Sample preparation procedure for determination of dopamine sulfate isomers in human urine by high-performance liquid chromatography with dual-electrode electrochemical detection

We developed a procedure utilizing small columns of solid-phase extraction material for sample preparation for the determination of dopamine sulfate (DAS) isomers in human urine. Processed sample is then subjected to high-performance liquid chromatography (HPLC) with dual-series-electrode electrochemical detection. Dopamine 3-O-sulfate (DA-3-S) and dopamine 4-O-sulfate (DA-4-S) were determined using two different HPLC systems. The ratio of the urinary excretion rate of DA-3-S to DA-4-S was relatively constant, but the 24-h excretion rates of total DAS varied widely among individuals. This method should prove useful in future studies concerning the metabolic and physiologic roles of DAS isomers.     

Determination of dopamine-3- and 4-O-sulphate in human plasma and urine by anion-exchange high-performance liquid chromatography with fluorimetric detection

A high-performance liquid chromatographic (HPLC) method is reported for the determination of dopamine-3- and -4-O-sulphate isomers in human plasma and urine using an anion exchanger coupled with post-column hydrolysis and fluorimetric detection. Samples of plasma or urine are partially purified on Dowex 1 and Dowex 50 columns and separated using HPLC. These compounds are then hydrolysed and determined automatically by the p-aminobenzoic acid method in a continuous-flow reaction system. As the p-aminobenzoic acid method is very specific for dopamine, it is also possible to determine the isomers by injecting 5-20 microliter of urine or 100-200 microliter of deproteinized plasma directly into the HPLC system without clean-up. The detection limit of the method for both isomers is 0.3 pmol. In normal subjects, the plasma levels of dopamine-3- and -4-O-sulphate are 26.5 (S.D. 11.1) and 2.68 (S.D. 0.34) pmol/ml, and their urinary excretion rates are 1.73 (S.D. 0.56) and 0.27 (S.D. 0.04) nmol/min, respectively. Thus the two isomers are present in both plasma and urine and their urinary excretions reflect directly their plasma levels.     

Electron paramagnetic resonance study on free radical scavenging and/or generating activity of dopamine-4-O-sulfate

The free radical scavenging and/or generating activity of dopamine-4-O-sulfate was examined and compared with that of dopamine. In humans, dopamine mostly exists in two isomeric forms of sulfate ester conjugates as metabolites; i.e., dopamine-3-O-sulfate and dopamine-4-O-sulfate in the circulation. Dopamine is generally believed to be oxidized by molecular oxygen or another reactive oxygen species under physiological conditions, to form oxidized dopamine derivatives that are cytotoxic. However, it is not known whether dopamine conjugates are generated on interaction with reactive oxygen species or not. In the present study, we measured the susceptibility to oxidization of dopamine-4-O-sulfate by using electron paramagnetic resonance (EPR) spectroscopy and optical absorption spectrometry. Dopamine was easily oxidized and dopamine-derived radicals appeared, whereas dopamine-4-O-sulfate was not oxidized under physiological conditions. Furthermore, dopamine-4-O-sulfate did not react with a strong oxidizing agent, sodium periodate. These results suggest that dopamine-4-O-sulfate has resistance against autoxidation, and seems to be a stable metabolite of dopamine.     

Determination of the anti-allergenic agent, 2-methoxy-11-oxo-11H-pyrido[2,1-b]quinazoline-8-carboxylic acid, in biological fluids by high-performance liquid chromatography.

A rapid, sensitive, and specific high performance liquid chromatographic (HPLC) assay was developed for the determination of 2-methoxy-11-oxo-11H-pyrido-[2,1-b]quinazoline-8-carboxylic acid (I) from biological fluids. The overall recovery from blood and plasma is 69 +/- 10% (S.D.) and 84 +/- 6% (S.D.), respectively, and the sensitivity limit of quantitation is 100 ng/ml by UV absorption and 5 ng/ml by fluorescence detection using a 1 ml specimen. The assay was used in the determination of blood levels of compound in the Rhesus monkey following intravenous administration of a 10 mg/kg dose, and of blood and urine levels of compound I in a dog following intravenous and oral administration of a 1 mg/kg dose.     

