Liquid Chromatography With Electrochemical Detection Research Articles

Dual Electrode Detection in LC‐EC Analysis of Sanger‐tagged Amino Acids: Electrochemical Reduction of Aromatic Nitro Groups

AbstractAnalysis of amino acids is crucial to protein structure elucidation. Amino acids, amenable to separation by liquid chromatography (LC), can be detected by sensitive detectors used in liquid chromatograph including absorbance, fluorescence or electrochemical detector when derivatized. Derivatization of amino acids using suitable reagents enhances the separation and detection of amino acids using liquid chromatography. Sanger's reagent (1‐fluoro‐2,4‐dinitrobenzene, DNFB) makes amino acids suitable for absorbance detection. However, little has been done in terms of liquid chromatography with electrochemical detection (LC‐EC) of the DNFB derivatized amino acids. Furthermore, sensor development based on electrochemistry of nitroaromatics has increased dramatically for explosive detection. Since nitroaromatics are electrochemically active, it is essential to determine the reduction pathway. Cyclic voltammetry (CV), rotating disk electrode (RDE) and rotating ring disk electrode (RRDE) experiments have been performed on nitrobenzene (NB) and N‐methyl‐2,4‐dinitroaniline (NMDNA). The results showed that the nitro group was reduced to hydroxylamine in aqueous solutions by addition of four electrons and four protons per nitro group and the hydroxylamine can be reversibly oxidized into a nitroso by removal of two electrons and two protons. Electrochemical detection of the nitro groups is appropriate for dual electrode detection of DNFB labeled amino acids in which reduction can occur on an upstream electrode and corresponding oxidation of the reduction product occurs on the downstream electrode.

The Determination of Uric Acid in Human Saliva by Liquid Chromatography with Electrochemical Detection

A method employing high-performance liquid chromatography with electrochemical detection (LC-ED) has been successfully developed for the determination of uric acid in human salvia. The optimal chromatographic conditions were found to comprise a mobile phase containing 5 % methanol, 95 % 50 mM trichloroacetic acid, adjusted to pH 2.7, in conjunction with a Hypersil C18 250 mm x 4.6 mm column at a flow rate of 0.6 mL min-1. Hydrodynamic voltammetric studies were undertaken to optimize the operating potentials required for electrochemical detection. It was found that an applied potential of +1.2 V was optimum for the determination of uric acid. The proposed method was evaluated by carrying out replicate uric acid determinations on spiked and unspiked human saliva samples (%CV = 8.8 %). The data suggest that the method holds promise for clinical applications.

Evaluation of catecholamines in erythrocytes in hemodialysis patients

Chronic kidney disease (CKD) is a syndrome developing as a result of congenital or acquired diseases of the urinary tract, which is a consequence of end-stage renal failure. There are a variety of metabolic dysfunction, and accumulation of toxic substances, this leads to neurological disorders, in particular dysfunction of the sympathetic nervous system. Raised sympathetic activity is now recognized as an important mechanism involved in cardiovascular complication in humans. The main mediators of the sympathetic nervous system in the effectors part are catecholamines (CA), which are involved in various physiological processes. CA released into the circulation, as one of many substances, may penetrate across the cellular membranes of erytrocytes. The aim of this study was to evaluate concentrations of catecholamines in erythrocytes (RBC) and used it as prediction of mortality in the haemodialysis patients. Material and Methods: The studies were performed among 37 haemodialysed patients. Extractions of catecholamines from erythrocytes lysate was carried out with a ChromSystems Reagent kit for HPLC (Germany) analysis. Intracellular concentration of catecholamines were measured prior to and following haemodialysis by the method of high performance liquid chromatography with electrochemical detection (HPLC-ED). Results: Diagnostic sensitivity and specificity of delta A RBC positive and negative predictive values for cut-off value 44.87 pg/ mL were 77.78% and 82.14%, respectively. The area under the curve equal (AUC) to 0.73 (p<0.01) indicates a good diagnostic usefulness of RBC adrenaline as an independent predictor of mortality. Conclusions: The results suggest that the dynamic changes in concentration of erythrocytes adrenaline are an independent predictor of mortality in haemodialysis patients.

