Determination of matairesinol in flax seed by HPLC with coulometric electrode array detection

A HPLC method coupled with coulometric electrode array detection for the determination of matairesinol in flax seed is described. The defatted sample was spiked with bisphenol A (internal standard), refluxed for 75 min in a mixture of ethanol-bidistilled water-12 M hydrochloric acid (2:2:1, v/v/v) to extract matairesinol conjugates and to hydrolyze them simultaneously. The extract was diluted with mobile phase [250 ml acetonitrile-750 ml buffer (730 ml bidistilled water, 20 ml glacial acetic acid adjusted to pH 3 with 5 M sodium hydroxide)] and injected into the HPLC system. Matairesinol was separated from other compounds on a reversed-phase column (Lichrospher 60 RP-Select B, 250 x 4 mm, 5 micro m) and detected in a coulometric electrode array detector using a flow-rate of 0.8 ml/min. The potentials of the eight electrodes were set on +150, +200, +250, +300, +350, +400, +550 and +600 mV against modified palladium electrodes. The content of matairesinol determined in seven samples varies between 7 and 28.5 micro g/g. The limit of quantitation is 5 micro g/g.     

Application of the dual-cell coulometric detector: a method for assaying monoamines and their metabolites

A new HPLC assay technique for monoamines and their metabolites, using a controlled potential coulometric detector equipped with a dual working electrode cell of fully porous graphite through which the samples flow, is described in comparison with a classical amperometric detector equipped with a glassy carbon electrode. Different potentials can be applied at each cell of the coulometric detector to improve sample resolution and detection sensitivity. The signal-to-noise ratio (s/n) calculated in similar conditions was 10 times lower for the coulometric detector than for the amperometric one. The dual-coulometric detector does not undergo daily decay or variation, and needs no particular care or preparation. It is therefore possible to achieve stable routine sensitivity in a range of 10 fmol. This new technique has been applied for assaying monoamines and their precursors and metabolites by direct injection of clear supernatant after centrifugation and for determination of catecholamine turnover in rat pineal gland and neuro- and adenohypophysis in samples purified by Al2O3 adsorption.     

Sensitive high-performance liquid chromatographic method for profiling phytoestrogens using coulometric electrode array detection: application to plasma analysis.

An HPLC method for profiling 13 phytoestrogens and their metabolites using coulometric electrode array detection was developed. Sensitivity of the method was slightly less than that of our GC-MS method, but significantly higher compared to the HPLC methods using diode-array or UV detection. Detection limits varied from 3.4 (secoisolariciresinol) to 40.3 (genistin) pg on column. Signal linearities ranged from the detection limits to 61 ng on column. Resolution values for the peak pairs varied from 1.1 (O-desmethylangolensin-anhydrosecoisolariciresinol) to 16 (daidzin-genistin). Intra- and interassay retention time variations were negligible and detector response variation was eliminated by frequent calibration. Chromatographic method was applied to plasma analyses and 6 of the 13 compounds were detected. Method accuracy for those six analytes varied from 69% (enterodiol) to 118% (genistein). Intraassay precision CVs ranged from 1.5% (enterolactone, 12.4 nmol/liter) to 14% (genistein, 245 nmol/liter) and interassay precision CVs ranged from 9.9% (daidzein, 67.4 nmol/liter) to 44% (enterodiol, 1.20 nmol/liter).     

Determination of heterocyclic aromatic amines in beef extract, cooked meat and rat urine by liquid chromatography with coulometric electrode array detection

This paper describes a method for the determination of heterocyclic aromatic amines (HAs; DMIP, IQ, MeIQ, MeIQx, 4,8-DiMeIQx, 7,8-DiMeIQx, AalphaC, PhIP) by high-performance liquid chromatography (HPLC) with coulometric electrode array detection. The compounds are separated on reversed phase columns (LiChroCart Superspher 60 RP-select B, 250 mm x 2 mm, 4 microm and LiChrospher 60 RP-select B, 250 mm x 4 mm, 5 microm) using mobile phases consisting of acetonitrile/buffer/distilled water and detected at eight working electrodes at potentials between +190 and +680 mV against modified palladium electrodes. In the context of an EU-interlaboratory exercise, the method was applied to analyse a standardised test solution and--after isolation of the analytes by several clean-up steps--for the analysis of standardised beef extract and grilled meat. Further, the method could be applied for the analysis of HAs in suspensions of bacteria and rat urine without any sample preparation step beyond sample dilution. The data obtained show that HPLC with coulometric electrode array detection gives accurate results.     

