Identification of 6-hydroxymelatonin in normal human urine by gas chromatography-mass spectrometry.

The major melatonin metabolite, 6-hydroxymelatonin, has been identified in normal human urine by gas chromatography-mass spectrometry (gc-ms) after rapid acid hydrolysis, extraction, and derivatization. An estimate of the amount detected (approximately 20 ng/ml) is consistent with calculated rates of melatonin synthesis and turnover.     

Study on metabolites of 2,5-dimethoxy-4-bromamphetamine (DOB) in human urine using gas chromatography-mass spectrometry

The study of the biotransformation of a new synthetic drug 2,5-dimethoxy-4-bromamphetamine (DOB) and identification of its metabolites in urine of a poisoned person is described using gas chromatography mass spectrometry (GC-MS) with various ways of derivatization. It has been confirmed that one of its metabolic pathways leads to the corresponding 2-O-desmethyl and 5-O-desmethyl metabolites when the latter is prevailing. It is important to know the metabolism of this neurotoxic and hallucinogenic substance as it is a prerequisite for developing reliable toxicological diagnostic procedures and for assessment of toxicological risks.     

Determination of dihydroxynaphthalenes in human urine by gas chromatography-mass spectrometry

A gas chromatography-mass spectrometry (GC-MS) method was developed for measuring 1,2-dihydroxynaphthalene (1,2-DHN) and 1,4-dihydroxynaphthalene (1,4-DHN) in urine. The method involves enzymatic digestion of urinary conjugates to release the DHNs which were then analyzed as trimethylsilyl derivatives by GC-MS. For 1,2-DHN and 1,4-DHN, respectively, the assay limits of detection were 0.21 and 0.15 microg/l, the assay limits of quantitation were 0.69 and 0.44 microg/l, and the coefficients of variation were 14.7 and 10.9%. This method was successfully applied to determine urinary levels of 1,2-DHN and 1,4-DHN in coke workers (14 top workers and 13 side-bottom workers) and 21 matching control workers from the steel industry of northern China. The geometric mean (GM) levels of 1,2-DHN were approximately 100 and 30 times higher than those of 1,4-DHN in exposed and control subjects, respectively. The GM levels 1,2-DHN and 1,4-DHN were significantly higher for coke workers (1,2-DHN: top workers--552 microg/l, side-bottom workers--260 microg/l; 1,4-DHN: top workers--3.42 microg/l, side-bottom workers--3.56 microg/l) than for controls (1,2-DHN: 38.8 microg/l; 1,4-DHN: 1.21 microg/l) (por=0.623; p<0.0001). Also, levels of 1,2-DHN were significantly correlated with those of serum albumin adducts of l,2-naphthoquinone (rs=0.492, p=0.0004). These results indicate that 1,2- and 1,4-DHN are good biomarkers for assessment of naphthalene exposure in coke workers. Since the DHNs are precursors of the naphthoquinones, which have been implicated as toxic products of naphthalene metabolism, measurements of urinary DHNs may have toxicological significance.     

Simultaneous determination of various aromatic amines and metabolites of aromatic nitro compounds in urine for low level exposure using gas chromatography-mass spectrometry