Analysis of plasma 3-methoxytyramine, normetanephrine and metanephrine by ultraperformance liquid chromatographytandem mass spectrometry: utility for diagnosis of dopamine-producing metastatic phaeochromocytoma

Measurements of plasma normetanephrine (NMN) and metanephrine (MN) provide a sensitive test for diagnosis of phaeochromocytomas and paragangliomas (PPGLs), but do not allow detection of dopamine-producing tumours. Here we introduce a novel mass spectrometric based method coupled to ultraperformance liquid chromatography (LC-MS/MS) for measuring NMN, MN and 3-methoxytyramine (MTY), the O-methylated metabolite of dopamine. Specific collision-induced fragment ions assessed by multireaction monitoring transitions were used for identification, with quantification according to signal intensities of analytes relative to stable isotope labelled internal standards. Results for solid-phase extracted samples from 196 subjects analysed by LC-MS/MS were compared with those analysed by liquid chromatography with electrochemical detection (LC-ECD). Concentration ranges in 125 volunteers were compared with those from 63 patients with PPGLs, including 14 with metastatic disease. The LC-MS/MS method showed linearity over four orders of magnitude with analytical sensitivity sufficient to measure to 0.02 nmol/L. Intra- and inter-assay coefficients of variation ranged from 2.8% to 13.5%. NMN and MN were respectively measured 17% and 10% higher and MTY 26% lower by LC-MS/MS than by LC-ECD. Medians and ranges for 3-methoxytramine, NMN and MN were respectively 0.08 (0.03-0.13), 0.35 (0.13-0.95) and 0.15 (0.07-0.33) nmol/L in volunteers. Among patients with PPGLs, plasma methoxytyramine was six-fold higher in patients with than without metastastases (1.09 versus 0.19 nmol/L) and in three patients was the only metabolite increased. The LC-MS/MS method enables accurate, selective and sensitive measurements of catecholamine O-methylated metabolites that should be particularly useful for screening and management of dopamine-producing metastatic PPGLs.

LC-ED with an Acetylene Black–Dihexadecyl Hydrogen Phosphate Composite Film-Modified Electrode for in Vivo Analysis of Thiols in Rat Striatal Microdialysate

Liquid chromatography with electrochemical detection (LC-ED), coupled with in vivo microdialysis sampling, has been used for analysis of thiols. An acetylene black–dihexadecyl hydrogen phosphate (AB–DHP) composite film-modified electrode was used as working electrode. The AB–DHP-modified electrode enabled efficient electrocatalytic oxidation of l-cysteine (l-Cys) and glutathione (GSH) with relatively high sensitivity, stability, and longevity. The peak currents of l-Cys and GSH were linear in the concentrations ranges 2.0 × 10−7–2.0 × 10−4 and 3.0 × 10−7–5.0 × 10−4 mol L−1, respectively, with calculated detection limits (S/N = 3) of 1.0 × 10−7 and 2.0 × 10−7 mol L−1, respectively. The method has been successfully used to measure the amounts of l-Cys and GSH in striatal microdialysate of freely moving rats.

Contamination of the Norepinephrine Prodrug Droxidopa by Dihydroxyphenylacetaldehyde

L-threo-3,4-dihydroxyphenylserine (L-DOPS, droxidopa) is a norepinephrine (NE) prodrug under development to treat orthostatic hypotension. 3,4-Dihydroxyphenylacetaldehyde (DOPAL), an endogenous catecholaldehyde produced by enzymatic oxidative deamination of dopamine, is toxic to catecholaminergic neurons. Based on the observation of increasing plasma DOPAL after oral administration of L-DOPS to a patient, we examined whether other subjects also had DOPAL in their plasma after droxidopa administration, and whether droxidopa is contaminated with DOPAL. Thirteen subjects took 400 mg droxidopa orally. We sampled venous blood at baseline and 1, 2, 3, 6, 24, and 48 h after drug administration and assayed L-DOPS, NE, and DOPAL by use of liquid chromatography with electrochemical detection (LC-ED). Droxidopa in acidic solution (20:80 mixture of 0.04 mol/L phosphoric acid:0.20 mol/L acetic acid) was vacuum centrifuged for 1 h at 30 degrees C and then assayed by LC-ED. Droxidopa contained 0.01% DOPAL. At 6 h after droxidopa, all subjects had detectable DOPAL in plasma (1.89 nmol/L, P = 0.0001). Across the sampling times, plasma DOPAL correlated with plasma L-DOPS (r = 0.996). The mean increment in plasma DOPAL was more than 4 times that in plasma NE (0.39 nmol/L). In 2 patients with Parkinson disease and orthostatic hypotension, DOPAL was detected in plasma at baseline (0.12 nmol/L) and increased by about 70-fold after droxidopa. Vacuum concentration of droxidopa in the acid solution converted L-DOPS to DOPAL completely. Droxidopa is contaminated with DOPAL. After oral droxidopa administration, DOPAL is detected in plasma of humans. Droxidopa is susceptible to extensive nonenzymatic conversion to DOPAL.

