Routine determination of urinary free catecholamines by high-performance liquid chromatography with electrochemical detection

A simple and reliable high-performance liquid chromatographic method is described for the routine determination of the free catecholamines (norepinephrine, epinephrine and dopamine) in urine. The catecholamines are isolated from urine samples using small affinity chromatography columns prepacked with immobilised m-aminophenylboronic acid, separated by ion-pair reversed-phase liquid chromatography and quantified by electrochemical detection. Total analysis, including sample preparation time, is achieved in less than 30 min with analytical recoveries of 92-96% for all three catecholamines. Long-term stability and reproducibility of the liquid chromatographic system is attained by selection of optimised conditions for chromatographic separation with a formate mobile phase and produces detection limits of 1.4, 1.8 and 2.2 nmol/l for norepinephrine, epinephrine and dopamine, respectively, in urine samples and day-to-day coefficients of variation of less than 6%. Furthermore, the affinity isolation gels can be reused a minimum of ten times providing a rapid and cost-effective means of sample preparation.     

High performance liquid chromatographic determination of aluminium in natural waters in the form of its lumogallion chelate

A high performance liquid chromatographic method for the determination of ultra trace amount of aluminium in natural waters has been developed using lumogallion as a precolumn reagent for fluorimetric detection. The highly fluorescent Al-lumogallion chelate (lambda(ex) 500 nm, lambda(em) 574 nm) was separated on a LiChrosorb RP 18 column with an eluent containing 3:7 acetonitrile/0.02M potassium hydrogen phthalate buffer (pH 4.7) containing 10(-5)M lumogallion. The proposed system provides a simple, quick, selective and sensitive method for the determination of ultra-trace amount of aluminium in water samples. The detection limit defined as three times the standard deviation of the blank signal, was 0.05 mug/l. in water samples for 100 mul injection. The tolerance limits were 5 mg/l. for Fe(III) and F(-) and over 10 mg/l. for other foreign ions. The sensitivity of the method was independent of salinity. This method had been used for the direct determination of aluminium in both tap and coastal sea-waters without any preconcentration steps.     

Determination of 2-naphthylamine in urine by a novel reversed-phase high-performance liquid chromatography method.

A high-performance liquid chromatographic method for the determination of 2-naphthylamine in urine using fluorescence detection was developed. The method validation analysis showed the method to be in analytical control, i.e. the distribution of the difference between the observed and true values of the method evaluation samples did not deviate significantly from the normal distribution. The recovery of the method was 85%. The entire run time of chromatography was 10 min using isocratic elution (acetonitrile-water, 35:65), and the retention time for 2-naphthylamine was 5.8 min. The relative short time of analysis in combination with the low limit of detection (0.272 nmol/l) makes the method potentially applicable for surveillance of occupational and environmental exposure to 2-nitronaphthalene. The developed method is presently used for measurement of 2-naphthylamine in urine samples from workers employed at factories, characterized by a low airborne exposure level of polycyclic aromatic hydrocarbons, i.e. in general less than 25 micrograms/m3. The urine samples of exposed workers (n = 95) showed a 2-naphthylamine range of up to 9.4 nmol/l, whereas unexposed control individuals (n = 114) showed a range of up to 0.87 nmol/l.     

Exposure to 4,4'-methylenediphenyl diisocyanate (MDI) during moulding of rigid polyurethane foam: determination of airborne MDI and urinary 4,4'-methylenedianiline (MDA)

