Volunteer bias, sexuality, and personality

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.     

Screening for the detection of angiotensin-converting enzyme inhibitors, their metabolites, and AT II receptor antagonists

A gas chromatography-mass spectrometry (GC-MS) screening procedure was developed for the detection of angiotensin-converting enzyme (ACE) inhibitors, their metabolites, and angiotensin (AT) II receptor antagonists in urine as part of a systematic toxicologic 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 157, 160, 172, 192, 204, 220, 234, 248, 249, and 262, the possible presence of ACE inhibitors, their metabolites, and AT II antagonists 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 detection of therapeutic concentrations of ACE inhibitors (benazepril, enalapril, perindopril, quinapril, ramipril, trandolapril, their metabolites, or both) and therapeutic concentrations of the AT II antagonist, valsartan, in human urine samples. Human urine samples were not available for testing cilazapril, moexipril, and losartan; they were detected only in rat urine. The overall recoveries of ACE inhibitors ranged between 80% and 88%, with a coefficient of variation (CV) of less than 10% and the limit of detection of at least 10 ng/ml (signal to noise ratio 3) in the full-scan mode. The overall recovery of the valsartan was 68%, with a CV of less than 10%; the limit of detection was at least 10 ng/ml (S/N 3) in the full scan mode.     

Severe meningoencephalitis in Borrelia burgdorferi infection]

A gas chromatographic-mass spectrometric method was used to separate quinine and its metabolites present in urine after oral dosing of 300 mg quinine in humans. The technique allowed the separation of quinine and ten metabolites. Four of these metabolites were definitely identified as 3-hydroxyquinine, 2'-quinone, O-desmethylquinine and 10,11-dihydroxydihydroquinine, by comparing their methane chemical ionization mass spectra with those of authentic standards prepared by organic synthesis. Six other metabolites are described for the first time in human urine. From their electron impact and chemical ionization mass spectra, we propose these compounds to be 3-hydroxy-2'-quinone, O-desmethyl-2'-quinone, O-desmethyl-3-hydroxyquinine, O-desmethyl-3-hydroxy-2'-quinone, 10,11-dihydroxydihydro-2'-quinone and 10,11-dihydroxydihydro-O-desmethylquinine. These secondary metabolites probably arose from further biotransformation of the four primary metabolites.     

Electron capture gas chromatographic analysis of the amine metabolites of pesticides: derivatization of anilines

A number of amines have been shown to result from metabolism of various pesticides. From an epidemiological standpoint, it may be possible to monitor human exposure to these pesticides through the excretion of their corresponding amines in urine. An investigation has been initiated to develop and apply methods of analysis of amines in human urine. The results of a survey of derivatization techniques involving several substituted anilines are presented. These include conditions for derivatization, utilizing a number of halo- and nitro- substituted reagents; electron capture and gas chromatographic properties of the derivatives; and stability of the derivatives to extraction and column chromatography for purposes of separation and cleanup. The recoveries of anilines from spiked water and urine samples at the 1.0 ppm and 0.1 ppm levels were between 85 and 90%. The advantages and disadvantages of the various derivatives and techniques are discussed and a rationale is presented for the preliminary selection of a particular derivative for application of the analysis of aniline metabolites in urine.     

Metabolism of 6-chloro n-butyl phthalide in rat

6-Chloro n-butyl phthalide (CBP) was orally administered to healthy, male Wistar rats pretreated with or without 3-methylcholanthrene (3-MC) by a single dose of 150 mg/kg, and urine samples were collected for 0-24 h. The urine sample was hydrolyzed with beta-glucuronidase, extracted and concentrated for TMS derivatization, and analysed on a GC-MS system for identification of CBP metabolities. Mass spectral analysis suggests that 7 CBP metabolites were present in the urine sample, and similar metabolism patterns were viewed in rats with or without pretreatment with 3-MC. Four main metabolites of CBP in rat urine were identified as alpha-beta oxolate, beta-gamma oxolate, beta-hydroxylate and gamma-hydroxylate, based on their chromatographic and mass spectral properties. Two hydroxylates have been previously identified in CBP metabolism by rat liver microsomes. The other two metabolites with higher polarity were tentatively identified as dihydroxylation products on the n-butyl side chain by the mass spectra of their TMS derivatives. One minor metabolite was found by the isotopic effect of chlorine, but its specific structure was undetermined. The difference between in vivo and in vitro metabolic profiles of CBP is also discussed.     

