Detection of 4-hydroxycoumarin anticoagulants and their metabolites in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons by gas chromatography–mass spectrometry after extractive methylation

A gas chromatography–mass spectrometry (GC–MS) procedure was developed for the detection of 4-hydroxycoumarin anticoagulants and their metabolites in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons after extractive methylation. The part of the phase-transfer catalyst remaining in the organic phase was removed by solid-phase extraction on a diol phase. The compounds were separated by capillary GC and identified by computerized MS in the full scan mode. Using mass chromatography with the ions m/z 291, 294, 295, 309, 313, 322, 324, 336, 343 and 354, the possible presence of 4-hydroxycoumarin anticoagulants and/or their metabolites could be indicated. The identity of positive signals in such mass chromatograms was confirmed by comparison of the peaks underlying full mass spectra with the reference spectra recorded during this study. This method allowed the detection of therapeutic concentrations of phenprocoumon and warfarin in human urine samples. In absence of human urine, acenocoumarol, coumachlor, coumatetrayl, pyranocoumarin (cyclocumarol) could be detected only in rat urine.     

Identification of 2-guanidinoethanol in human urine

Guanidino compounds in normal human urine were analyzed by high-performance liquid chromatography; an unknown peak was observed in the chromatogram that was identical to the peak of synthetic 2-guanidinoethanol. In another experiment, the substance was purified from human urine by successive use of strongly acidic ion-exchanger, thin-layer chromatography and then weakly acidic ion-exchanger. After this it was reacted with acetylacetone to form dimethylpyrimidyl derivative. After further reaction of this derivative with trifluoroacetic anhydrate, it was analyzed by gas chromatography/mass spectrometry. The mass chromatogram and mass spectrum were identical to those of the trifluoroacetylated dimethylpyrimidyl derivative of synthetic 2-guanidinoethanol. This is the first report on the identification of 2-guanidinoethanol in human urine. The concentration of 2-guanidinoethanol in the urine of healthy humans was 5.7 +/- 1.8 (mean +/- SD) mumol/g creatinine.     

Qualitative Profiling and Quantification of Neonicotinoid Metabolites in Human Urine by Liquid Chromatography Coupled with Mass Spectrometry

Neonicotinoid pesticides have been widely applied for the production of fruits and vegetables, and occasionally detected in conventionally grown produce. Thus oral exposure to neonicotinoid pesticides may exist in the general population; however, neonicotinoid metabolites in human body fluids have not been investigated comprehensively. The purpose of this study is the qualitative profiling and quantitative analysis of neonicotinoid metabolites in the human spot urine by liquid chromatography coupled with mass spectrometry (LC/MS). Human urine samples were collected from three patients suspected of subacute exposure to neonicotinoid pesticides. A qualitative profiling of urinary metabolites was performed using liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) with a database of nominal molecular weights of 57 known metabolites of three neonicotinoid pesticides (acetamiprid, Imidacloprid, and clothianidin), as well as the parent compounds. Then a quantitative analysis of selected urinary metabolites was performed using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with a standard pesticide and metabolite, which were detected by the qualitative profiling. The result of qualitative profiling showed that seven metabolites, i.e. an acetamiprid metabolite, N-desmethyl-acetamiprid; three Imidacloprid metabolites, 5-hydroxy-Imidacloprid, 4,5-dihydroxy-imidacloprid, 4,5-dehydro-Imidacloprid; a common metabolite of acetamiprid and Imidacloprid, N-(6-chloronicotinoyl)-glycine; and two clothianidin metabolites, N-desmethyl-clothianidin, N-(2-(methylsulfanyl)thiazole-5-carboxyl)-glycine, as well as acetamiprid, were detected in the urine of three cases. The result of the quantitative analysis showed N-desmethyl-acetamiprid was determined in the urine of one case, which had been collected on the first visit, at a concentration of 3.2 ng/mL. This is the first report on the qualitative and quantitative detection of N-desmethyl-acetamiprid in the human urine. The results suggest that the one case with detection of N-desmethyl-acetamiprid was exposed to acetamiprid through the consumption of contaminated foods. Urinary N-desmethyl-acetamiprid, as well as 5-hydroxy-Imidacloprid and N-desmethyl-clothianidin, may be a good biomarker for neonicotinoid exposure in humans and warrants further investigation.     