Occupational exposure to 4,4'-methylenediphenyl diisocyanate (MDI) was measured during moulding of rigid polyurethane foam. The aim of the study was to find out whether an MDI-derived urinary amine metabolite could be detected in the urine of workers exposed to apparently low levels of MDI. Airborne MDI was sampled on 1-(2-methoxyphenyl)-piperazine (2MP)-impregnated glass fibre filters and determined by high-performance liquid chromatography (HPLC) using ultraviolet (UV) and electrochemical (EC) detection. The limit of detection of MDI was 3 ng ml-1 for a 20 microliters injection. The precision of sample preparation, expressed as relative standard deviation (RSD), was 1.3% with UV detection and 2.1% with EC detection at a concentration of 70 ng MDI ml-1 (n = 6). The 2MP-MDI derivative was stable at +4 degrees C up to eight weeks. The accuracy of the method was validated in an international quality control programme. Workers (n = 57) from three different factories participated in the study. Urinary 4,4'-methylenedianiline (MDA) metabolite was determined after acid hydrolysis as heptafluorobutyric anhydride derivatives by gas chromatography-mass spectrometry using chemical ionisation and monitoring negative ions. The limit of detection in urine was 0.2 nmol l-1. The precision of six analyses for a urine sample spiked to a concentration of 1 nmol l-1 was 29% (RSD). The MDI concentrations were below the limit of detection in most (64%) of the air samples collected in the worker's breathing zone. Still, detectable amounts of MDA were found in 97% of the urine samples. Monitoring of urinary MDA appears to be an appropriate method of assessing MDI exposure in work environments with low or undetectable MDI concentrations in the workplace air.     

Excretion of iodide in 24-h urine as determined by ion-pair reversed-phase liquid chromatography with electrochemical detection

A simple method is presented for the routine analysis of iodide in urine. After a one-step sample clean-up, iodide was separated by ion-pair reversed-phase liquid chromatography and detected electrochemically with a silver electrode. The coefficient of variation of a single analysis of iodide in a pooled urine sample (530 nmol/l) was 7.6%. The detection limit, derived from a signal-to-noise ratio of 3, was 3 pmol, corresponding to 0.06 mumol/l. The recovery of iodide added to urine was 96 +/- 7%. The accuracy of the method was assessed by analysing ten different samples with neutron activation analysis. The data obtained with the two methods showed a high correlation (r = 0.991) and did not differ significantly. Excretion of iodide in samples of 24-h urine from a free-living population was shown to have a log-normal distribution and to be higher in men than in women. The iodide/creatinine ratio was independent of sex and increased with age.     

High-performance liquid chromatographic determination of 4,4'-methylenedianiline in human urine

A method is described for the determination of urinary 4,4'-methylenedianiline (MDA) by high-performance liquid chromatography (HPLC). MDA was extracted from hydrolyzed urine using C18 solid-phase extraction columns. The extract was analyzed by reversed-phase HPLC with electrochemical detection at a cell potential of 0.8 V. The method was very sensitive (detection limit 2.5 micrograms/l) and quantitation using 4,4'-ethylenedianiline as an internal standard correlated well with results by gas chromatography-mas spectrometry. Run-to-run precision (n = 25) averaged 8.9%. In analysis of more than 160 potentially exposed workers, MDA was detected in less than 20% of the urines and concentrations ranged up to 210 micrograms MDA per g of creatinine.     

‘Wrong‐way‐round ionization’ and screening for doping substances in human urine by high‐performance liquid chromatography/orbitrap mass spectrometry

To free analytical resources for new classes of doping substances, such as banned proteins, maximization of the number of compounds that can be determined with high sensitivity in a single run is highly urgent. This study demonstrates an application of ‘wrong‐way‐round ionization’ for the simultaneous detection of multiple classes of doping substances without the need to switch the polarity. A screening method for the detection of 137 compounds from various classes of prohibited substances (stimulants, diuretics, β₂‐agonists, β‐blockers, antiestrogens, glucocorticosteroids and anabolic agents) has been developed. The method involves an enzymatic hydrolysis, liquid–liquid extraction and detection by liquid chromatography/orbitrap mass spectrometry with wrong‐way‐round ionization. Up to 64% of compounds had a 10‐fold lower limit of detection (LOD) than the minimum required performance limit. To compare the efficiency of conventional ionization relative to wrong‐way‐round ionization of doping substances in + ESI, a fortified blank urine sample at the minimum required performance limit was analyzed using two ESI approaches. All compounds were detected with markedly better S/N in a high‐pH mobile phase, with the exception of acetazolamide (minimal change in S/N, < 20%).The method was validated by spiking 10 different blank urine samples at five different concentrations. Validation parameters included the LOD, selectivity, ion suppression, extraction recovery and repeatability. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of 6-N-trimethyllysine in urine by high-performance liquid chromatography