Determination of amdinocillin in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of amdinocillin (formerly mecillinam) in human plasma and urine. The assay is performed by direct injection of a plasma protein-free supernatant or a dilution of urine. A 10 micrometer muBondapak phenyl column with an eluting solvent of water--methanol--1 M phosphate buffer, pH 7 (70:30:0.5) was used, with UV detection of the effluent at 220 nm. Azidocillin potassium salt [potassium-6-(D-(-)-alpha-azidophenyacetamido)-penicillanate] was used as the internal standard and quantitation was based on peak height ratio of amdinocillin to that of the internal standard. The assay has a recovery of 74.4 +/- 6.3% (S.D.) in the concentration ranges of 0.1-20 microgram per 0.2 ml of plasma with a limit of detection equivalent to 0.5 microgram/ml plasma. The urine assay was validated over a concentration range of 0.025-5 mg/ml of urine, and has a limit of detection of 0.025 mg/ml (25 microgram/ml) using a 0.1-ml urine specimen per assay. The assay was applied to the determination of plasma and urine concentrations of amdinocillin following intravenous administration of a 10 mg/kg dose of amdinocillin to two human subjects. The HPLC and microbiological assays were shown to correlate well for these samples.     

Determination of the anxiolytic agent 8-chloro-6-(2-chlorophenyl)-4H-imidazo-[1,5-alpha] [1,4]-benzodiazepine-3-carboxamide in whole blood, plasma or urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of 8-chloro-6-(2-chlorophenyl)-4H-imidazo-[1,5-alpha]-[1,4]-benzodiazepine-3-carboxamide [I] and its 4-hydroxy metabolite, 8-chloro-6-(2-chlorophenyl)-4-hydroxy-4H-imidazo-[1,5-alpha] [1,4]-benzodiazepine-3-carboxamide [II] in whole blood, plasma or urine. The assay for both compounds involves extraction into diethyl ether-methylene chloride (70:30) from blood, plasma, or urine buffered to pH 9.0. The overall recoveries of [I] and [II] are 92.0 +/- 5.4% (S.D.) and 90.3 +/- 4.9% (S.D.), respectively. The sensitivity limit of detection is 50 ng/ml of blood, plasma, or urine using a UV detector at 254 nm. The HPLC assay was used to monitor the blood concentration-time fall-off profiles, and urinary excretion profiles in the dog following single 1 mg/kg intravenous and 5 mg/kg oral doses, and following multiple oral doses of 100 mg/kg/day of compound [I].     

Assessment of the biotransformation of the cardiotonic agent piroximone by high-performance liquid chromatography and gas chromatography-mass spectrometry.

14C-labelled piroximone was administered to rats at a dose of 10 mg/kg body weight. Of the total radioactivity administered, 74.9 +/- 7.9% (n = 4) and 87.8 +/- 1.7 (n = 3) were recovered in the 8-h urine collection after oral and intravenous administration, respectively. Two major metabolites, M1 and M2, were detected in methanol extracts and accounted for 7.1 +/- 1.2% (n = 4) (M1) and 4.3 +/- 0.4% (n = 4) (M2) in response to oral administration and 5.7 +/- 0.8% (n = 3) (M1) and 6.7 +/- 2.0% (n = 3) (M2) in response to intravenous administration. In addition, three minor metabolites were detected; M3 and M4 in the 8-h urine collection and M5 in the 12-h urine collection. Separation of piroximone and metabolites was achieved by high-performance liquid chromatography on a C18 column by gradient elution with 0.05 M ammonium acetate (pH 7) using 0-60% methanol over 20 min at a flow-rate of 1 ml/min, followed by isocratic elution with 60% methanol for 10 min. M1 and M2 were isolated by fraction collection following the addition of 1 mM tetrabutylammonium acetate in the mobile phase. Between each injection a column re-equilibration time of 45 min was necessary to achieve optimum collection of M1 and M2 fractions. Gas chromatography-mass spectrometry of M1 provided evidence for a molecular structure consistent with isonicotinic acid methyl ester. Corroborative evidence for this identification was obtained by comparison with a synthetic standard. Isonicotinic acid is assumed to be the actual metabolite while esterification with methanol had occurred as a result of the work-up procedure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of 2-mercaptopropionylglycine in plasma and urine by high-performance liquid chromatography

