Sensitive biomarker of polycyclic aromatic hydrocarbons (PAHs): urinary 1-hydroxyprene glucuronide in relation to smoking and low ambient levels of exposure.

The study was conducted in a Chinese population with occupational or environmental exposures to polycyclic aromatic hydrocarbons (PAHs). A total of 106 subjects were recruited from coke-oven workers (workers), residents in a metropolitan area (residents) and suburban gardeners (gardeners). All subjects were monitored twice for their personal exposures to PAHs. The biological samples were collected for measurements of 1-hydroxypyrene (1-OHP) and cotinine in urine. The geometric means of personal exposure levels of pyrene, benz(a)anthracene (BaA) and benzo(a)pyrene (BaP) in workers were 1.470, 0.978 and 0.805 microg m-3, respectively. The corresponding levels in residents were 0.050, 0.034 and 0.025 microg m-3; and those in gardeners were 0.011, 0.020 and 0.008 microg m-3, respectively. The conjugate of 1-OHP with glucuronide (1-OHP-G) is the predominant form of pyrene metabolite in urine and it showed strong associations with exposures not only to pyrene, but also to BaA, BaP and total PAHs. Most importantly, a significant difference in 1-OHP-G was even detected between the subgroups with exposures to BaP at < 0.010 and > 0.010 but < 0.020 microg m-3, suggesting that 1-OHP-G is a good marker that can be used for the risk assessment of BaP exposure at levels currently encountered in ambient air. Furthermore, multiple regression analyses of 1-OHP-G on PAHs exposure indicated that cigarette smoke was a major confounding factor and should be considered and adjusted for while using 1-OHP to estimate PAHs exposure.     

Determination of ticarcillin epimers in plasma and urine with high-performance liquid chromatography

A high-performance liquid chromatogaphic method was developed for determining the concentrations of ticarcillin (TIPC) epimers in human plasma and urine. Samples were prepared for HPLC analysis with a solid-phase extraction method and the concentrations of TIPC epimers were determined using reversed-phase HPLC. The mobile phase was a mixture of 0.005 M phosphate buffer (pH 7.0) and methanol (12:1, v/v) with a flow-rate of 1.0 ml/min. TIPC epimers were detected at 254 nm. Baseline separation of the two epimers was observed for both plasma and urine samples with a detection limit of ca. 1 μg/ml with a S/N ratio of 3. No peaks interfering with either of the TIPC epimers were observed on the HPLC chromatograms for blank plasma and urine. The recovery was more than 80% for both plasma and urine samples. C.V. values for intra- and inter-day variabilities were 0.9–2.1 and 1.1–6.4%, respectively, at concentrations ranging between 5 and 200 μg/ml. The present method was used to determine the concentrations of TIPC epimers in plasma and urine following intravenous injection of TIPC to a human volunteer. It was found that both epimers were actively secreted into urine and that the secretion of TIPC was not stereoselective. Plasma protein binding was also measured, which revealed stereoselective binding of TIPC in human plasma.     

A sensitive high-performance liquid chromatographic procedure with fluorometric detection for the analysis of decarboxylated S-adenosylmethionine and analogs in urine samples

A highly sensitive HPLC method for the determination of decarboxylated S-adenosylmethionine (dc-SAM) by fluorometric detection was developed. The reaction of dc-SAM and its analogs with chloroacetaldehyde leads to the corresponding 1,N6-etheno derivatives. These highly fluorescent derivatives were fully characterized through their proton nuclear magnetic resonance spectra and/or mass spectra. This derivatization procedure has been applied to the analysis of dc-SAM in rat and human urine. After a simple cation exchange column prepurification, the urine extracts were derivatized with chloroacetaldehyde and analyzed by reversed-phase HPLC with fluorometric detection. The method allowed the determination of subpicomole amounts of dc-SAM and was shown to be highly reproducible with the use of decarboxylated S-adenosylethionine as internal standard. The application of the method to the analysis of urine of rats treated with MDL 72175, a potent ornithine decarboxylase inhibitor, showed that the dc-SAM levels increased in a dose-related fashion.     

High-performance liquid chromatographic determination of the new quinolone antibacterial agent DU-6859a in human serum and urine using solid-phase extraction with photolysis—fluorescence detection

A sensitive and specific HPLC method for the determination of DU-6859a (I), a fluoroquinolone antibacterial agent, in human serum and urine was developed. Compound I and the internal standard extracted from serum and urine by means of a Bond Elut C₈ LRC cartridge showed recoveries of 96%. The extracts were chromatographed on a reversed-phase column with photolysis—fluorescence detection. This unique detection method was 42.5 times more sensitive than intrinsic fluorescence detection, the limits of detection being in 3.43 ng/ml for serum and 4.35 ng/ml for urine. In addition, I was stable in serum and urine for at least 1 month at −20°C. The proposed method was sensitive and selective enough to apply to pharmacokinetic studies of I in humans after a single oral dose of 100 mg.     