A method for determination of 6-N-trimethyllysine in urine is described. Trimethyllysine and the chemically analogous 6-N-triethyllysine internal standard were isolated from aqueous samples by microcolumn ion-exclusion chromatography. The specimens were derivatized by reaction with 1-fluoro-2,4-dinitrobenzene and reaction byproducts extracted by organic solvents. The trimethyllysine and internal standard derivatives were separated easily from other sample constituents by reversed-phase paired-ion high-performance liquid chromatography with spectrophotometric detection at 405 nm. Standard curves were linear over a sample concentration range of 10-150 nmol/ml; the detection limit corresponded with 0.1 nmol trimethyllysine injected into the chromatograph. The procedure was used for determination of trimethyllysine in human urine.     

Plutonium determination in bioassay samples using radiochemical thermal ionization mass spectrometry

Summary A new high-sensitivity plutonium bioassay program employing thermal ionization mass spectrometry (TIMS) has been developed to monitor Savannah River Site employees for intakes of PuO₂. The U.S. Department of Energy requires bioassay laboratories which have the ability to detect a 100 mRem, 50-year committed effective dose equivalent (CEDE) intake of radioactive material. For PuO₂, traditional alpha-spectrometry methods are not sensitive enough to meet this specification. To comply with this requirement, a radiochemical TIMS method was developed to determine Pu in urine bioassay samples. Four radiochemical separation steps were used to purify Pu from urine to ensure samples were free from matrix effects that interfere with TIMS analysis. These included precipitation, ion-extraction chromatography, electrodeposition, and ion-exchange chromatography. A batch of reagent blanks determined the detection limit for this method was 0.59 fg ²³⁹Pu/l (1.3 µBq ²³⁹Pu/l). The ²³⁹Pu concentration was also measured in 20 urine blank samples to determine the minimum ²³⁹Pu concentration that would indicate an occupational intake. A Probit plot was constructed for the results and the 99 th percentile of the urine blanks showed that the minimum ²³⁹Pu concentration that would indicate an uptake was 2.4 fg/l (5.5 µBq/l).     

Determination of urinary 8‐hydroxy‐2′‐deoxyguanosine by a combination of on‐line molecularly imprinted monolithic solid phase microextraction with high performance liquid chromatography–ultraviolet detection

8‐Hydroxy‐2′‐deoxyguanosine (8‐OHdG) is a sensitive biomarker for DNA oxidative damage. However, its determination in human urine is confounded by trace level and complex matrix. In this study, a new configuration of on‐line solid phase microextraction coupled to high performance liquid chromatography‐ultraviolet detection was established with molecularly imprinted monolithic column as extraction sorbent. The tailor made monolith exhibited high extraction efficiency with the enrichment factor 101.84 for 8‐OHdG owing to its special porous structure and inherent selectivity. Under optimal condition, appreciable sensitivity had been achieved for this incorporation with limit of detection 2.04 nmol/L (S/N = 3) and limit of quantification 7.12 nmol/L (S/N = 10), respectively. Precise determination with wide range linearity (0.007–5.00 μmol/L) afforded a practical alternative in urinary 8‐OHdG analysis and 107 different subjects had been successfully analyzed. This newly developed method embodied useful prospect for the investigation of DNA oxidative damage with less expense, convenient maintenance and ease of operation     

Bio-analysis of vinorelbine by high-performance liquid chromatography with fluorescence detection.

A simple and selective procedure for the determination of vinorelbine, a new semi-synthetic vinca alkaloid, is presented. The method is based on ion-exchange high-performance liquid chromatography on normal-phase silica with fluorescence detection, combined with liquid-liquid extraction using diethyl ether for sample clean-up. The absence of endogenous interferences and the excellent chromatographic behaviour of vinca alkaloids provides accurate results even at low concentrations. The limit of determination in plasma is 1.5 micrograms/l (500-microliters sample). Reproducible recoveries in urine were obtained if 10-50 microliters of sample were processed supplemented with 500 microliters of blank plasma.     

