Determination of alpha-tocopherol and alpha-tocopherylquinone in rat tissues and plasma by high-performance liquid chromatography with electrochemical detection

Alpha-tocopherol and alpha-tocopherylquinone in rat tissues and plasma were determined simultaneously by using high-performance liquid chromatography-electrochemical detection (HPLC-ED) with dual electrodes in the series mode. Biological samples were saponified in the presence of a mixture of butylated hydroxytoluene, ascorbic acid, and pyrogallol and then extracted with hexane. The compounds were separated on a C18 column using a mobile phase containing 95% methanol and 0.05 M sodium perchlorate as the supporting electrolyte. After HPLC separation, alpha-tocopherylquinone was first reduced at an upstream electrode at -500 mV Both alpha-tocopherol and the reduction product of alpha-tocopherylquinone were then oxidized downstream at +600 mV. Only the downstream electrode current was monitored for the determination. Linearity of the standard curves was obtained over the range 5-30 pmol for alpha-tocopherol and alpha-tocopherylquinone. Minimum detectable quantities (S/N of 3) were 0.25 pmol for alpha-tocopherol and 0.31 pmol for alpha-tocopherylquinone. The method was applied to analysis of the contents of alpha-tocopherol and alpha-tocopherylquinone in rat tissues and plasma. By hyperoxia, the content of alpha-tocopherol was decreased remarkably in lung, and in contrast, the contents of alpha-tocopherylquinone were increased in all tissues studied with the exception of plasma, though the content of alpha-tocopherylquinone in normal rats is quite small. The technique is particularly useful in the quantitation of the oxidation of alpha-tocopherol in biological samples.     

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.     

Determination of urinary 5-S-cysteinyldopamine by high-performance liquid chromatography with electrochemical detection

Summary 5-S-Cysteinyldopamine, a new metabolite of dopamine, was determined in urine by high-performance liquid chromatography with electrochemical detection. The catechol was detected in 14 of 21 melanoma patients and 7 of 21 normal subjects; the highest values were 657 μg/day for melanoma patients and 44 μg/day for normal subjects. These results suggest that the cysteine conjugate may arise from autoxidation of dopamine but tyrosinase may also participate in the oxidation.     

Determination of thiamazole in serum by high-performance liquid chromatography with electrochemical detection

A simple and sensitive method for the determination of thiamazole in serum by high-performance liquid chromatography with electrochemical detection is described. Thiamazole in serum was quantified without an extraction procedure at concentrations down to 10 ng/ml. This method was applied to determine the serum concentration of the drug in two healthy volunteers given a single oral dose of 10 mg of thiamazole. The concentration of the drug reached a maximum at 3-4 h after the oral dose and two elimination phases were observed.     

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.     

Determination of acetaldehyde in fuel ethanol by high-performance liquid chromatography with electrochemical detection.

The cyclic voltammetric behavior of acetaldehyde and the derivatized product with 2,4-dinitrophenylhydrazine (DNPHi) has been studied at a glassy carbon electrode. This study was used to optimize the best experimental conditions for its determination by high-performance liquid chromatographic (HPLC) separation coupled with electrochemical detection. The acetaldehyde-2,4-dinitrophenylhydrazone (ADNPH) was eluted and separated by a reversed-phase column, C18, under isocratic conditions with the mobile phase containing a binary mixture of methanol/LiCl(aq) at a concentration of 1.0 x 10(-3) M (80:20 v/v) and a flow rate of 1.0 mL min(-1). The optimum condition for the electrochemical detection of ADNPH was +1.0 V vs. Ag/AgCl as a reference electrode. The proposed method was simple, rapid (analysis time 7 min) and sensitive (detection limit 3.80 microg L(-1)) at a signal-to-noise ratio of 3:1. It was also highly selective and reproducible [standard deviation 8.2% +/- 0.36 (n = 5)]. The analytical curve of ADNPH was linear over the range of 3-300 mg L(-1) per injection (20 microL), and the analytical recovery was > 99%.     

Determination of sulphite and ascorbic acid by high-performance liquid chromatography with electrochemical detection

A rapid HPLC method with electrochemical detection for the determination of free and total sulphite and ascorbic acid in beer and other beverages is presented. Interferences of these compounds are discussed, in addition to the behaviour in buffer solutions of different pH. Only a dilution step is required before injecting the sample into the chromatographic system. To obtain better specificity for these compounds, two different working electrodes (platinum for sulphite and carbon glass for ascorbic acid) with distinct potentials are used.     

Determination of cabergoline in plasma and urine by high-performance liquid chromatography with electrochemical detection.

A sensitive and selective high-performance liquid chromatographic method for the determination of cabergoline in plasma and urine has been developed. After buffering plasma and urine samples, cabergoline was extracted with a methylene chloride-isooctane mixture, back-extracted into 0.1 M phosphoric acid, then analysed by reversed-phase high-performance liquid chromatography. Quantitation was achieved by electrochemical detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from the biological matrices (human plasma and urine) was observed. The assay was still inadequate in terms of sensitivity for the quantitation of cabergoline plasma concentrations after a single oral dose of 1 mg of the drug to humans, but was successfully used in the determination of the urinary excretion of the drug.     