A newly developed method permits the simultaneous quantitative determination of various aromatic amines (or metabolites of aromatic nitro compounds, respectively) in human urine in one analytical run. Applying this method it is possible to determine aniline, toluidines, 4-isopropylaniline, o-anisidine, 3- and 4-chloroaniline, 4-bromoaniline, aminonitrotoluenes, aminodinitrotoluenes, 3,5- and 3,4-dichloroaniline, alpha- and beta-naphtylamine and 4-aminodiphenyl. After separation from the urinary matrix by a simple liquid-liquid extraction at pH 6.2-6.4 the analytes are converted into their pentafluoropropionic acid amides. Separation and quantitative analysis is carried out by capillary gas chromatography and mass-selective detection in the single ion monitoring mode. The limits of detection were within the range from 0.05 microg/l (4-aminobiphenyl, o-anisidine, 3,5-dichloroaniline) to 2 microg/l urine (4-amino-2,6-dinitrotoluene). The relative standard deviation of the within-series imprecision (determined at spiked concentrations of 2.0 microg/l and 10 microg/l) was between 2.9 and 13.6% depending on analyte and concentration. The relative recovery rates were in the range of 70-121%. The analytes that do not contain a nitro function showed better performance regarding the analytical reliability criteria. In order to determine the suitability of this new method for biological monitoring we analysed 20 12-h urine samples of persons without known exposure to aromatic amines, nitroaromatics or precursors in a pilot study. In these samples various aromatic amines could be clearly identified. The general population renally excretes aniline (median: 3.5 microg/l; 95th percentile: 7.9 microg/l), o- (0.12 microg/l; 2.7 microg/l), m- (0.17 microg/l; 2.2 microg/l) and p-toluidine (0.11 microg/l; 0.43 microg/l), and o-anisidine (0.22 microg/l; 0.68 microg/l). Additionally, we found that the persons investigated also excrete 3- (<0.05 microg/l; 0.55 microg/l) and 4-chloroaniline (0.11 microg/l; 0.57 microg/l) as well as 3,5-dichloroaniline (0.18 microg/l; 1.5 microg/l). 3,4-Dichloroaniline was found in some specimens (20%) in concentrations near the limit of detection (<0.05 microg/l; 0.12 microg/l). We did not detect alpha- or beta-naphtylamine, 4-aminobiphenyl or metabolites of explosives in the samples.     

Determination of selenium in urine by isotope dilution gas chromatography-mass spectrometry using 4-nitro-o-phenylenediamine, 3,5-dibromo-o-phenylenediamine, and 4-trifluoromethyl-o-phenylenediamine as derivatizing reagents.

An isotope dilution gas chromatography-mass spectrometry method for selenium determination in urine using 76Se as an internal standard is described. Three different derivatizing reagents, 4-nitro-o-phenylenediamine (NPD), 3,5-dibromo-o-phenylenediamine (DBPD), and 4-trifluoromethyl-o-phenylenediamine (TFMPD) were investigated for their gas chromatographic behavior including memory and precision and accuracy of isotope ratio measurements. By these criteria, the performance of these reagents was TFMPD greater than DBPD greater than NPD. Overall precision values of 1 to 7% were observed in determining Se isotope ratios at the 10-ng level, with no significant difference in using any of the three reagents. Memory effect was observed in the order NPD greater than DBPD greater than TFMPD with TFMPD showing no measurable memory effect. Accuracy of the GC-MS method was verified by quantitation of selenium in the NIST freeze-dried urine reference material SRM 2670.     

Determination of glyphosate, glyphosate metabolites, and glufosinate in human serum by gas chromatography-mass spectrometry

This paper describes an assay for the determination of glyphosate (GLYP), glyphosate metabolites [(aminomethyl) phosphonic acid] (AMPA), and glufosinate (GLUF) in human serum. After protein precipitation using acetonitrile and solid-phase extraction, serum samples were derivatized and analyzed by gas chromatography-mass spectrometry (GC-MS). The assay was linear over a concentration range of 3-100.0 microg/ml for GLYP, AMPA, and GLUF. The overall recoveries for the three compounds were >73%. The intra- and inter-day variations were <15%. Precision and accuracy were 6.4-10.6% and 88.2-103.7%, respectively. The validated method was applied to quantify the GLYP and AMPA content in the serum of a GLYP-poisoned patient. In conclusion, the method was successfully applied for the determination of GLYP and its metabolite AMPA in serum obtained from patient of GLYP-poisoning.     