Simultaneous determination of chlorpromazine and levomepromazine in human plasma and urine by high-performance liquid chromatography using electrochemical detection

A rapid, selective and sensitive method for the simultaneous determination of chlorpromazine and levomepromazine in human plasma and urine has been developed using high-performance liquid chromatography with electrochemical detection.The unchanged drugs and internal standard extracted from plasma and urine were separated by reversed-phase high-performance liquid chromatography. The influence of acetonitrile concentration and of the pH of the mobile phase were investigated. The detection limits were 100 pg for chlorpromazine and for levomepromazine. In comparison with three other detection systems this was found to be the most sensitive method.This method was successfully applied to the simultaneous determination of chlorpromazine and levomepromazine in human plasma and urine for pharmacokinetic studies.     

Determination of some benzodiazepines and metabolites in serum,urine and saliva by high-performance liquid chromatography

The performance of a number of liquid—solid systems, consisting of mixtures of buffers (0.05 M) and methanol as mobile phase and methyl-silica as stationary phase, were investigated with respect to their use in the separation of 1,4-benzodiazepines by reversed-phase high-performance liquid chromatography with UV detection at 254 nm. Phase system selectivities and column efficiencies were determined. A nomogram is presented from which the chromatographic parameters can be calculated.A complete separation of nine benzodiazepines within 12 min has been achieved, using methyl-silica as the stationary phase and 50% methanol as the eluent.The results were applied to the development of a method for the determination of therapeutic levels of diazepam and its metabolites in human serum, urine and saliva. The first step in the analysis, the extraction of diazepam and its metabolites from serum and urine, was also investigated and good recoveries were achieved. A low detection limit (0.2 ng) and high precision were obtained. The concentrations of diazepam and its metabolites in human serum, urine and saliva were determined after both single and multiple oral doses of diazepam (and oxazepam).     

Analysis of oxazepam in urine using solid-phase extraction and high-performance liquid chromatography with fluorescence detection by post-column derivatization

A reversed-phase high-performance liquid chromatographic method for oxazepam in human urine samples has been developed. The sample preparation consists of an enzymatic hydrolysis with β-glucuronidase, followed by a solid-phase extraction process using Bond-Elut C₂ cartridges. The mobile phase used was a methanol—water (60:40, v/v) mixture at a flow-rate of 0.50 ml/min. The column was a 3.5 cm × 4.6 mm I.D. C₁₈ reversed-phase column. The detection system was based on a fluorescence post-column derivatization of oxazepam in mixtures of methanol and acetic acid. A linear range from 0.01 to 1 μg/ml of urine and a limit of detection of 4 ng/ml of urine were attained. Within-day recoveries and reproducibilities from urine samples spiked with 0.2 and 0.02 μg/ml oxazepam were 97.9 and 95.0 and 2.1 and 9.4%, respectively.     

Fluorescence derivatizing procedure for 5-hydroxytryptamine and 5-hydroxyindoleacetic acid using 1,2-diphenylethylenediamine reagent and their sensitive liquid chromatographic determination

A pre-column derivatization method using a fluorogenic reagent, 1,2-diphenylethylenediamine (DPE) was studied for the sensitive HPLC determination of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), which are biosubstances used in the diagnosis of several diseases. For the quantitative determination, the biogenic indole compounds were converted to their corresponding fluorescent derivatives with DPE in the presence of potassium hexacyanoferrate (III) at room temperature, and then the derivatives were separated by reversed-phase liquid chromatography with fluorescence detection. The chromatographic detection limits of the fluorescent peaks at a signal-to-noise ratio of 3 were 0.3 fmol for 5-HT and 0.2 fmol for 5-HIAA. The proposed method permits the simultaneous quantification of 5-HT and 5-HIAA at concentrations higher than 2.4 nM in human urine without a clean-up procedure.     

Simultaneous determination of F-β-alanine and β-alanine in plasma and urine with dual-column reversed-phase high-performance liquid chromatography

F-β-Alanine and β-alanine were detected in plasma and urine samples with fluorescence detection of orthophthaldialdehyde derivatives of F-β-alanine and β-alanine after separation with dual-column reversed-phase HPLC. The detection limits of F-β-alanine and β-alanine in the HPLC system were approximately 0.3 and 0.7 pmol, respectively. The procedure proved to be very reproducible with intra-assay RSDs and inter-assay RSDs being less than 8%. The usefulness of the method was demonstrated by the analysis of the F-β-alanine and β-alanine concentrations in plasma and urine samples from tumor patients treated with S-1 (Tegafur, 5-chloro-2,4-dihydroxypyridine and potassium oxonate in a molar ratio of 1:0.4:1).     