Metabolism and toxicological detection of the new designer drug 3',4'-methylenedioxy-alpha-pyrrolidinopropiophenone studied in urine using gas chromatography-mass spectrometry

R,S-3',4'-Methylenedioxy-alpha-pyrrolidinopropiophenone (MDPPP) is a new designer drug with assumed amphetamine-like effects, which has appeared on the illicit drug market. The aim of this study was to identify the MDPPP metabolites using solid-phase extraction, ethylation or acetylation as well as to develop a toxicological detection procedure in urine using solid-phase extraction, trimethylsilylation and GC-MS. Analysis of urine samples of rats treated with MDPPP revealed that MDPPP was completely metabolized by demethylenation of the methylenedioxy group followed by partial 3'-methylation of the resulting catechol, oxidative desamination to the corresponding diketo compounds and/or hydroxylation of the pyrrolidine ring with subsequent dehydrogenation to the corresponding lactam. The hydroxy groups were found to be partly conjugated. Based on these data, MDPPP could be detected in urine via its metabolites by full-scan GC-MS using mass chromatography for screening and library search for identification by comparison of the spectra with reference spectra.     

New polar acid metabolites in human urine

A sequence of chromatographic methods (thin-layer chromatography, high-performance liquid chromatography and glass capillary gas chromatography) was used to separate the acid fraction of human urine. The power of this method to separate and detect previously unknown compounds and the elucidation of their final structure with mass spectrometry is exemplified by the identification of N-acetyl-2-aminooctanoic acid as a metabolic compound in the urine of healthy individuals.In addition, the conjugate of glycine with indolepropionic acid, N-formylanthranilic acid, succinoylphenylalanine, δ-hydroxyvaleric acid, δ-hydroxycapric acid, 3-hydroxyadipic acid, and higher homologues were detected in a polar fraction of human urine.     

Characterization of odorants in human urine using a combined chemo-analytical and human-sensory approach: a potential diagnostic strategy

The volatile and odorous profile of human urine may be a rich source for physiological information and could increase our understanding of metabolization and excretion processes of low-molecular weight compounds originating from, for example, dietary or endogenous sources. However, the diagnostic potential of the urinary volatile fraction is not yet fully understood, probably due to the limited application of modern analytical tools in urine volatile analysis. Accordingly, the aim of this study was to evaluate a combined chemo-analytical and human-sensory approach for characterization of the human urine odorant composition. We used one- and two-dimensional high resolution gas chromatography–olfactometry/mass spectrometry to identify commonly occurring and potent odorants in human urine. The studies were carried out on both native urine and on urine that had been treated by glucuronidase assays, with analysis of the liberated odor-active compounds using the same techniques. Based on retention indices, odor qualities and intensities, and mass spectra compared to references, a total of 14 odorants were detected in the majority of the untreated urine samples, and 24 odorants in the glucuronidase-treated samples. A major part of the identified substances are reported here for the first time. Our results show that chemosensory approaches are a useful strategy for the characterization of the odorant profile of human urine.     

Arsenic speciation in human urine reference materials using high-performance liquid chromatography with inductively coupled plasma mass spectrometric detection.

Six arsenic compounds including arsenocholine, arsenobetaine, dimethylarsinic acid, methylarsonic acid, arsenous acid and arsenic acid were separated by high-performance liquid chromatography (HPLC) on a Hamilton PRP-X100 anion-exchange column using isocratic elution and detected by inductively coupled plasma mass spectrometry (ICP-MS). This analytical procedure was applied to the speciation of arsenic compounds in human urine. The influence of urine matrix on the separation of arsenic compounds was evaluated and the determination of arsenic compounds was not hampered by the ArCl interference which has often been encountered in ICP-MS. Three human urine reference materials, SRM 2670 normal level, SRM 2670 elevated level and Lyphocheck urine metal control 1, were analyzed with respect to arsenic compounds by HPLC-ICP-MS. The results were found to be in good agreement with the certified total arsenic concentration in the reference materials. Six arsenic compounds were detected. Arsenobetaine was found to be present in all of the investigated human urine reference materials.     

Determination of vanilmandelic acid in urine by coupled-column liquid chromatography combining affinity to boronate and separation by anion exchange

An automated liquid chromatographic method for assaying vanilmandelic acid in urine is described. Vanilmandelic acid and potential interfering substances, such as catechol compounds and their metabolites, have been tested for affinity to boronic acid-substituted silica at various pH values. Vanilmandelic acid and the internal standard, isovanilmandelic acid, were bound to the boronate matrix at an acidic pH, whereas for instance catecholamines were unretained and passed through the column. The α-hydroxycarboxylic acids were then desorbed by another mobile phase (pH 6.0) and transferred to an anion exchanger for chromatography and electrochemical detection. A relative standard deviation of 2.8% was obtained for the analysis of human urine samples containing 6.6 μM vanilmandelic acid.     