Simultaneous Determination of Six Phenolic Compounds in Jujube by LC-ECD

A simple, sensitive and accurate liquid chromatography with electrochemical detection (LC-ECD) method for simultaneous separation and determination of six phenolic compounds, including gallic acid, protocatechuic acid, caffeic acid, p-coumaric acid, rutin and quercetin in jujube has been established. ECD is more sensitive than diode-array detection (DAD) for the detection of the six phenolic compounds. All calibration curves of the phenolic compounds showed good linearity (r ≧ 0.9995) within the test ranges. The recoveries were higher than 98%. This approach can provide a reference to determine phenolic compounds in other products.

Simultaneous determination of eight estrogens and their metabolites in serum using liquid chromatography with electrochemical detection

The liquid chromatography (LC) with electrochemical detection allows to determine, evaluate and validate the level of estrogens and their metabolites in serum. The method is fast and sensitive, and the hormones can be determined simultaneously, from one serum sample. The proposed method was successfully applied to the determination of following estrogens: estrone (E(1)), 17beta-estradiol (E(2)), estriol (E(3)) and following catecholestrogens: 2-hydroxyestradiol (2-OHE(2)), 4-hydroxyestradiol (4-OHE(2)), 4-hydroxyestrone (4-OHE(1)), 2-methoxyestradiol (2-MOE(2)) and 2-methoxyestrone (2-MOE(1)). The method of LC with electrochemical detection (LC/EC) was applied for the determination of catecholestrogens in serum sample taken from pregnant women. Estrogens and catecholestrogens were extracted from 1 mL of serum by applying diethyl ether under the specified conditions. The parameters of the procedure included using a specific mobile phase, applying the column Symmetry C18 (5 microm, 3.0 mm x 150 mm), equipping the applied electrochemical detector with the working glassy-carbon electrode, as well as applying the reference electrode Ag/AgCl. The calibration studies on this study were performed, and a good analytical performance for E(1), E(2), E(3), 2-OHE(2), 4-OHE(2), 4-OHE(1), 2-MOE(2) and 2-MOE(1) was attained, along with low limits of detection (LOD of 0.18-0.30 ng/mL), satisfactory limit of quantification (LOQ of 0.23-0.92 ng/mL) and excellent linear dynamic range (0.6-8.0 ng/mL). In conclusion, the presented methodology is the sensitive method of the simultaneous measurement of eight estrogens and their metabolites from one sample of blood and it might be clinically applied for testing serum of pregnant women, as well as for further studies on estrogens and their metabolites.

Selective Analysis of Secondary Amines Using Liquid Chromatography with Electrochemical Detection (LC‐EC)

AbstractIn a mixture of primary and secondary aliphatic amines, the primary amines were derivatized (masked) with o‐phthalaldehyde (OPA) followed by derivatization of the remaining secondary amines with ferrocenecarboxylic acid chloride (FAC). The “tagged” amines were analyzed by LC‐EC (liquid chromatography with electrochemical detection) using in‐series dual electrode detection. Chemically‐reversible oxidation of the FAC tagged secondary amines and their subsequent complementary oxidation and reduction signals coupled with chemically‐irreversible oxidation of OPA tagged primary amines provided the selectivity for quantitative secondary amine analysis. The procedure was also applied for the selective identification of fragment 4–11 (N‐terminus‐proline) of Substance P in the presence of other Substance P fragments with primary amino acids as their N‐termini.