Sensitive high-performance liquid chromatographic determination of cyclizine and its demethylated metabolite,norcyclizine, in biological fluids using coulometric detection

An accurate, sensitive, selective and reproducible high-performance liquid chromatographic method with coulometric detection for the determination of cyclizine and its inactive demethylated metabolite, norcyclizine, in biological fluids has been developed. The drugs were separated using a custom packed reversed-phase C₁₈ analytical column and phosphate buffer (0.05 M, pH 3)-acetonitrile (7:3) as mobile phase. The dual electrode coulometric detector was operated in the “oxidative-screen” mode with the upstream electrode (detector 1) set at 0.55 V and the downstream electrode (detector 2) set at 0.90 V. Serum and urine samples were prepared for analysis by solid-phase extraction, followed by a simple phase-separation step. The limit of quantitation was 1 ng/ml for both cyclizine and norcyclizine in serum and urine.     

Measurement of kynurenic acid in mammalian brain extracts and cerebrospinal fluid by high-performance liquid chromatography with fluorometric and coulometric electrode array detection

Kynurenic acid is a broad-spectrum excitatory amino acid (EAA) receptor antagonist which is present in the mammalian central nervous system. We describe a method for the measurement of kynurenic acid using isocratic reverse-phase high-performance liquid chromatography (HPLC) with fluorometric detection enhanced by Zn2+ as a postcolumn reagent. The method requires no prior sample preparation procedures other than extraction with 0.1 M HClO4. The reliability of the primary fluorometric method was verified by comparing measurements of tissue concentrations of kynurenic acid in human cerebral cortex and putamen using three different methods of separation with fluorometric detection, as well as four methods utilizing HPLC with coulometric electrode array system (CEAS) detection. All seven methods produced comparable results. The concentration of kynurenic acid in human cerebral cortex was 2.07 +/- 0.61 pmol/mg protein, and in human putamen, 3.38 +/- 0.81 pmol/mg protein. Kynurenic acid was also found to be present in human cerebrospinal fluid (CSF) at a concentration of 5.09 +/- 1.04 nM. The regional distribution of kynurenic acid in the rat brain was examined. Kynurenic acid concentrations were highest in brainstem (149.6 fmol/mg protein) and olfactory bulb (103.9 fmol/mg protein) and lowest in thalamus (26.0 fmol/mg protein). There were no significant postmortem changes in kynurenic acid concentrations in cerebral cortex, hippocampus, and striatum at intervals ranging from 0 to 24 h. Perfusion of the cerebral vasculature with normal saline prior to sacrifice did not significantly alter kynurenic acid content in rat hippocampus, cerebral cortex, or striatum. The analytical methods described are the most sensitive (10-30 fmol injection-1) and specific (utilizing both excitation and emissions properties and electrochemical reaction potentials, respectively) methods for determining kynurenic acid in brain tissue extracts and CSF. These methods should prove useful in examining whether kynurenic acid modulates EAA-mediated neurotransmission under physiologic conditions, as well as in determining the role of kynurenic acid in excitotoxic neuronal death.     

Development of a fingerprint of Salvia miltiorrhiza Bunge by high-performance liquid chromatography with a coulometric electrode array system

To standardize and control herbal medicines, a feasible approach and control system is necessary. In this paper, a high-performance liquid chromatography with a coulometric electrode array detector (HPLC-CEAD) system was applied to fingerprint Salvia miltiorrhiza Bunge (S. miltiorrhiza Bunge), a popular herbal medicine, for the first time. pH of mobile phase, working potentials and sample preparation were included in our research. Twenty-five common peaks were obtained from extracts of S. miltiorrhiza Bunge (Shandong province), more than that obtained in previous report. Fingerprints of S. miltiorrhiza Bunge from different locations were also studied. The content of main components varied in different samples. Overlapping ratio of peaks (ORP) in 10 batches of S. miltiorrhiza Bunge (Shandong province) was not less than 72.46%. In method validation, relative standard deviation (RSD) of relative retention times and relative peak areas were of not more than 3%. It was concluded that HPLC-CEAD system can be applied in fingerprinting herbal medicines.     