Methods for quantitative analysis of total and non-protein-bound 2-mercaptopropionylglycine (2-MPG) in plasma, and total 2-MPG in urine, have been developed. By reduction of urine, plasma or deproteinized plasma samples with tributylphosphine, 2-MPG is liberated from its disulphides, and after clean-up of the sample, 2-MPG is derivatized with N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide (DACM). The 2-MPG-DACM derivative is then quantified by high-performance liquid chromatography (HPLC) with fluorimetric detection. Both ion-suppression and ion-pair HPLC gave satisfactory chromatograms. The precision of the methods was satisfactory (coefficient of variation 3.1-5.8%), analytical recovery was quantitative (85-99%) and the two HPLC techniques were well correlated (r = 0.99). Five healthy subjects receiving 500 mg of 2-MPG showed maximal total plasma concentration of 13.8-26.9 mumol/l at 3-5 h after intake, and their non-protein-bound 2-MPG was, at the same time, 62-77% of the total 2-MPG. The urinary excretion was 27.8 +/- 3.8% (mean +/- S.D.) of the given dose, most of it excreted within 12 h after intake.     

Mass fragmentographic determination of prostaglandin F2 alpha in human and rabbit urine

Analysis of prostaglandin F2 alpha (PGF2 alpha) in urine is a useful indicator of renal prostaglandin synthesis. A mass fragmentographic method for PGF2 alpha analysis in human urine was developed using [3,3,4,4-2H4]PGF2 alpha as an internal standard and carrier. PGF2 alpha was extracted from urine (20 ml) with chloroform, purified by preparative thin-layer chromatography and converted to the methyl ester trimethylsilyl ether before analysis by gas chromatography--mass spectrometry. The specificity of the urine analysis was demonstrated by retention time and the use of two pairs of fragments m/e 494/498 and 513/517 with the same results. The coefficient of variation for duplicate analysis averaged 12.6%, n = 17. Urine from recumbent women contained 4.9 +/- 2.6 (S.D.) ng/ml or 4.1 +/- 1.0 ng PGF2 alpha per mg creatinine (n = 10) with little diurnal variation. Male urine contained 5.0 +/- 2.7 (S.D.) ng/ml or 3.7 +/- 2.1 ng/mg creatinine (n = 10). Similar concentrations were found in boys and in girls. These observations indicate that urinary PGF2 alpha originates from the kidneys with little contribution from the male accessory sexual glands. This method can also be applied to analysis of PGF2 alpha in rabbit urine.     

Multiple inverse isotope dilution assay for the stereospecific quantitative determination of R(-) and S(+)-oxaprotiline in biological fluids

An isotope dilution assay for the determination of both oxaprotiline enantiomers in biological samples after administration of the racemic mixture has been developed. The enantiomers were reacted with synthetically prepared, optically pure N-trifluoroacetyl-S(-)-prolyl chloride, followed by high-performance liquid chromatographic separation of the diastereoisomers formed. Quantitation was performed by on-line UV detection at 260 nm and off-line radiometry by liquid scintillation counting. Endogenous compounds and metabolites do not interfere in the assay. Analysis of water and the blood and urine of rats spiked with [14C]oxaprotiline X HCl showed recoveries for S(+)-oxaprotiline X HCl (mean +/- coefficient of variation, n = 4-6) of 98.0 +/- 1.0% (water), 100.5 +/- 0.6% (blood) and 101.5 +/- 2.0% (urine), and for R(-)-oxaprotiline X HCl of 101.3 +/- 2.0% (water), 102.2 +/- 2.1% (blood) and 103.2 +/- 0.2% (urine). A pilot study to determine blood levels of the two enantiomers in two rats dosed with racemic [14C]oxaprotiline X HCl (10 mg/kg i.v.) was carried out to test the method. The results indicated stereoselective disposition of oxaprotiline enantiomers in the rat. The ratio of the areas under the blood concentration curves for R(-)-to S(+)-oxaprotiline X HCl was 1.14.     