Quantitative analysis of the sulfur mustard hydrolysis product thiodiglycol (2,2'-sulfobisethanol) in in vivo microdialysates using gas chromatography coupled with pulsed flame photometric detection

An analytical method employing gas chromatography is presented for assessing the concentrations of the sulfur mustard hydrolysis product thiodiglycol (TDG) in cutaneous in vivo microdialysates. The use of a pulsed flame photometric detector allows for selective detection of the analyte following solvent exchange and derivatization with heptafluorobutyric anhydride. Quantitative assessment is performed using thiodipropanol (TDP) as a surrogate internal standard. A linear relationship and a very significant correlation (r2 = 0.9982) between the ratio of TDG and TDP concentrations and the ratio of the square root of peak heights is demonstrated. The suitability of the analytical method is verified by the evaluation of blank in vivo microdialysates spiked with known amounts of TDG. The limit of detection in microdialysates is 0.200 nmol/mL (24.4 ng/mL) and the limit of quantitation was 0.364 nmol/mL (44.4 ng/mL). The presented method provides selective, sensitive, rapid, and high-throughput analysis of microdialysates containing TDG, providing an efficient alternative for high-performance liquid chromatography and capillary electrophoresis techniques.     

A highly sensitive method for the determination of 7‐aminonitrazepam,a metabolite of nitrazepam,in human urine using high‐performance electrospray liquid chromatography tandem mass spectrometry

A highly sensitive method has been developed for the determination of urinary 7‐aminonitrazepam (7‐ANZP), the major metabolite of nitrazepam, using high‐performance electrospray liquid chromatography tandem mass spectrometry. The samples were prepared for analysis by adding 7‐aminoclonazepam (7‐ACZP, internal standard), hydrolysis with β‐glucuronidase and liquid–liquid extraction. Mass spectral acquisition was achieved by selectively monitoring the reaction between the two diagnostic transition reactions. Qualitative analysis was based on the retention time, and the quantitation was carried out by comparison with the internal standard. The recoveries of different concentrations of 7‐ANZP from spiked blank samples was 89.0–95.2%, and the relative standard deviation was below 6.4%. The limit of determination in urine was 0.07 ng/mL, and the limit of quantitation was 0.5 ng/mL in the linear range of 1–50 ng/mL. This method possesses the merits of convenient operation, high sensitivity and good repeatability, making it a practical method for analysis of 7‐ANZP in urine. Copyright © 2009 John Wiley & Sons, Ltd.     

Combination of a sub‐3 μm superficially porous particle packed column with charged aerosol detection for the simple and sensitive measurement of nine macrolides in human urine

Due to the lack of chromophores in many macrolides, analytical methods based on mass spectrometry and electrochemical detection coupled to liquid chromatography have been suggested to be suitable for the quantification of macrolides in complex matrices. In this study, a simple and sensitive analytical method was established for the simultaneous measurement of nine macrolides in human urine by combining a sub‐3 μm superficially porous particle packed column with charged aerosol detection. After thorough investigation of various sample preparation methods, including two liquid–liquid extraction methods and four solid‐phase extraction methods, HLB solid‐phase extraction was selected and further optimized. Absolute recovery of the optimized sample preparation method ranged from 99.5–110.2%, indicating its very high extraction/clean‐up efficiency. For chromatography, parameters influencing macrolide separation were systematically optimized, and the resulting conditions allowed baseline separation of nine macrolides within 24 min using a very simple mobile phase. The established method was validated for linearity, limit of detection, limit of quantification, absolute recovery, and precision. Based on its limit of detection (0.025–0.100 μg/mL), the method had similar or greater sensitivity than most methods based on electrochemical detection. It was found that the current method was appropriate for application to real human urine samples after drug administration.     