Enantioselective determination of tramadol and its main phase I metabolites in human plasma by high-performance liquid chromatography

A sensitive and relatively rapid reversed-phase HPLC method was applied to the enantiomeric separation of tramadol and its two main metabolites, O-desmethyltramadol (M1) and N-desmethyltramadol (M2) in plasma samples. Chromatography was performed on an AGP column containing alpha1-acid glycoprotein as chiral selector with a mobile phase of 30 mM diammonium hydrogen phosphate buffer-acetonitrile-triethylamine (98.9:1:0.1, v/v), adjusted to pH 7 by phosphoric acid, and a flow rate of 0.5 ml/min. The fluorescence of analytes was detected at excitation and emission wavelengths of 200 and 301 nm, respectively. The sample preparation was a simple extraction with ethyl acetate using fluconazol as internal standard (IS). The enantiomers of all analytes and IS peaks eluted within 32 min, without any endogenous interference. The calibration curves were linear (r(2) > 0.993) in the concentration range of 2-200, 2.5-100 and 2.5-75 ng/ml for tramadol, M1, and M2 enantiomers, respectively. The within- and between-day variation determined by the measurement of quality control samples at four tested concentrations, showed acceptable values. The lower limit of quantitation was 2 ng/ml for tramadol enantiomers and 2.5 ng/ml for M1 or M2 enantiomers. Mean recoveries of enantiomers from plasma samples were > 81% for all analytes. The procedure was applied to assess the pharmacokinetics of the enantiomers of tramadol and its two main metabolites following oral administration of single 100-mg doses to healthy volunteers.     

Combination of high-performance liquid chromatography and radioimmunoassay for the measurement of urodilatin and alpha-hANP in the urine of healthy males

Urodilatin (ANP-(95-126)), a natriuretic peptide in urine, and alpha-hANP (ANP-(99-126)) are crossreactive in the radioimmunoassay of alpha-hANP (ANP-RIA). We therefore developed a method to separate physiological amounts of urodilatin and alpha-hANP in urine by high-performance liquid chromatography (HPLC) followed by ANP-RIA of the separated fractions. We studied urine samples of 10 healthy adult males with a plasma alpha-hANP level of 41 +/- 21 pg/ml (mean +/- SD) and a total urinary ANP-RIA reactivity of 40 +/- 21 pg/ml. In all urine samples we found three peaks of ANP-RIA reactivity, the first one coeluting with synthetic urodilatin, the second one with the retention time of alpha-hANP and a late eluting ANP-RIA-reactive peak, possibly containing degradation products. The ratio of urodilatin/alpha-hANP was 0.77 +/- 0.17.     

Concentrations of ciclosporin in allogeneic bone marrow recipients. Comparison of assay methods

Thirteen patients in complete remission from acute nonlymphoblastic leukaemia or in chronic phase of chronic myelocytic leukaemia were treated with total body irradiation, cyclophosphamide and allogeneic bone marrow transplantation (BMT). Ciclosporin (CS) was administered for the prevention and the treatment of Graft versus Host Disease. Blood concentrations of CS were determined by Radioimmunoassay (RIA) and by High Performance Liquid Chromatography (HPLC). Trough levels of CS in peripheral blood as measured by RIA exceeded HPLC derived levels in nearly all (56/58) samples with a ratio of RIA:HPLC ranging from 2.43 +/- 1.42 at day 12 to 3.65 +/- 1.86 at day 26 after BMT (means +/- SD). A comparable ratio was found as regards the peak concentrations of CS in peripheral blood. Neither the dose of CS (0.5-3.0 mg/kg/day intravenously; 3.0-5.0 mg/kg/day per os) nor the duration of treatment (12, 19, 26 or 33 days after start of CS) were a significant factor as regards the ratio between HPLC and RIA. Concentrations of CS were also determined in bone marrow nucleated cells at 1 hour after the drug infusion had started. Here the ratio of RIA versus HPLC varied upon the duration of CS treatment with a highest ratio of 8.75 +/- 8.74 at day 12 after BMT. Bone marrow levels corresponded well with blood trough concentrations (p less than 0.01). It is concluded that the concentrations of CS in blood and bone marrow as determined by RIA and HPLC differ significantly, though consistently. At present, no advantage can be attributed to either method of analysis for routine clinical monitoring, as long as detailed information on the immunosuppressive and the toxic characteristics of CS metabolites in humans is lacking.     