Characterization of a novel diclofenac metabolite in human urine by capillary gas chromatography-negative chemical ionization mass spectrometry

A sensitive analytical method was developed to characterize diclofenac metabolites in small amounts of body fluids. Desalted and lyophilized urine samples were extracted with supercritical carbon dioxide directly or after acidic hydrolysis. The extracts were derivatized with N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamide. The derivatives were separated by capillary gas chromatography and identified by negative chemical ionization mass spectrometry. Full mass spectra were obtained at a level of 1·10⁻⁹ g/ml. With direct extraction, the metabolites could be analysed in one step as open-chained acids and as (cyclic) oxindoles. By acidic hydrolysis the conjugates were transformed to the oxindoles. With both methods, a new main metabolite, [2-[(2,6-dichloro-4-hydroxy-3-methoxyphenyl)amino]phenyl]acetic acid, was identified. The mechanism of its formation is discussed.     

Chromatographic profile of high boiling point organic acids in human urine

The profile of high boiling point organic acids in urine samples from both normal subjects and patients suspected of having some form of metabolic disorder has been determined by combined gas chromatography—mass spectrometry. Fifteen different compounds eluting after hippuric acid have been identified, including two, cinnamoylglycine and acetyltributylcitrate, which have not been recognised previously. Relative retention times and abbreviated mass spectra of the identified compounds are presented.     

Mass spectrometry of dimethylthiophosphinates of aromatic hydroxy compounds

The mass spectra of 20 differently substituted dimethylthiophosphinic esters of aromatic hydroxy compounds are presented. Fragmentation routes were investigated using high resolution mass measurements, decoupled metastable determinations and deuterium labelling. All compounds exhibited abundant molecular ions and typical phosphorus-containing ions. Characteristic elimination processes strongly dependent upon the respective type of substitution were observed. Due to their high stability, their great ease of formation and their good gas chromatographic properties these new types of derivatives are of special interest for establishing gas chromatography mass spectrometry profiles of acidic catecholamine metabolites.     

Isolation of polyketides from Prymnesium parvum (Haptophyta) and their detection by liquid chromatography/mass spectrometry metabolic fingerprint analysis

Prymnesium parvum is a microalga that forms blooms coupled with the presence of potent exotoxins; however, no chemical standards are currently available for the toxins. Streamlined methods are presented for the separation and enrichment of polyketide toxins, prymnesin-1 (prym1) and prymnesin-2 (prym2). Prymnesins were separated by reversed-phase chromatography and detected by positive-mode electrospray ionization MS to generate a unique metabolic fingerprint. More than 10 ions were detected and mass assignments were in agreement with predicted isotopic distributions for the intact compounds and related fragments; ions occurred as multiply protonated species and with common salt adducts. The most prevalent ion was observed at 919.88 m/z, which represents the aglycone [prym_agly + 2H]²⁺ backbone structure common to both molecules. Expanded mass spectra for this and related ions were in excellent agreement (<0.5 ppm) with empirically derived spectra based on elemental composition and naturally occurring isotopes. These investigations have confirmed the isolation of polyketide prymnesins from whole cells, which heretofore has not been reproduced since their original characterization. Moreover, this study represents the first time these compounds have been verified in aqueous materials. These tools should allow the direct identification and analysis of polyketide prymnesins, which will greatly improve our understanding of these toxins in P. parvum.     

Isolation and Preliminary Characterization of Hydroxamic Acids Formed by Nitrogen-Fixing Azotobacter chroococcum B-8

The free-living diazotroph Azotobacter chroococcum B-8 responded to iron-limited growth conditions by forming hydroxamic acids and an 85,000-dalton outer membrane protein. The Fe(III)-binding hydroxamate compounds stimulated the growth of Arthrobacter flavescens JG-9 and gave a positive Csaky reaction for bound hydroxylamines. The hydroxamates were isolated from liquid cultures by benzyl alcohol extraction and purified by size exclusion chromatography and high-performance liquid chromatography. Four high-performance liquid chromatography fractions, designated A, B, C, and D, had the characteristic hydroxamate absorption maximum at 420 to 423 nm, which did not shift over a pH range from 3.0 to 9.0. Cyclic voltammograms of the iron-hydroxamate complexes exhibited reduction potentials of −0.426 to −0.442 V for fractions A, B, and D and of −0.56 V for fraction C versus the normal hydrogen electrode at pH 8.0. Based on mass spectra, nominal molecular weights of 800 and 844 were assigned to ferrated compounds A and B, respectively. Reductive hydrolysis of compounds A and B in 57% hydriodic acid yielded ornithine as detected by gas chromatography-mass spectrometry. All of these physiological and chemical data strongly support the hypothesis that the high-affinity iron-binding compounds isolated from A. chroococcum B-8 are hydroxamic acids and probably function as siderophores for this diazotroph.     