Glutathione Redox State Regulates Mitochondrial Reactive OxygenProduction

Oxidative stress induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) is poorly understood. Following one dose of TCDD (5 microg/kg body weight), mitochondrial succinate-dependent production of superoxide and H2O2 in mouse liver doubled at 7-28 days, then subsided by day 56; concomitantly, levels of GSH and GSSG increased in both cytosol and mitochondria. Cytosol displayed a typical oxidative stress response, consisting of diminished GSH relative to GSSG, decreased potential to reduce protein-SSG mixed disulfide bonds (type 1 thiol redox switch) or protein-SS-protein disulfide bonds (type 2 thiol redox switch), and a +10 mV change in GSSG/2GSH reduction potential. In contrast, mitochondria showed a rise in reduction state, consisting of increased GSH relative to GSSG, increases in type 1 and type 2 thiol redox switches, and a -25 mV change in GSSG/2GSH reduction potential. Comparing Ahr(-/-) knock-out and wild-type mice, we found that TCDD-induced thiol changes in both cytosol and mitochondria were dependent on the aromatic hydrocarbon receptor (AHR). GSH was rapidly taken up by mitochondria and stimulated succinate-dependent H2O2 production. A linear dependence of H2O2 production on the reduction potential for GSSG/2GSH exists between -150 and -300 mV. The TCDD-stimulated increase in succinate-dependent and thiol-stimulated production of reactive oxygen paralleled a four-fold increase in formamidopyrimidine DNA N-glycosylase (FPG)-sensitive cleavage sites in mitochondrial DNA, compared with a two-fold increase in nuclear DNA. These results suggest that TCDD produces an AHR-dependent oxidative stress in mitochondria, with concomitant mitochondrial DNA damage mediated, at least in part, by an increase in the mitochondrial thiol reduction state.

Determination of myocardial norepinephrine in freely moving rats using in vivo microdialysis sampling and liquid chromatography with dual-electrode amperometric detection.

Myocardial norepinephrine (NE) is considered a meaningful parameter for estimation of cardiac function. Long lasting changes in myocardial NE appear to be not only a consequence of pathologic processes in the myocardium, but may be a factor responsible for some diseases (e.g. increased propensity for arrhythmias or negative effect on left ventricular contractility in congestive heart failure). In this respect monitoring of myocardial NE is of great importance. A microdialysis sampling technique coupled with liquid chromatography with electrochemical detection (LCEC) was developed to measure the in vivo NE concentration in the myocardial interstitium of conscious, freely moving rats. LCEC using a dual-electrode amperometric detection in the series configuration provided detection limits for NE of 10 pg/ml in 20 microl microdialysis samples. Microdialysis probes of the linear design were implanted in the myocardial tissue in the periphery of the left descending coronary artery. The basal steady-state concentration of NE in myocardial dialysate of awake, freely moving rats was found to be 0.17+/-0.026 ng/ml. Delivery through the microdialysis probe of the NE reuptake inhibitor desipramine (DMI) at a concentration of 0.1 mM increased NE release to 153+/-13% of control. If the concentration of DMI in the perfusate was increased to 1.0 mM, NE release increased to only 166+/-21% of control.

In vivo monitoring of the monoamine neurotransmitters in rat brain using microdialysis sampling with liquid chromatography electrochemical detection

The fabrication and application of a novel electrochemical detector (ED) with the new ion-exchange polymer-overoxidized polypyrrole (OPPy) film chemically modified electrode (CME) for liquid chromatography (LC) are described. The electrochemical behavior of dopamine (DA) and ascorbate (AA) at the OPPy CME is investigated by cyclic voltammetry (CV). It is found that the CME can permselective dopamine cations and exile the ascorbate anions, which could be used to determine the monoamine neurotransmitters and avoid the interference of ascorbate in liquid chromatography. The thickness of the OPPy film has an important effect on the sensitivity and selectivity for dopamine with respect to ascorbate. In liquid chromatography with electrochemical detection (LC-ED), DA, norephinephrine (NE), 5-hydroxytryptamine (5-HT), epinephrine (E) and 3,4-dihydroxyphenylacetic acid (DOPAC) have good and stable current responses at the CME. The linear ranges of five analytes are over three orders of magnitude and the limits of detection are 0.05 pmol for DA, 0.05 pmol for NE, 0.05 pmol E, 0.1 pmol for 5-HT and 1.0 pmol for DOPAC. The applications of this method coupled with microdialysis sampling for the in vivo determination of the monoamine neurotransmitters in the different areas of rat brain is satisfactory.

Liquid chromatography — electrochemical detector for the determination of glucose in rat brain combined with in vivo microdialysis

In this paper, microdialysis sampling was combined with liquid chromatography with electrochemical detection (LC-ECD) and glucose in rat brain was determined. A cobalt tetraaminophthalocyanine (CoTAPC)/glucose oxidase (GOD)/Nafion film-coated glassy carbon electrode was used to selectively detect the hydrogen peroxide generated enzymatically in the enzyme reactors, without any interference from electroactive species such as l-ascorbate, urate, proteins. The signal current was linearly related to the glucose concentration in the range of 1.0×10 −6–5.0×10 −3 mol/l at +0.5 V (versus Ag/AgCl) with a detection limit of 5.0×10 −7 mol/l. To measure glucose in rat brain, microdialysis sampling was employed. The dialysate from the microdialysis system was injected into system. The result was 0.74±0.10 mmol/l ( n=4).