Novel fiber coated with β-cyclodextrin derivatives used for headspace solid-phase microextraction of ephedrine and methamphetamine in human urine

Heptakis (2,6-di-O-methyl)-β-cyclodextrin (DM-β-CD) blending with hydroxy-terminated silicone oil (OH-TSO) coated solid-phase microextraction (SPME) fiber (DM-β-CD/OH-TSO) was first prepared with sol-gel technology and applied to headspace SPME for analysis of ephedrine (EP) and methamphetamine (MA) in human urine by gas chromatography (GC). By exploiting the advantages of the unique cavity-shaped cyclic molecular structure of CD and the superiorities of sol-gel coating technique, the novel fiber showed desirable extraction ability and operational stability. Influence of relevant experimental parameters (extraction time, extraction temperature, basicity, ionic strength, etc.) was systematically investigated. In the optimal conditions the proposed headspace SPME-GC method provided good linearity over four orders of magnitude with limit of detection (LOD) of ng/ml (0.33 ng/ml for EP, 0.60 ng/ml for MA). The recoveries of EP and MA in urine were 98.0% and 98.2%. And the relative standard deviations (R.S.D., n = 6) for EP and MA were 3.9% and 5.0%, respectively.     

Four new andrographolide metabolites in human urine

Andrographolide is one of principal components of a famous traditional Chinese herbal medicine Andrographis paniculate (BURM) NEES. Four new metabolites of andrographolide were isolated from human urine. All of them were characterized as sulfate and one of them also as a cysteine S-conjugate. The structures were determined to be andrographolide-3-O-sulfate (M-1), isoandrographolide-3-O-sulfate (M-2), 14-deoxyandrographolide-3-O-sulfate (M-3), 14-deoxy-12-(cysteine-S-yl)-andrographolide-3-O-sulfate (M-4), respectively, based on chemical evidence and spectroscopic analyses.     

Simultaneous determination of tranilast and metabolites in plasma and urine using high-performance liquid chromatography

A method has been developed for the simultaneous determination of Tranilast, N-(3',4'-dimethoxycinnamoyl)anthranilic acid (N-5'), and metabolites in plasma and urine from humans, dogs and rodents administered N-5'. Total N-5' and metabolite N-3 conjugates were determined in human urine. Detection limits in plasma were 0.2 micrograms/ml for metabolite N-3-S and N-5' and 0.1 micrograms/ml for metabolites N-3 and N-4. In urine, detection limits were 2 micrograms/ml for metabolite N-3-S and N-5' and 1 micrograms/ml for metabolites N-3 and N-4. Metabolite N-4 was not identified in any sample assayed.     

An in vitro and in vivo investigation into the suitability of bacterially triggered delivery system for colon targeting

The colon specific drug delivery systems based on polysaccharides; locust bean gum and chitosan in the ratio of 2 : 3, 3 : 2 and 4 : 1 were evaluated using in vitro and in vivo methods. The in vitro studies in pH 6.8 phosphate buffer containing 2% w/v rat caecal contents showed that the cumulative percentage release of mesalazine after 26 h were 31.25+/-0.56, 46.25+/-0.96, 97.5+/-0.26 (mean+/-S.D.), respectively. The in vivo studies conducted in nine healthy male human volunteers for the various formulations revealed that, the drug release was initiated only after 5 h (i.e.) transit time of small intestine and the bioavailability (AUC(0-->t*)) of the drug was found to be 85.24+/-0.10, 196.08+/-0.12, 498.62+/-0.10 microg x h/ml 26 (mean+/-S.D.), respectively. These studies on the polysaccharides demonstrated that the combination of locust bean gum and chitosan as a coating material proved capable of protecting the core tablet containing mesalazine during the condition mimicking mouth to colon transit. In particular, the formulation containing locust bean gum and chitosan in the ratio of 4 : 1 held a better dissolution profile, higher bioavailability and hence a potential carrier for drug targeting to colon.     

Determination of clorazepate and its major metabolites in blood and urine by electron capture gas-liquid chromatography.

