Determination of catechol-O-methyltransferase activity in erythrocytes by high performance liquid chromatography with electrochemical detection

A rapid assay employing HPLC with electrochemical detection for catechol-O-methyltransferase (COMT) activity in red blood cells is described. Enzyme activity is determined from erythrocyte lysates using S-adenosyl-L-methionine as methyl donor and 3,4-dihydroxybenzoic acid as substrate. The 3-O- and 4-O-methylated reaction products are measured by high-performance liquid chromatography with electrochemical detection. Human erythrocyte soluble form of COMT had Km values of 6.1 microM and 26.0 microM for S-adenosyl-L-methionine and dihydroxybenzoic acid, respectively. The mean O-methylation ratio for the soluble form of COMT was 5.3. An O-methylation ratio of 15.5 was estimated in the membrane fraction of an erythrocyte pool from three samples. The activities of soluble COMT in erythrocytes of some animal species are also reported. The procedure is easily automated, and a large number of samples can be processed during one working day.     

Determination of hydralazine and metabolites in urine by liquid chromatography with electrochemical detection

The cyclic voltammetric behavior of hydralazine and its primary metabolites, the pyruvate and acetone hydrazones, was examined in the positive potential range at both conventional and electrochemically pretreated glassy carbon electrodes. The enhanced oxidations observed at the treated surface were used as the basis of amperometric electrochemical detection of the compounds following reversed-phase liquid chromatography. The detection limits so obtained at +0.75 V vs. Ag/AgCl (1, 3, and 5 ng injected, respectively) were comparable to those previously reported for absorption and fluorescence detection approaches employing derivatization/preconcentration procedures. For liquid chromatography with electrochemical detection, however, direct quantitation of all three species in urine samples was readily accomplished without any chemical derivatization or sample treatment operations other than particulate filtration.     

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 intraoperative plasma catecholamine concentrations using liquid chromatography with electrochemical detection

Liquid chromatography with electrochemical detection was used for the determination of norepinephrine and epinephrine in human plasma samples obtained prior to, after, and six times during the course of spinal fusion surgery for the correction of scoliosis. The catecholamines were extracted from plasma by alumina adsorption and chromatographed isocratically using a reversed-phase, ion-pairing system. Data obtained are compared to those obtained intraoperatively by other authors using a radioenzymatic method, and the mechanism of sympathetic activation during surgery is discussed. Preliminary data using 3-micron particle size columns and dual-parallel electrochemical detection are presented.     

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

A simple and sensitive HPLC method that does not require derivatization for determining cholesterol has been developed. Investigation of voltammetric behavior of cholesterol showed that cholesterol could be oxidized on a glassy carbon electrode in non-aqueous solvents. This was applied to the development of a method by HPLC with electrochemical detection (HPLC-ED). The HPLC-ED was optimized using the separation of cholesterol and oxysterols including 26-hydroxycholesterol and 24S-hydroxycholesterol. The separation was carried out with a Develosil C30-UG-3 column; acetonitrile-2-propanol (9:1, v/v) containing 50mM LiClO(4) as a mobile phase; and an applied potential at 1.9V versus Ag/AgCl. The current peak height was linearly related to the amount of cholesterol injected from 0.5-100 microM (r>0.999). The detection limit (S/N=3) of cholesterol was 0.36 microM (1.8 pmol). Cholesterol at 100 microM was directly detected with a relative standard deviation (RSD) of less than 1.0% (n=8). Total cholesterol and free cholesterol in control human serum were determined by the present method with the recovery of more than 90% and the RSD (n=6) of less than 3.0%.     

Measurement of naltrexone in rat brain regions and serum by high performance liquid chromatography with electrochemical detection

Summary A method is described for the determination of naltrex-one in discrete rat brain regions and serum. Separation and quantitation are performed by reversed-phase high-performance liquid chromatography with electrochemical detection. The drug gave a linear response over the range of 12.5–100ng for either 1.0ml for cerebellar homogenate or 25l for serum (1.25–10ng on column). Recoveries of naltrexone, added to the homogenate or serum, were 94±7 and 86±6%, respectively, when compared with naltrexone dissolved in water. The mean between-run relative standard deviations were 5.8 and 6.8% for cerebellar homogenate and serum, respectively, over the concentration range studied. The within-run relative standard deviations for cerebellar homogenate (12.5ng/ml) or serum (12.5ng/25l) were 7.7%. The application of the method to an in vivo animal experiment is demonstrated.     

Determination of dopamine,serotonin, and their metabolites in pediatric cerebrospinal fluid by isocratic high performance liquid chromatography coupled with electrochemical detection

A method to rapidly measure dopamine (DA), dihydroxyindolphenylacetic acid, homovanillic acid, serotonin (5‐HT) and 5‐hydroxyindoleacetic acid concentrations in cerebrospinal fluid (CSF) has not yet been reported. A rapid, sensitive, and specific HPLC method was therefore developed using electrochemical detection. CSF was mixed with an antioxidant solution prior to freezing to prevent neurotransmitter degradation. Separation of the five analytes was obtained on an ESA MD‐150 × 3.2 mm column with a flow rate of 0.37 mL/min and an acetonitrile–aqueous (5 : 95, v/v) mobile phase with 75 mM monobasic sodium phosphate buffer, 0.5 mM EDTA, 0.81 mM sodium octylsulfonate and 5% tetrahydrofuran. The optimal electrical potential settings were: guard cell +325 mV, E1 ?100 mV and E2 +300 mV. Within‐day and between‐day precisions were <10% for all analytes and accuracies ranged from 91.0 to 106.7%. DA, 5‐HT, and their metabolites were stable in CSF with antioxidant solution at 4°C for 8 h in the autoinjector. This method was used to measure neurotransmitters in CSF obtained from children enrolled on an institutional medulloblastoma treatment protocol. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of 6-mercaptopurine and its metabolites in plasma or serum by high performance liquid chromatography