Detection of cocaine exposure in the neonate. Analyses of urine, meconium, and amniotic fluid from mothers and infants exposed to cocaine

Cocaine and its metabolites were measured in urine, meconium, and amniotic fluid specimens collected from 30 maternal-infant pairs with histories of prenatal cocaine use. Cocaine, benzoylecgonine, and ecgonine methyl ester were measured by isotope dilution gas chromatography-mass spectrometry. Mothers were interviewed at delivery regarding their cocaine use during pregnancy. There was qualitative agreement between the results of drug determinations in maternal urine, amniotic fluid, infant urine, and meconium. Although all of the mothers in this study admitted to using cocaine during their pregnancy, cocaine or its metabolites were detected only in the 20 cases in which cocaine was used within 3 weeks before delivery. We conclude that when sufficiently sensitive analytic methods are used, maternal urine, infant urine, and meconium analyses yield equivalent results for detection of prenatal cocaine exposure. Importantly, neither meconium nor urinary drug measurements detected cocaine exposure when the last reported use was prior to 3 weeks before delivery.     

Expression of the von Hippel-Lindau-binding protein-1 (Vbp1) in fetal and adult mouse tissues

BACKGROUND: Tetrahydropalmatine (THP) is a neuroactive alkaloid with analgesic and hypnotic action. Its analysis is important because cases of human poisonings have emerged as a result of unregulated use of some proprietary biopharmaceuticals containing purified THP. METHODS: We established analytical parameters for HPLC with diode-array detection (HPLC-DAD) and gas chromatography-mass spectrometry (GC-MS) for the detection of THP in serum and urine. Nine acutely THP-poisoned adults were thus screened over 16 months. RESULTS: All patients recovered quickly after mild neurological disturbance. In general, THP was metabolized rapidly and excreted as polar metabolites in urine. Serum THP was measured in five cases and found to be <0.1-1.2 mg/L (<0.3-3.4 micromol/L). Paired analyses of urine with and without glucuronidase treatment clarified the disposition of THP. Our GC-MS method with trimethylsilane derivatization identified O-desmethyl metabolites. With a uniform solid-phase extraction, the HPLC-DAD procedure detected intact glucuronide metabolites. CONCLUSION: Intact glucuronide metabolites of THP are sensitive markers for THP exposures. Our methods and findings provide practical tools and information for surveillance of intoxication caused by excessive THP intake.     

Molecular characterisation of Cryptosporidium parvum from two large suspected waterborne outbreaks. Outbreak Control Team South and West Devon 1995, Incident Management Team and Further Epidemiological and Microbiological Studies Subgroup North Thames 1997

Routine paper chromatographic screening of the urine of racing greyhounds exposed to BIGELOIL, a veterinary counter-irritant, revealed metabolites suggestive of menthol, an ingredient of BIGELOIL. To determine whether BIGELOIL use caused these metabolites, 2 Dalmatian dogs were exposed to BIGELOIL. Thin-layer chromatographic screening of their urine confirmed that exposure to BIGELOIL by either dermal or oral routes causes the same metabolites as those observed in the racing greyhounds. Metabolites suggestive of thymol were also present in some samples. We conclude that, if metabolites suggestive of menthol are detected in urine of animal athletes, further analysis for the other performance-affecting ingredients of BIGELOIL should be undertaken.     