ACUTE EFFECTS OF CAFFEINE INTAKE ON THE 30-S WINGATE TEST PERFORMANCE

1348 The purpose of this study was to investigate whether a single, caffeine ingestion affected anaerobic performance and serum catecholamine concentration during the 30-s Wingate test. Fourteen college-male cyclists (n=7) and students (n=7) volunteered as subjects. The subjects completed two 30-s Wingate tests after a single administration of either caffeine or placebo (a double-blind method) 60 min prior to the test. A washout-period of 6 days was allowed between the trials. Sample of venous blood (10 ml) was taken just before and immediately after the Wingate test for lactate and catecholamine analysis. Serum norepinephrine and epinephrine were analyzed using high performance liquid chromatography with electrochemical detection (LCEC). A 2 × 3 ANOVA with repeated measures on the time factor, followed by the Student Newman Kuels post hoc test, showed that maximal anaerobic power (TP, MP, PP) in both groups was significantly increased with caffeine administration (p<0.05). In addition, caffeine administration increased the serum levels of both catecholamine and lactate at exhaustion (p<0.05). Based on the nutritional survey and urinary excretion of caffeine (undetectable), the subjects' daily caffeine intake was minimal, and they all recognized whether a caffeine or placebo trial was done when asked immediately after the tests. These data suggest that a single administration of caffeine can increase anaerobic performance.

Liquid Chromatography with Electrochemical Detection (LC-EC): An Experiment Using 4-Aminophenol

The combination of liquid chromatography with electrochemical detection (LC-EC) is a powerful analytical tool for determining electroactive compounds in complex matrices. It has found numerous applications especially in the pharmaceutical and clinical areas. This experiment is intended to give students a practical experience with the LC-EC technique. The first part is designed to explore the electrochemistry of p-aminophenol (PAP), the analyte, while the second part deals with separation and identification of PAP in the presence of ascorbic acid and catechol. The improvement in detection limit with electrochemical detection compared to ultraviolet detection is also illustrated.

Determination of phenols in water by on-line solid-phase disk extraction and liquid chromatography with electrochemical detection

Poly(styrene-divinylbenzene) (PS-DVB) membrane extraction disks were used as sorbents for the on-line solid phase extraction of 13 phenols (nitro and chlorophenols) in river and tap waters. Determination was performed by liquid chromatography with electrochemical detection (LC-ED). An acetate buffer-acetonitrile-methanol mixture as mobile phase and amperometric detection at +1100 mV were used. High water volumes, up to 250 ml, can be preconcentrated without loss of phenols (recoveries between 80% and 100%) except for the more polar ones. Moreover, detection limits between 0.01 and 0.1 μg l −1 in tap water and between 0.1 and 1.0 μg −1 in river water were obtained. The method has been applied to the analysis of two river water samples.

Norepinephrine content in primary and secondary lymphoid organs is altered in rats with adjuvant-induced arthritis

Chemical sympathectomy of secondary lymphoid organs with sparing of the hind limbs exacerbates adjuvant-induced arthritis (AA) in Lewis rats supporting a role for noradrenergic (NA) innervation of the immune system in AA pathology. The present study examines sympathetic innervation of lymphoid organs from Lewis rats 32 days after treatment with complete Freund's adjuvant (CFA) or vehicle using fluorescence histochemistry for localization of catecholamines (CA) and high-performance liquid chromatography with electrochemical detection (LCEC) for measurement for norepinephrine. The thymus from AA rats was significantly reduced in size, while secondary lymphoid organs, i.e. spleen and draining lymph nodes (DLN), were significantly enlarged compared with that seen in vehicle-treated controls. Fluorescence histochemistry revealed no apparent differences in the density of NA innervation, or the intensity of staining in sympathetic nerves in any of the secondary lymphoid organs from AA rats compared with that observed in control animals. However, there was an apparent increase in the density of NA nerve fibers in the thymus of AA rats. Norepinephrine (NE) concentration (pmol NE per g or mg wet weight), in the thymus from AA rats was significantly increased. Conversely, a significant decrease in splenic and lymph node NE concentration was measured in adjuvant-treated animals compared with that seen in vehicle-treated rats. Total NE content (pmol NE per whole organ weight) in lymphoid organs was not altered, except in popliteal lymph nodes (PLN), where it was increased. Collectively, our findings suggest that changes in NA innervation of lymphoid organs from AA rats result largely from increases or decreases in organ mass. Since NE released from NA nerves acts in a paracrine fashion, changes in lymphoid tissue volume that result from enhanced proliferation, migration, or cell death can make a significant difference in the availability of NE for interaction with immune target cells in these organs, even in the absence of a change in NE metabolism. Decreased thymic weight and increased spleen and lymph node weight should increase and decrease NE availability for interaction with target cells, respectively. Additionally, in PLN (a site where the highest concentration of antigen is encountered) an increase in total NE content suggests compensatory changes in NE metabolism.