Monoamines and Their Precursors and Metabolites in the Chicken Brain, Pineal, and Retina: Regional Distribution and Day/Night Variations

Levels of norepinephrine, epinephrine, dopamine, and serotonin (5-HT) and their precursors [tyrosine, L-3,4-dihydroxyphenylalanine, tryptophan, and 5-hydroxytryptophan (5-HTP)] and metabolites [3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT), homovanillic acid, 3-methoxy-4-hydroxyphenylglycol, and 5-hydroxyindoleacetic acid (5-HIAA)] were determined concurrently in samples of chick retina, pineal gland, and nine selected areas of the brain (optic lobes, thalamus, hypothalamus, optic chiasm, pons/medulla, cerebellum, neostriatum/ectostriatum, hyperstriatum, and basal forebrain) using HPLC coupled with a coulometric electrode array detection system. The norepinephrine level was highest in the pineal gland, but it was also widely distributed throughout the chick brain, with the thalamus and hypothalamus showing substantial levels. The dopamine level was highest in the basal forebrain. The epinephrine level was highest in the hypothalamus. The thalamus and hypothalamus showed the highest levels of 5-HT. Daytime levels (1100 h) of these compounds were compared with levels in chicks killed in the middle of the dark phase (2300 h). In the brain areas examined, no day/night variations in levels of norepinephrine, epinephrine, dopamine, or 5-HT were seen, although significant nocturnal changes in levels of their metabolites were observed in some areas. Pineal levels of 5-HIAA decreased significantly at night. The retina showed significant nocturnal increases in 5-HTP, 5-HT, and 5-HIAA levels. Retinal levels of 3-MT and DOPAC were significantly decreased at night.     

High-performance liquid chromatographic determination of alpha-acetyldigoxin in Digitalis lanata leaves.

An analytical method for the determination of alpha-acetyldigoxin in Digitalis lanata leaves by HPLC has been developed. The procedure consists of extraction of dry leaf powder with 50% methanol and cleanup by a Sep-Pak C18 cartridge prior to HPLC analysis. The quantitation is carried out by the incorporation of beta-methyldigoxin as an internal standard. HPLC is performed on an octylsilyl bonded silica column with acetonitrile/methanol/water (100/11/188, v/v). The effluent is monitored by uv absorption at 220 nm. The amount of alpha-acetyldigoxin per 100 mg of dry leaf powder is estimated at 5.55 +/- 0.21 micrograms (mean +/- SD). The average recovery of alpha-acetyldigoxin from added samples is 97.2%. The present method is sensitive, reliable, and relatively simple. Application of this HPLC method to the analysis of samples obtained by fermentation of the leaf powder is also demonstrated.     

Simultaneous determination of theophylline and its major metabolites in urine by reversed-phase ion-pair high-performance liquid chromatography

A new, highly selective high-performance liquid-chromatographic (HPLC) assay for theophylline and its major metabolites in urine is described. The method utilizes an ion-pair extraction followed by separation and quantitation by reversed-phase ion-pair gradient-elution HPLC. Comparison with several other methods showed that interferences were present in too many blank urine samples to allow for the accurate quantitation of the metabolites of theophylline by direct injection-isocratic HPLC assays. Sample processing involving ion-pair complexing and extraction together with gradient-elution systems is recommended for accurate pharmacokinetic studies.     