Determination of grepafloxacin in plasma and urine by a simple and rapid high-performance liquid chromatographic method

A rapid, specific, sensitive and economical method has been developed and validated for the determination of grepafloxacin in human plasma and urine. The assay consisted of reversed-phase HPLC with UV detection. Plasma proteins were removed by a fast and efficient procedure that has eliminated the need for costly extraction and evaporation. For the urine samples, the only required sample preparation was dilution. Separation was achieved on a reversed-phase TSK gel column with an isocratic mobile system. The method had a quantification limit of 0.05 μg/ml in plasma and 0.5 μg/ml in urine. The coefficients of variation (C.V.) were less than 4% for within- and between-day analyses. The method was successfully applied to a pharmacokinetic study, and was proved to be simple, fast and reproducible.     

Validation of a simplified method for determination of cimetidine in human plasma and urine by liquid chromatography with ultraviolet detection

A HPLC method was developed for determination of cimetidine in human plasma and urine. Plasma samples were alkalinized followed by liquid extraction with water-saturated ethyl acetate then evaporated under nitrogen. The extracts were reconstituted in mobile phase and injected onto a C(18) reversed-phase column; UV detection was set at 228 nm. Urine samples were diluted with an internal standard/mobile phase mixture (1:9) prior to injection. The lower limit of quantification in plasma and urine were 100 ng/ml and 10 microg/ml, respectively; intra- and inter-day coefficients of variation were 相似文献   

Simultaneous determination of nefiracetam and its metabolites by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic assay was developed to simultaneously quantitate nefiracetam (NEF), a novel nootropic agent, and its three known oxidized metabolites (N-[(2,6-dimethylphenylcarbamoyl)methyl]succinamic acid (5-COOH-NEF), 4-hydroxy-NEF and 5-hydroxy-NEF) in human serum and urine. The quantitative procedure was based on solid-phase extraction with Sep-Pak C₁₈ and ultraviolet detection at 210 nm. The calibration curves of NEF and the metabolites were linear over a wide range of concentrations (0.5–21.5 nmol/ml for NEF and 0.4–9.5 nmol/ml for metabolites in serum and 4–86 nmol/ml for NEF and 8–190 nmol/ml for metabolites in urine). Intra- and inter-day assay coefficients of variation for the compounds were less than 10%. The limit of detection was 0.1 nmol/ml for NEF, 5-COOH-NEF and 4-hydroxy-NEF, and 0.2 nmol/ml for 5-hydroxy-NEF in both serum and urine. This method is applicable for the determination of NEF and its metabolites in human serum and urine with satisfactory accuracy and precision.     

Determination of dexamethasone in urine by gas chromatography with negative chemical ionization mass spectrometry

Dexamethasone, as some other synthetic corticosteroids, is licensed for therapy in veterinary practice, but its misuse as a growth promotor, often in combination with beta-agonists, is forbidden. In this report an analytical method is described for the detection and confirmation of very low concentrations of dexamethasone in urine. The influence of enzymatic hydrolysis time of samples with glucuronidase was studied. The proposed method consisted of the enzymatic hydrolysis of urine samples, which were then extracted and concentrated using solid-phase cartridges with mixed reversed-phase materials (OASIS). No further clean-up step was found to be necessary. Eluates were derivatized following a previously described method [Analyst 119 (1994) 2557]. Detection, identification and quantification of residues of this compound was carried out by gas chromatography with mass spectrometry in the negative chemical ionization mode. The proposed procedure permits the determination of dexamethasone in urine at levels as low as 0.2 ng ml(-1)     

Gas chromatographic method with mass-selective detection for the determination of 2-isopropoxyphenol in human urine

Human metabolism of the insecticide propoxur yields 2-isopropoxyphenol (IPP) which is excreted conjugated in urine. In this publication a sensitive and selective analytical method is described which permits the determination of IPP as a suitable parameter for biomonitoring. The clean-up of the hydrolysed urine samples consisted of steam distillation and solid-phase extraction using a reversed-phase column. IPP and the internal standard 2-ethoxyphenol were converted to their pentafluorobenzyl ethers. Excess of the derivatisation reagent was removed using deactivated silica gel. Separation and quantitative analysis was carried out by capillary gas chromatography and mass selective detection. Coefficients of variation were below 5% for concentrations from 6 to 300 μg/l. The detection limit was 0.5 μg/l. The method was checked by analysing six urine samples from pest controllers after indoor application of propoxur. The IPP concentrations ranged from 45 to 306 μg/g creatinine. IPP was not detected in urine specimens from 10 non-exposed persons. The sensitivity of the developed method permits the detection of latent exposure to propoxur.     