Identification of fendiline metabolites in human urine

After oral application of 14C labelled fendiline, 13 metabolites of this drug could be identified in human urine. Only traces of parent fendiline were excreted in the urine. The main pathway of metabolism is hydroxylation of phenyl groups with subsequent glucuronidation and sulphation. On the other hand, oxidative dealkylation occurs with the amino group remaining at the 3,3-diphenylpropyl moiety and p-hydroxyacetophenone being formed almost entirely from the 1-phenylethyl group.     

A sensitive method for 4-hydroxybutyric acid in urine using gas chromatography-mass spectrometry

4-Hydroxybutyric acid (4HB) was analyzed by gas chromatography-mass spectrometry. Under acidified conditions, 4HB is difficult to detect due to lactonization. Using a urine sample containing 0.01 mg creatinine, we performed trimethylsilyl derivatization without extraction, only adding dimethylsuccinic acid as an internal standard and 10 microl of 0.1 N NaOH methanol solution with adequate evaporation. Urine 4HB levels in a patient with 4-hydroxybutyric aciduria was determined to be 1258 mmol/mol Cr (control, 0.28-2.81 mmol/mol Cr) in this method. Direct derivatization of samples without extraction showed good reproducibility and linearity. Only a small sample of urine was required. Alkalinization by NaOH prevented not only lactonization of 4HB, but also loss of the compounds during evaporation.     

Measurement of 6-oxo-PGF1 alpha in human plasma using gas chromatography-mass spectrometry.

The plasma concentration of the prostacyclin (PGI2) hydration product 6-oxo-PGF1 alpha has been assayed by stable isotope dilution GC-MS in six normal volunteers infused with increasing doses of PGI2 intravenously. The predosing levels of 6-oxo-PGF1 alpha ranged between 114 and 266 pg/ml. Infusion of PGI2 increased 6-oxo-PGF1 alpha concentration in plasma but the increments were lower than expected suggesting less conversion of the PGI2 to 6-oxo-PGF1 alpha at high infusion rates.     

Identification and determination of propafenone and its principal metabolites in human urine using capillary gas chromatography/mass spectrometry

The capillary gas chromatography/mass spectrometry of trimethylsilyl-trifluoroacetyl, trifluoroacetyl and pentafluoropropionyl (PFP) derivatives of the antiarrhythmic agent propafenone (Rytmonorm), as well as its main metabolites N-despropyl-propafenone and 5-hydroxy-propafenone, have been investigated. Both electron impact and positive isobutane chemical ionization mass spectrometry using the Ion Trap Detector have been evaluated. The presence of propafenone and its co-extracted metabolites in human urine at time intervals after the oral administration of 150 mg Rytmonorm to healthy volunteers was established, and the urinary excretion of propafenone and 5-hydroxy-propafenone was calculated using selective chemical ionization mass spectrometric detection. Only a few per cent of the dose was excreted unchanged in the urine. Large intersubject variabilities had been observed also. The large dynamic range of the Ion Trap Detector and the high correlation coefficients (0.92-0.99) of the calibration curves were striking.     

Detection and identification of carprofen and its in vivo metabolites in greyhound urine by capillary gas chromatography-mass spectrometry

Rimadyl (carprofen) was administered orally to the racing greyhound at a dose of 2.2 mg kg(-1). Following both alkaline and enzymatic hydrolysis, postadministration urine samples were extracted by mixed mode solid-phase extraction (SPE) cartridges to identify target analyte(s) for forensic screening and confirmatory analysis methods. The acidic isolates were derivatised as trimethylsilyl ethers (TMS) and analysed by gas chromatography-mass spectrometry (GC-MS). Carprofen and five phase I metabolites were identified. Positive ion electron ionisation (EI(+)) mass spectra of the TMS derivatives of carprofen and its metabolites show a diagnostic base peak at M(+)*. -117 corresponding to the loss of COO-Si-(CH(3))(3) group as a radical. GC-MS with positive ion ammonia chemical ionisation (CI(+)) of the compounds provided both derivatised molecular mass and some structural information. Deutromethylation-TMS derivatisation was used to distinguish between aromatic and aliphatic oxidations of carprofen. The drug is rapidly absorbed, extensively metabolised and excreted as phase II conjugates in urine. Carprofen, three aromatic hydroxy and a minor N-hydroxy metabolite were detected for up to 48 h. For samples collected between 2 and 8 h after administration, the concentration of total carprofen ranged between 200 and 490 ng ml(-1). The major metabolite, alpha-hydroxycarprofen was detected for over 72 h and therefore can also be used as a marker for the forensic screening of carprofen in greyhound urine.     