Determination of 9-cis beta-carotene and zeta-carotene in biological samples

Concentrations of 9-cis beta-carotene (9-cis betaC) and zeta-carotene (zetaC) in biological samples may provide crucial information on the biological activities of these carotenoids. However, in high-performance liquid chromatography (HPLC) these carotenoids are often co-eluted. Therefore, there is an urgent need to develop a method for 9-cis betaC and zetaC quantitation. Both 9-cis betaC and zetaC have peak absorbance at 400 and 450 nm, respectively, whereas only 9-cis betaC has peak absorbance at 475 nm. We developed a HPLC method to quantitate 9-cis betaC and zetaC by using peak absorbance ratios. The 9-cis betaC/zetaC peak area was monitored at 475, 450 and 400 nm. The 9-cis betaC was quantified by using absorbance value at 475 nm; zetaC was then calculated from the 9-cis betaC/zetaC peak at 400 nm by subtracting 9-cis betaC contribution at 400 nm using the 400-nm/475-nm peak absorbance ratio of 9-cis betaC (0.39). This method was applied to determine 9-cis betaC and zetaC concentrations in serum and breast milk samples (n=12) from American lactating women and serum and breast adipose tissue samples (n=16) from Korean women with either benign or malignant breast tumors. 9-cis betaC concentrations in serum and breast milk of American women, and serum and adipose tissue of Korean women were 7.1+/-0.8 and 1.1+/-0.2 nM, and 15.6+/-1.1 nM and 0.2+/-0.1 nmol/g, respectively. zetaC concentrations in the above samples were 54.2+/-7.2 and 8.3+/-1.8 nM, and 49.0+/-3.9 nM and 0.3+/-0.1 nmol/g, respectively.     

Identification and quantitation of unique fatty acid oxidation products in human atherosclerotic plaque using high-performance liquid chromatography

Oxidation of lipoproteins, particularly low-density lipoprotein, is thought to play a major role in the development of atherosclerosis. We set out to identify and quantitate the major fatty acid oxidation products in human atherosclerotic plaque obtained from individuals undergoing carotid endarterectomy. Oxidized lipids were extracted from plaque homogenate under conditions to prevent artifactual oxidation. Identification and quantitation was performed using HPLC and GC-MS. High levels of hydroxyoctadecanoic acids (0.51 +/- 0.17 ng/microg of linoleic acid), 15-hydroxyeicosatetranoic acid (HETE) (0.66 +/- 0.24 ng/microg of arachidonic acid), and 11-HETE (0.84 +/- 0.24 ng/microg of arachidonic acid) were detected in all atherosclerotic plaques (n = 10). Low levels of 9-oxo-octadecanoic acid (oxoODE) (0.04 +/- 0.01 ng/microg of linoleic acid), were present in all samples, while 13-oxoODE (0.01 +/- 0.008 ng/microg of linoleic acid) was present in only 4 of the 10 plaque samples. Of interest was the identification of two previously unidentified compounds in atherosclerotic plaque, 11-oxo-eicosatetranoic acid in 9 of the 10 samples and 5,6-dihydroxyeicosatetranoic acid in 3 samples. Chiral analysis revealed that all the major compounds identified in this study are of a nonenzymatic origin. This study is the first to provide a convenient HPLC method to quantify all the products of both linoleic acid and arachidonic acid oxidation in human atherosclerotic plaque. The quantitation of lipid peroxidation products in plaque may be important given the potential biological activity of these compounds and their possible relationship to plaque pathogenesis and instability.     

Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection

We describe a method for the determination of the two major urinary metabolites of vitamin K as the methyl esters of their aglycone structures, 2-methyl-3-(3'-3'-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone) and 2-methyl-3-(5'-carboxy-3'-methyl-2'-pentenyl)-1,4-naphthoquinone (7C-aglycone), by HPLC with electrochemical detection (ECD) in the redox mode. Urinary salts were removed by reversed-phase (C18) solid-phase extraction (SPE), and the predominantly conjugated vitamin K metabolites were hydrolyzed with methanolic HCl. The resulting carboxylic acid aglycones were quantitatively methylated with diazomethane and fractionated by normal-phase (silica) SPE. Final analysis was by reversed-phase (C18) HPLC with a methanol-aqueous mobile phase. Metabolites were detected by amperometric, oxidative ECD of their quinol forms, which were generated by postcolumn coulometric reduction at an upstream electrode. The assay gave excellent linearity (typically, r2 > or = 0.999) and high sensitivity with an on-column detection limit of < 3.5 fmol (< 1 pg). The interassay precision was typically 10%. Metabolite recovery was compared with that of an internal standard [2-methyl-3-(7'-carboxy-heptyl)-1,4-naphthoquinone] added to urine samples just before analysis. Using this methodology, we confirmed that the 5C- and 7C-aglycones were major catabolites of both phylloquinone (vitamin K1) and menaquinones (vitamin K2) in humans. We propose that the measurement of urinary vitamin K metabolite excretion is a candidate noninvasive marker of total vitamin K status.     