Analysis of dissolved methane, ethane, and ethylene in ground water by a standard gas chromatographic technique

The measurement of dissolved gases such as methane, ethane, and ethylene in ground water is important in determining whether intrinsic bioremediation is occurring in a fuel- or solvent-contaminated aquifer. A simple procedure is described for the collection and subsequent analysis of ground water samples for these analytes. A helium headspace is generated above a water-filled bottle. Gases that are dissolved in the water partition between the gas and liquid phases and equilibrate rapidly. An aliquot of this headspace is analyzed by gas chromatography to determine the gases concentration in this phase. The concentration of the gas dissolved in the water can then be calculated based on its partitioning properties, as indicated by its Henry's Law constant.     

Fighting for balance

The proof of diacetylmorphine (heroin) application is based on the identification of its specific metabolite 6-monoacetylmorphine simultaneously detected with the main metabolite morphine in human urine. Codeine is another morphine derivative appearing in cases of diacetylmorphine abuse; it can be considered a metabolite of 6-acetylcodeine, a typical impurity found in raw heroin. An analytical procedure for detection of the mentioned morphine basesin urine is presented, including a screening method. A careful extraction method is required for 6-monoacetylmorphine. To classify it into the code system of the screening, its properties are expressed by a threecomponent code. Subsequent identity confirmation of the mentioned bases by means of thin layer chromatography uses mobile phases, in which optimal separation effects are achieved, even in the presence of nicotine or caffeine and its metabolites theobromine and theophylline. Circumstances of 6-monoacetylmorphine discovery in urine are discussed.     

Studies on the metabolism and toxicological detection of the amphetamine-like anorectic mefenorex in human urine by gas chromatography-mass spectrometry and fluorescence polarization immunoassay

Studies on the metabolism and on the toxicological analysis of mefenorex [R,S-N-(3-chloropropyl)-alpha-methylphenethylamine, MF] using gas chromatography-mass spectrometry (GC-MS) and fluorescence polarization immunoassay (FPIA) are described. The metabolites were identified in urine samples of volunteers by GC-MS. Besides MF, thirteen metabolites including amphetamine (AM) could be identified and three partially overlapping metabolic pathways could be postulated. For GC-MS detection, the systematic toxicological analysis procedure including acid hydrolysis, extraction at pH 8-9 and acetylation was suitable (detection limits 50 ng/ml for MF and 100 ng/ml for AM). Excretion studies showed, that only AM but neither MF nor its specific metabolites were detectable between 32 and 68 h after ingestion of 80 mg of MF. Therefore, misinterpretation can occur. The Abbott TDx FPIA amphetamine/methamphetamine II gave positive results up to 68 h. All the positive immunoassay results could be confirmed by the described GC-MS procedure.     

Nutrition education in rural Australia: why, who and how?

Acyl glucuronide conjugates of acidic drugs are chemically unstable metabolites, able to undergo a number of reactions including covalent binding interactions with proteins. The question of whether any toxicological or immunological responses result from such covalent modification of native proteins in vivo is topical. Study of acyl glucuronide reactivity thus requires a convenient source of these metabolites. The utility of the bile-exteriorized rat for this purpose is highlighted herein using the formerly marketed nonsteroidal antiinflammatory agent zomepirac. Zomepirac was injected i.v. at 60 mg/kg four times into bile-exteriorized rats at 6-h intervals. The 24-h bile samples contained ca. 24% of zomepirac doses as zomepirac acyl glucuronide (ZAG). Purification was achieved by washing of the acidified bile with etherhexane, extraction into ethyl acetate, semipreparative HPLC, and crystallization. Overall recovery through the purification procedure was ca. 50%. Identity as ZAG was confirmed by mass spectrometry. The approach takes advantage of the robust glucuronidation capacity of the rat, especially at higher drug doses, and of its ability to preferentially excrete hepatically formed drug glucuronides into bile rather than into urine via blood. Prior to this work, ZAG was presumed to be only a minor metabolite of zomepirac in rats, based on early urinary recovery studies. Thus, measurement of urinary acyl glucuronide conjugates in the rat may severely underestimate their true formation in this species.     