On-line clean-up and screening of oxacillin and cloxacillin in human urine and plasma with a weak ion exchange monolithic column

A weak ion exchange monolithic column prepared by modifying the GMA-MAA-EDMA (glycidyl methacrylate-methacrylic acid-ethylene glycol dimethacrylate) monoliths with ethylenediamine was applied to remove matrix compounds in biological fluid. Using this monolithic column, on-line clean-up and screening of oxacillin and cloxacillin in human urine and plasma samples had been investigated. Chromatography was performed by reversed-phase HPLC on a C(18) column with ultraviolet detection at 225 nm. Results showed that the ion exchange monolithic column could be used for deproteinization and retaining oxacillin and cloxacillin in human urine and plasma, which provided a simple and fast method for assaying drugs in human urine and plasma.     

Simultaneous determination of N2-(3-aminopropyl)biopterin (oncopterin), biopterin and neopterin by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method is described for the simultaneous determination of N²-(3-aminopropyl)biopterin (oncopterin, a newly found natural pteridine in urine from cancer patients), biopterin, and neopterin in urine. For the detection and quantification of the compounds, fluorometry was used. Using Develosil ODS K-5 and Develosil ODS HG-5 reversed-phase columns and a Nucleosil 100-5SA strong cation-exchange column, oncopterin, biopterin, and neopterin in urine were completely separated and assayed simultaneously by fluorescence detection. Similar values of oncopterin were obtained using each of the three columns, and the Develosil ODS K-5 reversed-phase column gave the most satisfactory separation. The sensitivity was high enough to measure 1 pmol of each pteridine. The HPLC method was highly reproducible. Our preliminary results indicate that oncopterin could be a most sensitive marker for cancer.     

Quantitative analysis of a model opioid peptide and its cyclic prodrugs in rat plasma using high-performance liquid chromatography with fluorescence and tandem mass spectrometric detection

An isocratic reversed-phase HPLC method for the simultaneous quantitation of alpha-lipoic acid and five of its metabolites in human plasma as well as in human urine employing solid-phase extraction and pulsed amperometric detection was developed and validated. The method was found to be sufficiently precise and accurate for the measurement of alpha-lipoic acid and its metabolites 6,8-bis(methylthio)octanoic acid, 4,6-bis(methylthio)hexanoic acid and 2,4-bis(methylthio)butanoic acid in plasma and urine samples, obtained from patients suffering from diabetic neuropathy as well as from healthy volunteers following daily oral administration of 600 mg alpha-lipoic acid. The quantitation of the metabolite bisnorlipoic acid was often impaired by interferences caused by an unidentified metabolite. However, bisnorlipoic acid was detected in few test samples and the concentrations were consistently low. Despite the poor recovery of the metabolite tetranorlipoic acid from plasma, reproducibility and accuracy were found to be from acceptable magnitude to assess concentration time courses. Thus, the obtained analytical results are considered as reliable and well suited for pharmacokinetic studies of alpha-lipoic acid and its metabolites.     

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

A rapid, reliable and specific reversed-phase high-performance liquid chromatographic procedure is described for the determination of diphenylpyraline hydrochloride at nanogram concentrations in plasma and urine. After extraction of the drug with n-pentane-2-propanol (50:1) from alkalinized samples, the organic extract was evaporated to dryness, reconstituted with methanol and chromatographed using a 5-μm Asahipak ODP-50 C₁₈ column with UV detection at 254 nm. The elution time for diphenylpyraline was 7.9 min. The overall recovery of diphenylpyraline from spiked plasma and urine samples at concentrations ranging from 53 to 740 ng/ml were 94.65% and 92.29%, respectively. Linearity and precision data for plasma and urine standards after extraction were acceptable. The limit of detection was 15 ng/ml for both plasma and urine samples at 0.002 AUFS.     

Determination of pilocarpine, isopilocarpine, pilocarpic acid and isopilocarpic acid in human plasma and urine by high-performance liquid chromatography with tandem mass spectrometric detection

A method is described for the determination of pilocarpine and its degradation products isopilocarpine, pilocarpic acid and isopilocarpic acid in human plasma and urine. The method is based on a simple sample preparation step – ultrafiltration for plasma and dilution for urine samples – followed by a reversed-phase liquid chromatographic separation of the analytes and detection by means of tandem mass spectrometry. Parameters affecting the performance of these steps are discussed. The high sensitivity and selectivity of the method allow low ng/ml concentrations to be determined for all compounds in plasma and undiluted urine, which enables the investigation of the metabolic fate and elimination of pilocarpine after oral administration to humans.