Metabolic studies of explosives. 5. Detection and analysis of 2,4,6-trinitrotoluene and its metabolites in urine of munition workers by micro liquid chromatography/mass spectrometry

Metabolites of 2,4,6-trinitrotoluene (TNT) were found in the urine of a group of TNT munition workers. The urine extracts were analysed by micro liquid chromatography/mass spectrometry. The metabolites found included 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, 2,4-diamino-6-nitrotoluene, 2,6-diamino-4-nitrotoluene and untransformed TNT. The detection limit of the metabolites in urine was 0.1 ng/ml for 20 ml urine samples.     

New extraction procedure and high-performance liquid chromatographic method for analyzing polyethylene glycol-400 in urine.

We describe a new, highly efficient method for extracting polyethylene glycol-400 from urine and for its analysis by isocratic reversed-phase high-performance liquid chromatography. This method is an improvement over previously published methods in that it does not require the use of ion-exchange resins and lyophilization prior to extraction, nor does it require the separation and analysis of the individual polymers of polyethylene glycol. The procedure described in this report entails extraction with a salt-solvent combination of ammonium sulfate and dichloromethane and analysis by reversed-phase high-performance liquid chromatography. The lower limit of detection was approximately 0.25 g/l with a 2-ml urine sample. Analytical recoveries of polyethylene glycol-400 added to urine at 2.5 and 5.0 g/l averaged 97 and 96%, respectively (n = 10). Within- and between-day coefficients of variation were less than 5% at 2.5 and 5.0 g/l. Studies of various urine samples from patients receiving polyethylene glycol-400 revealed no interferences from other urine substances.     

Determination of metoprolol and two major metabolites in plasma and urine by column liquid chromatography and fluorometric detection

Metoprolol and its alpha-hydroxy metabolite were determined in plasma down to 2 nmol/l (S.D. 10-15%) after solvent extraction and bonded-phase liquid chromatography with fluorometric detection. The major metabolite with a carboxylic function was also measured in plasma when liquid-solid extraction on a column activated with dodecyl sulphate was applied. In urine the three components were assayed by direct injection of a diluted sample.     

Determination of the heroin metabolite 6-acetylmorphine by high-performance liquid chromatography using automated pre-column derivatization and fluorescence detection

An improved method for the determination of 6-acetylmorphine in the urine of drug addicts receiving morphine was developed. A newly introduced reversed-phase high-performance liquid chromatographic system proved to be more sensitive than a normal-phase system used previously. By replacing the earlier manual derivatization procedure with an automated on-line pre-column method, both the reproducibility and efficiency were considerably improved. Coefficients of variation for repeated analyses typically ranged from 6 to 10% in the 1-100 micrograms/l concentration range. The detection limit was 1 microgram/l and the correction for recovery by calibration with blank urine samples spiked with 6-acetylmorphine was satisfactory. The analytical improvements achieved, however, did not increase the chance of detecting heroin use by drug addicts.     

Microanalytical high-performance liquid chromatographic assay for cefpirome in human milk and urine.

To permit the characterization of cefpirome disposition in lactating females, a previously published high-performance liquid chromatographic (HPLC) method for determining the drug in serum was adapted for use with milk and urine. This automated, microanalytical technique requires 50 microliters of biological matrix, which is subjected to an isopropanol extraction. Chromatography was accomplished using a microbore HPLC system, a reversed-phase C18 column and a mobile phase of 0.3% triethylamine in water (pH 5.1). Cefpirome and the internal standard (beta-hydroxypropyltheophylline) were monitored using UV detection at 240 nm and had retention times of 2.84 and 5.05 min, respectively. The method was linear up to 500 mg/l for both matrices and had a limit of detection of 0.6 mg/l. The interday variation (relative standard deviation) at concentrations of 5.0, 50.0 and 500.0 mg/l was consistently less than 5% in both urine and breast milk. The method was found to be free from interference by other commonly administered medications and readily adaptable for use in clinical investigations. The ease of sample preparation, small sample volume requirement, short chromatographic time, apparent lack of interferences, analytical sensitivity and high precision and accuracy make this method ideal for use in pharmacokinetic investigations involving the determination of cefpirome in human milk and urine.     

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.