Determination of isoproterenol sulfate on surfaces using high-performance liquid chromatography with electrochemical detection

Trace amounts of isoproterenol (IP) sulfate are determined on surfaces using high-performance liquid chromatography with electrochemical detection (LC-ED). The drug residues are removed from surfaces with cotton swabs using an aqueous buffer which is 0.05 M phosphate, 5 m M pentanesulfonic acid and 0.1 m M EDTA at pH 3.6. The drug substance is chromatographed using a 15 cm × 4.6 mm I.D. Nucleosil C 18 column and an eluent containing the same buffer and methanol as a modifier which are mixed in a 90:10 ratio. Detection is performed using an amperometric thin-layer cell with a glassy carbon working electrode operated at +0.65 V versus Ag/AgCl which gives a linear response ( r = >0.9999) for isoproterenol sulfate to at least 42.9 ng/ml. Repeatability of the chromatographic finish was demonstrated by replicately chromatographing IP solutions over several days. Repeatability (R.S.D. values) of the peak areas ranged from ±0.32% to ±3.0%. For additions of 40 to 120 ng of isoprotenol sulfate average recoveries from cotton swabs ranged from 82 to 98%. At the same addition levels. average recoveries ranged from 87% to 95% for stainless steel and glass surfaces. The precision for all recovery data (R.S.D. values) ranged from ±2.1% to 12.8%.

Na+, K(+)-ATPase, glutathione, and hydroxyl free radicals in cadmium chloride-induced testicular toxicity in mice.

Cadmium chloride (CdCl2)-induced biochemical changes were characterized in male, CD-1 mouse testes. CdCl2 inhibited the testes microsomal Na+,K(+)-ATPase activity in vitro and in vivo. The inhibitory range was 30-50 microns and the concentration for half maximal inhibition (IC50 value) was 90 microns over 5 min preincubation. CdCl2 (2mg/kg/day, s.c.) for 2 days significantly inhibited testes Na+,K(+)-ATPase (near 90% inhibition). The content of testicular GSH and the ratio of reduced glutathione (GSH)/GSSG (oxidized glutathione) decreased in CdCl2-treated groups. Using salicylate as a trapping agent and high pressure liquid chromatography with electrochemical detection (LCED), we measured the OH production in vivo. 2,5-dihydroxybenzoic acid (2,5-DHBA) and 2,3-dihydroxybenzoic acid (2,3-DHBA) as indices of hydroxyl free radical formation significantly increased after 5 days CdCl2 exposure. Pretreatment with vitamin E (20 mg/kg, s.i.d., i.m., 7d) protected CdCl2-induced increase in OH. generation in testes. From this study, it was demonstrated that CdCl2 induced testicular toxicity could possibly be mediated by a significant increase in hydroxyl free radical formation and a reduction in GSH content and Na+,K(+)-ATPase activity. Vitamin E seems to prevent the CdCl2 induced increase in hydroxyl free radical generation.

Reversed phase-liquid chromatography of primary and secondary aliphatic amines after derivatization combined with electrochemical detection

An analytical procedure has been developed for the determination of primary and secondary aliphatic amines by liquid chromatography with electrochemical detection (LC-ECD). Aliphatic amines, such as propylamine (PA), butylamine (BA), 2-pentylamine (2-PA), dimethylamine (DMA), diethylamine (DEA) and dipropylamine (DPA) react readily with salicylic acid chloride (SAC) to produce electroactive amide derivatives with good chemical stability. The isocratic elution pattern of synthetic mixtures of the investigated amines on a reversed-phase column is shown. Hydrodynamic voltammograms of standard compounds are presented. The detection limits monitored with an EC detector are compared with those of an UV detector. The method was sensitive enough to detect the derivatives in the picomol range. The method was also applied for the determination of amines in plasma and urine using 2-PA and DPA as model substances. Following a solid phase extraction and derivatization, the compounds could be detected in the lower ng range/ml biological material.