal-to-noise ratio of 3. Quantification of the compounds in human plasma and urine was possible down to 1 ng/ml (ca. 1 pmol). Pharmacokinetic results show that the sensitivity of the method is adequate for drug monitoring in clinical research.     

Determination of vitamin B6 using high-performance liquid chromatography with electrochemical detection

A chromatographic method for the determination of vitamin B₆ utilizing a thin-layer amperometric detector with a glassy carbon electrode has been described. The redox behavior of vitamin B₆ has been studied by means of cyclic voltammetry. The limit of detection is 4 ng for pyridoxine and 1 ng for pyridoxamine. Application of the technique for detection of vitamin B₆ in drugs was illustrated.     

Sensitive ferrocene reagents for derivatization of amines for high-performance liquid chromatography with electrochemical detection

Six reagents possessing ferrocene as an electrophore were prepared and evaluated for pre-column derivatization of amino compounds for their determination by high-performance liquid chromatography with electrochemical detection. The utility of these reagents was investigated employing phenethylamine as a model compound. Among these six, N-succinimidyl 3-ferrocenylpropionate was the best with respect to reactivity, stability and electrochemical properties. The developed method was applied to the determination of putrescine formed from ornithine by ornithine decarboxylase.     

Sensitive method for measuring hydrolysis of enkephalins in plasma, using high-performance liquid chromatography with electrochemical detection

This paper describes a simple and sensitive method for detection of [Leu]- and [Met]enkephalin and their N-terminal tyrosine-containing metabolic fragments (Tyr, Tyr-Gly, Tyr-Gly-Gly, and Tyr-Gly-Gly-Phe), using high-performance liquid chromatography with electrochemical detection. The method employs a carbon graphite working electrode with increased working electrode surface area (40 mm2). The procedures were applied to assay of the activities of enkephalin-degrading enzymes in whole plasma collected from rats, mice, and chicks.     

Determination of triazine pesticides by high-performance liquid chromatography with swept-potential electrochemical detection

A swept-potential electrochemical detector, operating in the square-wave voltammetric mode, is used to detect a mixture of five triazine pesticides separated on a reverse-phase resin column. Limits of detection are below 1 ng injected. Two compounds, not completely separated by the column, are resolved on the potential axis.     

Determination of diclofenac in plasma by high-performance liquid chromatography with electrochemical detection

A reversed-phase high-performance liquid chromatographic method with electrochemical detection for the quantitative determination of diclofenac potassium in plasma was developed. Naproxen was used as the internal standard. The drug and internal standard were isolated from plasma by extraction with dichloromethane and 2 M hydrochloric acid. Chromatographic separation was performed on a C18 column with methanol-water (68:32, v/v) adjusted to pH 3.2 with phosphoric acid as mobile phase. The oxidation potential for detection was established by constructing a voltammogram for diclofenac. The quantification limit for diclofenac in plasma was 5 ng mL(-1). Linearity of the method was confirmed in the range 5-2000 ng mL(-1), correlation coefficient 0.9998. Within-day relative standard deviations (RSDs) ranged from 0.66 to 14.00% and between-day RSDs from 0.59 to 15.78%. The method was successfully applied for the determination of pharmacokinetic parameters after ingestion of a 50 mg dose of diclofenac. Studies were performed on 18 healthy volunteers of both sexes.