Chromatographic determination of volatile solvents and their metabolites in urine for monitoring occupational exposure

The determination of volatile solvents and their metabolites in biological materials such as expired air, blood or urine allows the estimation of the degree of exposure of these chemicals. Chromatographic methods are now universally employed for this purpose and numerous analytical procedures are available for the determination of the most commonly used volatile solvents and their metabolites in urine. GC methods appear well adapted to the determination of the parent volatile solvents in blood and urine and may be used for the determination of their urinary metabolites, but these methods often require several prechromatographic steps. However, HPLC is becoming a powerful tool for the accurate and easy determination of urinary metabolites of volatile solvents, considering its decisive advantages for routine monitoring. Further, recent developments in HPLC could widen the usefulness of this method for most complex analytical problems that could be encountered during this measurement. However, despite the relative neglect of planar chromatography in this area of concern and considering the great interest in methods that could permit the simultaneous assay of numerous samples often required by routine monitoring, new approach using improved methods such as overpressured TLC could be very fruitful in the future.     

Application of gas chromatography-mass spectrometry and gas chromatography-tandem mass spectrometry to assess in vivo synthesis of prostaglandins, thromboxane, leukotrienes, isoprostanes and related compounds in humans

Prostaglandins, thromboxane, leukotrienes, isoprostanes and other arachidonic acid metabolites are structurally closely related, potent, biologically active compounds. One of the most challenging tasks in eicosanoids research has been to define the role of the various eicosanoids in human health and disease, and to monitor the effects of drugs on the in vivo synthesis of these lipid mediators in man. Great advances in instrumentation and ionization techniques, in particular the development of tandem mass spectrometry and negative-ion chemical ionization (NICI), in gas chromatography and also advances in methodologies for solid-phase extraction and sample purification by thin-layer chromatography and high-performance liquid chromatography have been made. Now gas chromatography-mass spectrometry (GC-MS) and GC-tandem MS in the NICI mode are currently indispensable analytical tools for reliable routine quantitation of eicosanoid formation in vivo in humans. In this article analytical methods for eicosanoids based on GC-MS and GC-tandem MS are reviewed emphasizing the quantitative measurement of specific index metabolites in human urine and its importance in clinical studies in man. Aspects of method validation and quality control are also discussed.     

Identification and measurement of endogenous beta-oxidation metabolites of 8-epi-Prostaglandin F2alpha

F2-isoprostanes are prostaglandin-like compounds derived from nonenzymatic free radical-catalyzed peroxidation of arachidonic acid. 8-epi-Prostaglandin (PG) F2alpha, a major component of the F2-isoprostane family, can be conveniently measured in urine to assess noninvasively lipid peroxidation in vivo. Measurement of major metabolites of endogenous 8-epi-PGF2alpha, in addition to the parent compound, may be useful to better define its formation in vivo. 2,3-Dinor-5,6-dihydro-8-epi-PGF2alpha is the only identified metabolite of 8-epi-PGF2alpha in man, but its endogenous levels are unknown. In addition to this metabolite, we have identified another major endogenous metabolite, 2,3-dinor-8-epi-PGF2alpha, in human and rat urine. The identity of these compounds, present at the pg/ml level in urine, was proven by a number of complementary approaches, based on: (a) immunoaffinity chromatography for selective extraction; (b) gas chromatography-mass spectrometry for structural analysis; (c) in vitro metabolism in isolated rat hepatocytes; and (d) chemical synthesis of the enantiomer of 2,3-dinor-5, 6-dihydro-8-epi-PGF2alpha as a reference standard. In humans, the urinary excretion rate of both dinor metabolites is comparable with that of 8-epi-PGF2alpha. Both metabolites increase in parallel with the parent compound in cigarette smokers, and they are not reduced during cyclooxygenase inhibition. Another beta-oxidation product, 2, 3,4,5-tetranor-8-epi-PGF2alpha, was identified as a major product of rat hepatocyte metabolism. In conclusion, at least two major beta-oxidation products of 8-epi-PGF2alpha are present in urine, which may be considered as additional analytical targets to evaluate 8-epi-PGF2alpha formation and degradation in vivo.     