Simultaneous determination of the histamine H1-receptor antagonist ebastine and its two metabolites,carebastine and hydroxyebastine,in human plasma using high-performance liquid chromatography

Ebastine (CAS 90729-43-4) is an antiallergic agent which selectively and potently blocks histamine H₁-receptors in vivo. A simple and sensitive high-performance liquid chromatography (HPLC) method is described for the simultaneous determination of ebastine and its two oxidized metabolites, carebastine (CAS 90729-42-3) and hydroxyebastine (M–OH), in human plasma. After a pretreatment of plasma sample by solid-phase extraction, ebastine and its metabolites were analyzed on an HPLC system with ultraviolet detection at 254 nm. Chromatography was performed on a cyano column (250×4.0 mm I.D.) at 40 °C with the mobile phase of acetonitrile–methanol–0.012 M ammonium acetate buffer (20:30:48, v/v/v) at a flow rate of 1.2 ml/min. Accurate determinations were possible over the concentration range of 3–1000 ng/ml for the three compounds using 1 ml plasma samples. The intra- and inter-day assay accuracy of this method were within 100±15% of nominal values and the precision did not exceed 12.4% of relative standard deviation. The lower limits of quantitation were 3 ng/ml for ebastine and its metabolites in human plasma. This method was satisfactorily applied to the determination of ebastine and its two oxidized metabolites in human plasma after oral administration of ebastine.     

Determination of benzethonium chloride in anthrax vaccine adsorbed by HPLC

A novel and sensitive HPLC method for the determination of benzethonium chloride (BZC) in anthrax vaccine was developed. Adjuvant Alhydrogel was removed by syringe filter after a simple sample pretreatment - acidification prior to injection. Chromatography was performed by isocratic reverse phase separation with methanol/262 mM ammonium acetate (80/20, v/v) on an endcapped C18 column with diode array detector (DAD). The method showed excellent recovery (100+/-1.5%). The results indicated that this method could accurately determine BZC at the limit of detection (LOD) of 0.5 ppm and the limit of quantitation (LOQ) of 1.5 ppm with dynamic range up to 100 ppm. The comparison of analysis between new HPLC and old titrimetric methods is also reported. The HPLC method is proven to be more accurate and precise with much less vaccine sample and human labor required.     

Kynurenine 3-hydroxylase inhibition in rats: effects on extracellular kynurenic acid concentration and N-methyl-D-aspartate-induced depolarisation in the striatum

Inhibition of kynurenine 3-hydroxylase suppresses quinolinic acid synthesis and, therefore, shunts all kynurenine metabolism toward kynurenic acid (KYNA) formation. This may be a pertinent antiexcitotoxic strategy because quinolinic acid is an agonist of NMDA receptors, whereas kynurenic acid antagonises all ionotropic glutamate receptors with preferential affinity for the NMDA receptor glycine site. We have examined whether the kynurenine 3-hydroxylase inhibitor Ro 61-8048 increases extracellular (KYNA) sufficiently to control excessive NMDA receptor function. Microdialysis probes incorporating an electrode were implanted into the striatum of anaesthetised rats, repeated NMDA stimuli were applied through the probe, and the resulting depolarisation was recorded. Changes in extracellular KYNA were assessed by HPLC analysis of consecutive dialysate samples. Ro 61-8048 (42 or 100 mg/kg) markedly increased the dialysate levels of KYNA. The maximum increase (from 3.0 +/- 1.0 to 31.0 +/- 6.0 nM; means +/- SEM, n = 6) was observed 4 h after administration of 100 mg/kg Ro 61-8048, but the magnitude of the NMDA-induced depolarisations was not reduced. A separate study suggested that extracellular KYNA would need to be increased further by two orders of magnitude to become effective in this preparation. These results challenge the notion that kynurenine 3-hydroxylase inhibition may be neuroprotective, primarily through accumulation of KYNA and subsequent attenuation of NMDA receptor function.