A sensitive and specific blood level method employing differential extraction was developed for the determination of clorazepate and its N-desmethyldiazepam metabolite by electron capture gas-liquid chromatography (GLC-ECD). The assay requires the initial extraction of N-desmethyldiazepam, the major metabolite, into benzene-methylene chloride (90:10) from the biological sample made alkaline with 0.1 N NaOH. The samples is then acidified with 2 N HCl to decarboxylate clorazepate to N-desmethyldiazepam, which is then extracted into benzene-methylene chloride (90:10) after adjusting the pH to 12.8 with NaOH. The two extracts are evaporated and the residues are dissolved in benzene which contains griseofulvin as the reference standard. These solutions are assayed by GLC-ECD. The overall recovery and sensitivity limit of the assay for clorazepate is 60+/-5% (S.D.) and 4.0 ng/ml blood, respectively, while that for N-desmethyldiazepam is 95+/-5% (S.D.) and 4.0 ng/ml blood, respectively. The urinary excretion of clorazepate was determined by the measurement of the levels of N-desmethyldiazepam and oxazepam, the major urinary metabolites of clorazepate, both prior to and after enzymatic deconjugation. These methods were applied to the measurement of clorazepate and its metabolites in blood and urine following a single 15-mg dose of clorazepate dipotassium.     

Determination of vigabatrin in human plasma and urine by high-performance liquid chromatography with UV-Vis detection

A simple and reliable high-performance liquid chromatographic (HPLC) method with UV-Vis detection has been developed and validated for the determination of vigabatrin (VG) in human plasma and urine. The samples were pre-column derivatizated with 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS). A good chromatographic separation was achieved on a C18 column with a mobile phase consisting of acetonitrile and 10 mM orthophosphoric acid (pH 2.5) gradient elution. Tranexamic acid was used as an internal standard (I.S.). The method was linear over the concentration range of 0.8-30.0 microg/ml for both samples. The method is precise (relative standard deviation (R.S.D.) <9.13%) and accurate (relative mean error (RME) <-8.75%); analytical recoveries were 81.07% for plasma and 83.05% for urine. The assay was applied to pharmacokinetic study in a healthy volunteer after a single oral administration of 1 g of vigabatrin.     

Determination of water soluble imidazo-1,4-benzodiazepines in blood by electron- capture gas--liquid chromatography and in urine by differential pulse polaragraphy.

A sensitive and specific electron-capture gas--liquid chromatographic (GLC--ECD) assay was developed for the determination of 8-chloro-6-(2'-fluorophenyl)-1-methyl-4H-imidazo(1,5a)(1,4)benzodiazepine (I) or 8-chloro-1,4-dimethyl-6-(2'-fluorophenyl)-4H-imidazo (1,5a)(1,4)benzodiazepine (II) in blood. The assay for both compounds involves extraction into benzene--methylene chloride (9:1) from blood buffered to pH 12.6 The overall recovery of I and II from blood is 86% +- 5.0 (S.D.) and the sensitivity limit of detection is of the order of 2 to 3 ng of I or II per milliltre of blood. The major urinary metabolite of I is 8-chloro-6-(2'-fluorophenyl)-1-hydroxymethyl-4H-imidazo(1,5a)(1,4)benzodiazepine, (IA) present as a glucuronide conjugate while 8-chloro-6-(2'-fluorophenyl)-4-hydroxyl-1-methyl-4H-imidazo(1,5a)(1,4)benzodiazepine, (IB) and 8-chloro-6-(2'-fluorophenyl)-4-hydroxy-1-hydroxymethyl-4H-imidazo(1,5a)(1,4) benzodiazepine, (IC) are minor metabolites. The major metabolite IA is extracted into benzene--methylene chloride (9:1) from urine buffered to pH 11.0 (after incubation with glucuronidase--sulfatase as pH 5.0), and analyzed by differential pulse polarography (DPP) in 0.1 M phosphate buffer PH 3). The overall recovery of IA is 84 +- 3.0% (S.D.) with a sensitivity limit of 50 ng per millilitre of urine. The metabolites of compound II have not as yet been elucidated. The GLC--ECD and DPP assays were applied to the determination of blood levels and urinary excretion in dogs following single 10 mg/kg intravenous and oral doses of I and following single 6 mg/kg intravenous and 10 mg/kg oral doses of II. Blood levels of compound I were also evaluated in man following intravenous infusion of single 10 mg doses.     