Isolation of 20 alpha and 20 beta 5 beta-pregnane-3 alpha,17 alpha,20,21-tetrol (hexahydro-Reichstein's compound-S) in a case of congenital adrenal hyperplasia

5β-Pregnane-3α, 17α, 20α, 21-tetrol (l) and 5β-pregnane-3α, 17α 20β, 21-tetrol (II) have been isolated and identified from the urine of a girl with congenital adrenal hyperplasia. The total 5β-pregnane-3α, 17α, 20(α+β),21-tetrol consisted of 60% of I and 40% of II. The final identity of the compounds was established by gas chromatography — mass spectrometry. The mass spectra of the two trimethylsilyl isomers were closely related to each other in contrast to the spectra of five other pairs of C₂₁-C-20(α and β)-hydroxy steroid-trimethylsilyl-ethers. The mass spectra of free I and II also exhibited many common features, but were less similar to each other than their trimethylsilyl derivatives.     

Screening of β-2 agonists and confirmation of fenoterol,orciprenaline, reproterol and terbutaline with gas chromatography–mass spectrometry as tetrahydroisoquinoline derivatives

A new method for a comprehensive screening and confirmation of β-2 agonists in human urine is presented based on gas chromatography–low-resolution mass spectrometry (GC–MS) using electron impact ionisation (EI). After hydrolysis of the conjugates with β-glucuronidase/arylsulfatase a derivatisation step with formaldehyde converts fenoterol, orciprenaline, reproterol and terbutaline to one derivative, a tetrahydroisoquinoline, while the other β-2 agonists remain unchanged. Liquid–liquid extraction and trimethylsilylation follow. The tetrahydroisoquinoline derivatives show good gas chromatographic and mass spectrometric behaviour. The detection limit of these four β-2 agonists in the screening using low-resolution mass spectrometry is 10 ng/ml of urine. The other β-2 agonists are detected as parent compounds with the same recovery after sample preparation with and without formaldehyde. The EI mass spectra of the tetrahydroisoquinoline derivatives are presented.     

Solid-phase extraction method for the determination of free and conjugated phenol compounds in human urine

A rapid flow system for automatic sample conditioning for the determination of phenol compounds in human urine has been developed and optimised. Free phenols are detected directly in urine samples while total phenols require acid hydrolysis to convert their conjugate fraction into free phenols, all compounds then being cleaned up and preconcentrated by solid-phase extraction. Separation and determination are done by gas chromatography, using mass spectrometry operating in the selective ion monitoring mode for quantitation. The linear range was 1-160 ng/ml of urine for most of the phenols. Limits of detection for phenol compounds (phenol, alkylphenols and chlorophenols) in the nanogram-per-millilitre range (0.3-0.6 ng/ml) are thus achieved by using 1 ml of urine; also, the repeatability, as RSD, is less than 6.5%. Based on the results for urine samples from unexposed individuals, 2-methylphenol, 2-chlorophenol and 2,4-dichlorophenol are largely detected in hydrolysed urine samples, whereas phenol and 4-methylphenol are detected in hydrolysed and unhydrolysed urine. Other chlorophenols such as trichlorophenols and pentachlorophenol are not detected. The results obtained in the analysis of urine from an individual before and after dietary intake reveal that the levels of phenol compounds in urine look related to food intake.     