Production of dihydrodaidzein and dihydrogenistein by a novel oxygen-tolerant bovine rumen bacterium in the presence of atmospheric oxygen

The original bovine rumen bacterial strain Niu-O16, capable of anaerobically bioconverting isoflavones daidzein and genistein to dihydrodaidzein (DHD) and dihydrogenistein (DHG), respectively, is a rod-shaped obligate anaerobic bacterium. After a long-term domestication, an oxygen-tolerant bacterium, which we named Aeroto-Niu-O16 was obtained. Strain Aeroto-Niu-O16, which can grow in the presence of atmospheric oxygen, differed from the original obligate anaerobic bacterium Niu-O16 by various characteristics, including a change in bacterial shape (from rod to filament), in biochemical traits (from indole negative to indole positive and from amylohydrolysis positive to negative), and point mutations in 16S rRNA gene (G398A and G438A). We found that strain Aeroto-Niu-O16 not only grew aerobically but also converted isoflavones daidzein and genistein to DHD and DHG in the presence of atmospheric oxygen. The bioconversion rate of daidzein and genistein by strain Aeroto-Niu-O16 was 60.3% and 74.1%, respectively. And the maximum bioconversion capacity for daidzein was 1.2 and 1.6 mM for genistein. Furthermore, when we added ascorbic acid (0.15%, m/v) in the cultural medium, the bioconversion rate of daidzein was increased from 60.3% to 71.7%, and that of genistein from 74.1% to 89.2%. This is the first reported oxygen-tolerant isoflavone biotransforming pure culture capable of both growing and executing the reductive activity under aerobic conditions.     

Identification of pheromones in mouse urine by head-space solid phase microextraction followed by gas chromatography-mass spectrometry

Transmission of HIV from mother to infant can be effectively prevented by zidovudine (3'-azido-3'-deoxythymidine; AZT) alone or in combination with other anti-retroviral drugs; however, significant evidence for genotoxicity, including transplacental carcinogenicity in mice, has been reported for AZT. A method, based upon solid phase extraction (SPE) in the 96-well format, gradient liquid chromatography (LC), and electrospray mass spectrometry (MS), was developed and validated to measure serum concentrations in maternal C57BL/6N and fetal B6C3F1 mice of the nucleoside analogs AZT, lamivudine ((-)2',3'-dideoxy-3'-thiacytidine; 3TC), and several metabolites selected based on importance in detoxification and bioactivation reactions. After intravenous (i.v.) and oral dosing with either 400 mg/kg AZT or 200 mg/kg 3TC, pharmacokinetics were determined for AZT, AZT-5'-glucuronide, 3'-amino-3'-deoxythymidine (AMT), AZT-5'-phosphate, 3TC, and 3TC-5'-phosphate in serum of adult female mice. Pharmacokinetics were also determined in spleen for AZT-5'-phosphate and 3TC-5'-phosphate following i.v. dosing. In addition, a preliminary assessment was made of placental transfer of AZT and 3TC and the presence of metabolites in the fetal compartment. The method described provides a means to evaluate thoroughly metabolism and disposition of anti-retroviral nucleoside analogs in maternal and fetal mice for comprehensive studies of genotoxicity.     