Determination of dosulepin and its metabolite: application of high-performance liquid chromatography with electrochemical detection

Dosulepin, 11-(3-dimethylaminopropylidene)-6,11-dihydrodibenzo [b,e]thiepin hydrochloride, is a thio analogue of amitriptyline and is used for the treatment of anxiety and affective disorders. The present study developed a simple and sensitive procedure for the determination of this compound and its metabolite, northiaden, by a combination of high-performance liquid chromatography with electrochemical detection. Hydrodynamic voltammograms demonstrated an optimal applied potential at 1300 mV for both dosulepin and northiaden. A mobile phase consisting of 0.1 M acetate buffer-acetonitrile-perchloric acid-trichloroacetic acid (50:50:2:1.5) provided the best separation of the drugs. The extraction procedure, which used a heptane-isoamyl alcohol (99:1) mixture, was successfully applied with a recovery of over 90%. A preliminary pharmacokinetic study was performed by the proposed method.     

Determination of nalbuphine by high-performance liquid chromatography with electrochemical detection: application to clinical samples from postoperative patients

A rapid, selective and reproducible high-performance liquid chromatographic assay with electrochemical detection was developed for the determination of nalbuphine in human plasma. The method involves extraction with chloroform-isopropanol at pH 9.4, back-extraction into dilute phosphoric acid and reversed-phase chromatography on a microBondapak phenyl column. The recovery of nalbuphine and naltrexone (internal standard) was greater than 90%. Calibration curves were linear over a concentration range of 3-36 ng/ml with coefficients of variation, within-day or between-day, not exceeding 8% at any level. Although the limit of detection was 0.3 ng/ml based on a signal-to-noise ratio of 3, the reliable limit of quantitation was 1 ng/ml (coefficient of variation 12%) using 1 ml of plasma. The dual-electrode detector was operated in the screening mode of oxidation (electrode 1, 0.3 V and electrode 2, 0.6 V), providing a greater specificity and reducing background noise. This procedure was applied to a large number of clinical samples in an intravenous dose-range pharmacokinetic study in patients.     

Determination of trimethoprim metabolites including conjugates in urine using high-performance liquid chromatography with combined ultraviolet and electrochemical detection

A high-performance liquid chromatographic method for the determination of trimethoprim metabolites in pig urine was developed. The metabolites-glucuronic acid and sulphuric acid conjugates of phenolic metabolites formed by demethylation of trimethoprim-were quantitated after treatment of urine with beta-glucuronidase (Escherichia coli). The sulphuric acid conjugate was not susceptible to enzymatic hydrolysis and was therefore assayed as the conjugate by use of ion-pair chromatography on the reversed-phase column. In order to find suitable conditions for enzymatic hydrolysis of the glucuronides, the conjugates were obtained by gel chromatography of urine from a [14C] trimethoprim-treated pig.     

Determination of glutathione in biological material by high-performance liquid chromatography with electrochemical detection

Glutathione in biological samples is extracted by perchloric acid and separated by ion-paier chromatography on a RP-18 phase. In a post-column reaction, glutathione is converted to an isoindole derivative by reaction with o-phthalaldehyde and detected at a galssy carbon electrode at 800 mV v. Ag/AgCl/3 M KCl. The detection limit is 40 pmol of glutathione injected.     

Determination of deoxynivalenol (vomitoxin) by high-performance liquid chromatography with electrochemical detection

A rapid method for the analysis of deoxynivalenol (DON) was developed using high-performance liquid chromatography (HPLC) with reductive electrochemical detection (ED). Deoxynivalenol produced by Fusarium roseum growing on solid cornmeal and rice substrates and from naturally contaminated wheat was extracted and quantitated via ED. DON levels in wheat were verified by gas chromatography and structurally confirmed by mass spectrometry. DON was optimally resolved by HPLC employing a radially compressed octadecylsilane column and a mobile phase of deoxygenated methanol-40 mM borate buffer (35:65) at a flow-rate of 1.0 ml/min. Under these conditions DON exhibited an average retention time of 3.6 min. Reductive ED (-1.4 V) allowed a 12-fold increase in sensitivity and greater selectivity than classical UV absorption at 224 nm. A detection limit for DON of 25 pg/microliter was achieved under these conditions. The determination of DON in crude grain extracts was hindered by extractable interfering substances, whereas ED was more functional-group selective (i.e. reduction of the carbonyl moiety). ED permits a direct quantitation of DON from crude grain extracts and may facilitate the determination of this agent and associated metabolites in biological samples.     

Determination of bisphenol A in air by high-performance liquid chromatography with electrochemical detection

An air-sampling method for bisphenol A utilizing 13-mm glass fibre filters was developed. The collection efficiency and desorption characteristics were determined. Liquid chromatographic conditions were optimized for the electrochemical detection of bisphenol A. Detection under acidic and alkaline conditions is discussed. The detection minimum is 25 ng/m3 for a 60-1 sample.