C15H24 Volatile Compounds Unique to Aflatoxigenic Strains of Aspergillus flavus

Headspace volatiles from eight strains of Aspergillus flavus (four aflatoxigenic strains and four nonaflatoxigenic strains), grown for 1, 2, 3, 4, 8, and 10 days in submerged cultures, were collected in Tenax GC traps. The traps were desorbed onto a 50-m gas-liquid chromatography capillary column by heat and gas purge from an external direct injector device. The column was interfaced with a mass spectrometer data acquisition system. Peaks were identified by comparing retention times and mass spectra with those obtained from authentic compounds and by using a computer-assisted mass spectral data base. Aflatoxigenic strains of A. flavus produced several C₁₅H₂₄ compounds (e.g., alpha-gurjunene, trans-caryophyllene, and cadinene) which peaked in 3-day cultures and were not present in earlier (1- and 2-day) or later (8- and 10-day) cultures. None of these volatiles were detected in nonaflatoxigenic strains of A. flavus. There was an apparent correlation between the release of C₁₅H₂₄ volatile compounds and the initiation of aflatoxin biosynthesis, and a correlation between decline of aflatoxin synthesis and the disappearance of the C₁₅H₂₄ compounds unique to aflatoxigenic A. flavus also existed.     

Combined gas chromatography-chemical ionization mass spectrometry of sphingolipids. I. Glucosyl sphingosine,ceramides and cerebrosides of the spleen in Gaucher's disease

Trimethylsilylated glucosyl sphingosine, ceramides and glucocerebrosides were analysed by combined gas chromatography (GC)-chemical ionization (CI) mass spectrometry. Isobutane, methane and ammonia were used as reactant gases for chemical ionization. Essentially the same fragment ions were detected in the spectra with the different reactant gases.Purified glucocerebrosides isolated from the spleen of a patient with Gaucher's disease were clearly separated into their 8 molecular species by gas chromatography. Three other minor components were detected by spectrometry, and these 11 molecular species of glucocerebrosides from the spleen of the patient with Gaucher's disease have been analysed.The ceramides obtained by periodate oxidation and then alkaline reduction of the glucocerebrosides were also separated into 11 molecular species by GC-CI mass spectrometry.Molecular weight could be determined using the major fragment ion of m/e (M⁺?90) in the spectra of ceramides and cerebrosides. The chemical ionization method is useful for structural analyses and determination of the molecular species of sphingoglycolipids.     

Detection of new exemestane metabolites by liquid chromatography interfaced to electrospray-tandem mass spectrometry

Exemestane is an irreversible aromatase inhibitor used for anticancer therapy. Unfortunately, this drug is also misused in sports to avoid some adverse effects caused by steroids administration. For this reason exemestane has been included in World Anti-Doping Agency prohibited list. Usually, doping control laboratories monitor prohibited substances through their metabolites, because parent compounds are readily metabolized. Thus metabolism studies of these substances are very important. Metabolism of exemestane in humans is not clearly reported and this drug is detected indirectly through analysis of its only known metabolite: 17β-hydroxyexemestane using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and gas chromatography coupled to mass spectrometry (GC-MS). This drug is extensively metabolized to several unknown oxidized metabolites. For this purpose LC-MS/MS has been used to propose new urinary exemestane metabolites, mainly oxidized in C6-exomethylene and simultaneously reduced in 17-keto group. Urine samples from four volunteers obtained after administration of a 25mg dose of exemestane were analyzed separately by LC-MS/MS. Urine samples of each volunteer were hydrolyzed followed by liquid-liquid extraction and injected into a LC-MS/MS system. Three unreported metabolites were detected in all urine samples by LC-MS/MS. The postulated structures of the detected metabolites were based on molecular formulae composition obtained through high accuracy mass determination by liquid chromatography coupled to hybrid quadrupole-time of flight mass spectrometry (LC-QTOF MS) (all mass errors below 2ppm), electrospray (ESI) product ion spectra and chromatographic behavior.     

Use of direct-probe mass spectrometry as a toxicology confirmation method for demoxepam in urine following high-performance liquid chromatography

The identification of the metabolite demoxepam in human urine establishes that chlordiazepoxide, a common benzodiazepine, has been administered. Like N-oxide metabolites of other drugs, demoxepam cannot be detected by gas chromatography—mass spectrometry (GC-MS), due to thermal decomposition, and the product, nordiazepam, is a metabolite common to many benzodiazepines. Demoxepam can be readily screened using a high-performance liquid chromatography (HPLC) system such as REMEDi HS; at 35°C, no thermal decomposition will occur. Currently, there is no confirmation method available for the detection of demoxepam in urine samples. In this study, we demonstrated that following collection of the HPLC fraction, demoxepam can be confirmed using the technique of direct-probe MS. The mass spectra of demoxepam and nordiazepam differ and are easily distinguishable from each other. Ten urine samples that were analyzed by HPLC and determined to contain demoxepam were evaluated; demoxepam was confirmed in each case by direct-probe MS.