A sensitive and accurate reversed phase liquid chromatographic assay was developed for the determination of 6-mercaptopurine (6MP) (the active metabolite of azathioprine) in human plasma. The assay involved extraction into acetonitrile and dichloromethane from plasma pretreated with 0.038 M of dithiothreitol solution. The residue was analyzed by isocratic chromatography on a C18 analytical column with UV detection at 326 nm. The average extraction recovery of 6MP was 85%. The method has been applied successfully to the determination of 6MP and its metabolites in pharmacokinetic studies.     

Determination of tetraethyllead and tetramethyllead using high performance liquid chromatography and electrochemical detection

Summary A new method for the separation of tetramethyllead (TML) and tetraethyllead (TEL) was developed using high-performance liquid chromatography. The electrochemical detection was examined with different electrodes. Amperometric and pulse-amperometric techniques were investigated and the optimal working potential for each electrode was determined. Linearity for the glassy carbon electrode was observed between 350 ng and 30 g; the detection limit is 310 ng (TML) resp. 340 ng (TEL). In case of the mercury gold electrode the linearity range was 300 g–3 g and the detection limit 1.5 m (TML) resp. 1.7 g (TEL).     

Analysis of hydroxyurea in human plasma by high performance liquid chromatography with electrochemical detection

An improved HPLC-electrochemical detection (ED) method is described for the analysis of hydroxyurea (HU) in human plasma. After extraction process, HU was determined on a C18 column ( mm) by the mobile phase (25 mM sodium acetate-acetonitrile, 97.5:2.5; pH 6.5). The regression equations were linear (r>0.9990). The precision and accuracy of intra- and inter-batches were all below 5% for relative standard deviation (R.S.D.) and relative error (R.E.). Based on 20 μl of plasma, the limit of detection was 0.3 μM for HU (S/N=3, injection 10 μl). This method was applied for the HU drug monitoring of patients with myelofibrosis or polycythemia vera.     

Determination of vinclozolin metabolites in human urine by high-performance liquid chromatography and electrochemical detection

A sensitive and reliable method to assess occupational exposure to vinclozolin based on biomonitoring principles has been elaborated. The conditions for pretreating the human urinary samples were chosen in such a way that vinclozolin metabolites containing the intact 3,5-dichloroaniline (3,5-DCA) moiety are completely degraded into this amine by means of basic hydrolysis. After addition of 3,4-DCA as an internal standard, steam distillation and extraction, the analysis is carried out by high-performance liquid chromatography and electrochemical detection. The determination limit is 5 g 3,5-DCA/l urine. The method turned out to be sensitive enough to quantify not only occupational but also nutritional excretions of 3,5-DCA containing metabolites to some extent. Interpreting these results, which are verified by an independent method, it must be considered that in addition to vinclozolin some further crop protection agents are also based on the 3,5-DCA moiety.     

Determination of vinclozolin metabolites in human urine by high-performance liquid chromatography and electrochemical detection

A sensitive and reliable method to assess occupational exposure to vinclozolin based on biomonitoring principles has been elaborated. The conditions for pretreating the human urinary samples were chosen in such a way that vinclozolin metabolites containing the intact 3,5-dichloroaniline (3,5-DCA) moiety are completely degraded into this amine by means of basic hydrolysis. After addition of 3,4-DCA as an internal standard, steam distillation and extraction, the analysis is carried out by high-performance liquid chromatography and electrochemical detection. The determination limit is 5 microg 3,5-DCA/l urine. The method turned out to be sensitive enough to quantify not only occupational but also nutritional excretions of 3,5-DCA containing metabolites to some extent. Interpreting these results, which are verified by an independent method, it must be considered that in addition to vinclozolin some further crop protection agents are also based on the 3,5-DCA moiety.     

Determination of serum cholestanol by semi‐micro high‐performance liquid chromatography with electrochemical detection

A simple and sensitive method that dose not require derivatization for determining cholestanol has been developed using HPLC with electrochemical detection (HPLC‐ECD). The current peak height was linearly related to the amount of cholestanol injected, ranging from 1 to 200 μM (r = 0.999). The detection limit (S/N = 3) of cholestanol was 0.23 μM (1.2 pmol). Total cholestanol in control human and mouse serum was determined by the present method with a recovery rate of more than 90% and an RSD (n = 5) of less than 7.3%. Further, this method was successfully applied to monitor experimental hypercholestanolemia in mice fed a high‐cholestanol diet, an animal model of cerebrotendinous xanthomatosis (CTX). In conclusion, we found this method to be both simple and useful for the determination of cholestanol in serum, helping in the diagnosis of CTX. Copyright © 2009 John Wiley & Sons, Ltd.