Simultaneous determination of bromvalerylurea and allylisopropylacetylurea in human blood and urine by gas chromatography-mass spectrometry

We devised a sensitive and simple method to simultaneously determine bromvalerylurea and allylisopropylacetylurea in human blood and urine by gas chromatography-mass spectrometry. Bromvalerylurea and allylisopropylacetylurea were extracted using an Extrelut column with an internal standard, 2-bromohexanoylurea, followed by derivatization with heptafluorobutyric anhydride. The derivatized extract was submitted to GC-MS analysis of EI-SIM mode. The calibration curves of both compounds were linear in the concentration range from 0.01 to 10 microg/ml in both blood and urine samples. The lower limits of detection of bromvalerylurea and allylisopropylacetylurea were 0.005 and 0.005 microg/ml, respectively. This method proved most useful in accurately identifying these drugs in blood and urine from an autopsied individual.     

Identification of several human urinary metabolites of 6-benzoyl benzoxazolinone by gas chromatography/mass spectrometry.

The biotransformation of 6-benzoyl benzoxazolinone (6-BB), a non-narcotic peripheral analgesic, was studied in eight healthy volunteers after oral administration of a single dose of 1 g. Urinary metabolites were extracted either with ethyl acetate at different pH values or by percolating at pH 5 through Amberlite XAD 2 ion-exchange resin. Eluates were concentrated under vacuum, purified by thin-layer chromatography and analysed by gas chromatography/mass spectrometry or direct insertion probe mass spectrometry. Metabolites were identified with reference to the mass spectra of various synthesized compounds assumed to be metabolites of 6-BB, as N-methylated or monohydroxylated compounds. Another metabolic pathway was cleavage of the benzoxazolinone heterocycle giving 2-amino-5-benzoyl phenol after hydrolysis and decarboxylation. N-methyl, N-acetyl and hydroxylated metabolites having an amino-5-benzoyl phenol structure were also found.     

Determination of pethidine and its major metabolites in human urine by gas chromatography

Procedures based on gas chromatography were established to determine pethidine and its major metabolites in human urine. The chromatographic system consisted of a glass column packed with 3% (w/w) SP2250 on Chromosorb W (80–100 mesh) linked to a nitrogen—phosphorus detector. Diethyl ether was used as the extraction solvent. Pethidinic and norpethidinic acids, and their conjugated metabolites (after β-glucuronidase treatment) were determined after conversion into pethidine and norpethidine by acid-catalysed esterification. The retention times of pethidine, norpethidine and chlorpheniramine (internal standard) were 3.3, 4.5 and 7.5 min, respectively. The amount of unchanged drugs and metabolites excreted varied considerably among the subjects. The mean 24-h urinary recoveries in eight patients of pethidine, norpethidine, pethidinic acid, norpethidinic acid, and the glucuronides of pethidinic and norpethidinic acids were 6.62 ± 5.05, 4.33 ± 1.19, 18.9 ± 6.29, 9.10 ± 4.26, 15.1 ± 3.02 and 7.57 ± 2.28%, respectively. This indicates that the major metyabolic pathways of pethidine in the eight patients were hydrolysis followed by conjugation. Over 60% of the dose was accounted for in 24 h after intramuscular administration of 1 mg/kg pethidine.     

Determination of human fibroblasts metabolism in vitro by gas chromatography-mass spectrometry of cell-excreted metabolites

In order to develop an approach to the study of cell metabolism in vitro, we undertook the determination of metabolites excreted by human skin diploid fibroblasts into culture medium using high-resolution gas chromatography in combination with mass spectrometry. Chromatographic and mass spectrometric characteristics of 29 metabolites have been obtained, and 11 of the metabolites have been identified. The excreted metabolites reflect the activity of certain metabolic processes in fibroblasts in vitro. A comparison of chromatographic and mass spectrometric characteristics of the cell metabolites and of those excreted from the body in urine showed most of the metabolites excreted by fibroblasts to be different from the urine metabolites. The possibility of secondary conversion of cell metabolites in the organism and the specificity of metabolism in cells of different tissues are discussed.     