Measurement of catecholamines, their precursor and metabolites in human urine and plasma by solid-phase extraction followed by high-performance liquid chromatography with fluorescence derivatization

A high-performance liquid chromatographic method is described for the determination in human urine and plasma of catecholamines, their precursor and metabolites [amino compounds (norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine, 3-methoxytyramine and L-DOPA), acidic compounds (3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, vanillylmandelic acid and homovanillic acid) and alcoholic compounds (3,4-dihydroxyphenylethyleneglycol and 4-hydroxy-3-methoxyphenylethyleneglycol)]. Urine (0.5 ml) containing 3,4-dihydroxybenzylamine and 4-hydroxy-3-methoxycinnamic acid (internal standards) is deproteinized with perchloric acid, and the resulting solution is fractionated by solid-phase extraction on a strong cation-exchange resin cartridge (Toyopak IC-SP S) into two fractions (amine fraction and acid-alcohol fraction), which include 3,4-dihydroxybenzylamine and 4-hydroxy-3-methoxycinnamic acid, respectively. Plasma (0.7 ml) is deproteinized in the presence of 3,4-dihydroxybenzylamine (internal standard) in the same manner, and the resulting solution is directly used as an acid-alcohol fraction, while an amine fraction is obtained as for urine. Each fraction is subjected to the previously established ion-pair reversed-phase chromatography with post-column derivatization involving coulometric oxidation followed by fluorescence reaction with 1,2-diphenylethylenediamine. The detection limits, at a signal-to-noise ratio of 5, of the compounds measured in urine are 300 pmol/ml for the two mandelic acids, 2-7 pmol/ml for the other acidic and alcoholic compounds, 12 pmol/ml for L-DOPA and 0.6-2 pmol/ml for the other amino compounds; the corresponding values for plasma samples are 80, 0.5-3, 10 and 0.6-3 pmol/ml, respectively.     

The fractal nature of Escherichia coli biological flocs

The fractal structures of Escherichia coli biological flocs were characterised in terms of fractal dimension, which is a measurement of how the bacteria in the flocs occupy space. The dimensional analysis methods, based on power law correlations between floc perimeter, projected area and maximum length, were used to determine the one- and two-dimensional fractal dimensions (D(1) and D(2)) of E. coli flocs formed by flocculation in chitosan solution with a concentration of 10.0 mg chitosan per g dry cell weight (DCW), giving D(1)=1.07+/-0.06 and D(2)=1.70+/-0.08 (+/-S.D.). The three-dimensional fractal dimension (D(3)) of the E. coli flocs was determined by the two-slopes method, using cumulative size distributions of floc length and solid volume, to be 1.99+/-0.08 (+/-S.D.), which is close to the value of D(3)=2.14+/-0.04 (+/-S.D.) measured by the small angle light scattering method. The results demonstrate that E. coli flocs flocculated with chitosan have a fractal nature, as their fractal dimensions D(1), D(2) and D(3) differ from the values of 1, 2 and 3 expected for the spherical Euclidean object, respectively.     

Chromatographic determination of N-acetyl-DL-tryptophan and octanoic acid in human albumin solutions

Chromatographic procedures have been developed for determination of the stabilizers N-acetyl-DL-tryptophan and octanoic acid in human albumin solutions. N-Acetyl-DL-tryptophan and the internal standard, N-formyl-DL-tryptophan, were separated by liquid chromatography on a reversed-phase column with UV detection at 280 nm. Deproteinization and extraction were carried out with methanol. The extraction recovery at the level of 4.9 mM was 92.5 +/- 2.5% (S.D.) (n = 10), and the average coefficient of variation (C.V.) for replicate analyses of albumin solutions (mean = 2.57, 10.44 and 17.10 mM) was 1.10% (n = 27). Octanoic acid was determined gas chromatographically as its methyl ester, with nonanoic acid as the internal standard. The sample pretreatment included acidification, extraction with hexane and derivatization with methanol-sulphuric acid. The relative recovery from albumin solutions was 89.7 +/- 5.8% (S.D.) (n = 6), and replicate determinations of the compound yielded a C.V. of 5.5% (mean = 14.82 mM, n = 9).