Determination of human plasma levels of levo-alpha-acetylmethadol and its metabolites by gas chromatography-mass spectrometry

A gas chromatography-mass spectrometry (GC-MS) method is presented which allows the simultaneous determination of the plasma concentrations of the levo-alpha-acetylmethadol (LAAM) and of its active metabolites (NorLAAM and DiNorLAAM), after derivatization with the reagent trifluoroacetic anhydride (TFAA). No interferences from endogenous compounds were observed following the extraction of plasma samples from 11 different human subjects. The standard curves were linear over a working range of 5-200ng/ml for the three compounds. Recoveries measured at three concentrations ranged from 47 to 67% for LAAM, from 50 to 69% for NorLAAM and from 28 to 50% for DiNorLAAM. Intra- and interday coefficients of variation determined at three concentrations ranged from 5 to 13% for LAAM, from 3 to 9% for NorLAAM and from 5 to 13% for DiNorLAAM. The limits of quantitation of the method were found to be 4ng/ml for the three compounds. No interference was noted from methadone. This sensitive and specific analytical method could be useful for assessing the in vivo relationship between LAAM's blood levels, clinical efficacy and/or cardiotoxicity     

Quantitative detection of ketamine, norketamine, and dehydronorketamine in urine using chemical derivatization followed by gas chromatography-mass spectrometry

A repeatable and highly sensitive analytical method using gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring mode (SIM) is developed for the simultaneous detection of ketamine (KT), norketamine (NK), and newly introduced dehydronorketamine (DHNK) in urine. The test specimen along with the deuterium analogues as internal standards (IS): d4-KT for KT and d4-NK for NK/DHNK, was extracted on an automatic solid-phase extraction (SPE) apparatus. The extracted eluate then was dried and derivatized with N-methyl-bis(trifluoroacetamide) (CF3CONCH3COCF3, MBTFA). Finally, the cooled derivatized solution was directly injected into the GC-MS system for analysis. The proposed process achieves high sensitivity for the detection of KT, NK, and DHNK. Correlation coefficients derived from typical calibration curves in the range of 20-2000 ng/mL are 1.000 for KT and NK, 0.999 for DHNK. The limits of detection (LODs) and limits of quantitation (LOQs) are 0.5-1.0 and 1.5-3.0, respectively. The overall method recoveries of KT, NK, and DHNK are 82.2-93.4. The intra- and inter-day run deviations are smaller than 5.0%. The analytical scheme was also applied to the determination of KT, NK, and DHNK in 20 KT suspected urine specimens, and the results reconfirm that DHNK is a main metabolite of KT.     

Studies on the metabolism and toxicological detection of the designer drug 4-methylthioamphetamine (4-MTA) in human urine using gas chromatography-mass spectrometry

4-Methylthioamphetamine (4-MTA) is a scheduled designer drug that has appeared on the illicit drug market and led to several non-fatal or even fatal poisonings. Only few data are available on its metabolism. The first aim of this study was to identify the 4-MTA metabolites in human urine and then to study whether the authors' STA procedure is suitable for screening for and identification of 4-MTA and/or its metabolites in urine. After enzymatic cleavage of conjugates, solid-phase extraction (SPE) and acetylation the following metabolites could be identified by full-scan gas chromatography-mass spectrometry (GC-MS): deamino-oxo 4-MTA, deamino-hydroxy 4-MTA, ring hydroxy and beta-hydroxy 4-MTA. 4-MTA sulfoxide could be identified as possible artifact. In urine samples after enzymatic hydrolysis, acidic extraction, and methylation, 4-methylthiobenzoic acid could be identified. The authors' systematical toxicological analysis (STA) procedure using full-scan GC-MS after acid hydrolysis, liquid-liquid extraction (LLE) and acetylation allowed detection of 4-MTA as target analyte plus all the above-mentioned metabolites with the exception of 4-methylthiobenzoic acid. The extraction efficiency of 4-MTA was approximately 70% and the limit of detection (LOD) was 30 ng/ml (S/N 3).