Determination of four anabolic steroid metabolites by gas chromatography/mass spectrometry with negative ion chemical ionization and tandem mass spectrometry

A gas chromatography/mass spectrometry method is described which uses negative ion chemical ionization and tandem mass spectrometry for the determination of anabolic steroid metabolites. Four anabolic steroid metabolites to be derivatized by Pentafluoropropionic anhydride (PFPA) were determined using gas chromatography/mass spectrometry (GC/MS) with negative chemical ionization (NCI) and NCI/MS/MS. The repeatability and reproducibility of this procedure were in the range of 5.3-9.7% and 6.1-10.2%, respectively. This method of derivatization with PFPA for NCI and NCI/MS/MS was useful to determine four metabolites of nandrolone, dromostanolone, methenolone and boldenone. The derivatized metabolites of boldenone could be detected to 2 ppb and the other three steroids could be detected to 25 ppb in urine at a signal-to-noise ratio of S/N = 3.     

A theoretical framework for sex-biased parental care

Strong anion-exchange (SAX) chromatography and reversed-phase liquid chromatography (RPLC) followed by different mass spectrometric techniques for the separation and identification of conjugated and unconjugated 14C-labelled eltanolone (5beta-Pregnan-3alpha-ol-20-one) metabolites in biological fluids are presented. Conjugates of estradiol were used as model compounds for the development of a SAX based group separation of neutral steroids, glucuronides, sulfates and di-conjugated steroids. The usefulness of the technique is demonstrated by the analysis of 14C-labelled eltanolone metabolites in dog urine. The analytical SAX column used prior to RPLC improved the capacity to separate the metabolites from each other and from endogenous components, compared to a single reversed-phase system. Liquid chromatography negative ion electrospray-mass spectrometry (LC-ESI-MS) was used for the molecular mass determination of conjugated eltanolone metabolites. Unconjugated metabolites and hydrolysed conjugates were identified using gas chromatography-mass spectrometry with an electron impact ion source (GC-MS) after trimethylsilyl (TMS) derivatization. An unexpected finding in dog urine was the diglucuronide formation of eltanolone (presumably after enolisation of its carbonyl group).     

Quantitation of o-, m- and p-cresol and deuterated analogs in human urine by gas chromatography with electron capture detection

A gas chromatographic method for the analysis of cresol metabolites of toluene and [2H8]toluene in urine was developed. Cresol glucuronides and sulfates in urine were hydrolyzed with beta-glucuronidase and arylsulfatase. Following extraction with tert.-butyl methyl ether and solvent exchange into benzene, the cresols were derivatized with heptafluorobutyric anhydride to form the heptafluorobutyrate esters. The derivatives were analyzed by gas chromatography with electron capture detection. Chromatographic resolution was achieved between all cresol isomers and their 2H7 analogs. Calibration ranged from 0.001 to 500 microg/ml. Recoveries were 55-97% and showed no trend with respect to analyte concentration. Within-day precision of analyses of benchmark urine samples had a coefficient of variation of less than 4%. The assay sensitivity was limited by chromatographic background but was sufficient for quantification of the unlabeled cresols in urine from men with only environmental exposure to toluene. Average levels in urine samples from 45 men were 0.023, 0.054 and 37 microg/ml for o-, m- and p-cresol, respectively.     

4-aminobiphenyl, 2-naphthylamine, and analogs: analytical properties and trace analysis in five substrates

Methods for monitoring trace levels of 4-aminobiphenyl, 2-naphthylamine, and their hydrochloride salts in waste water, microbiological growth media, potable water, human urine, and mouse blood utilizing spectrophotofluorometry (SPF) are described. The salient elements of the methods are extraction of the residues as the free amine with benzene, rapid cleanup on an alumina column, and quantification of the free amine in methanol via SPF. Potable water solutions of the salts are diluted with 0.01 N aqueous HCL and quantified directly by SPF. Ancillary analytical information concerning gas chromatography of the free amines, partitioning properties of the compounds between solvent pairs, their solubility and stability in water, and thin-layer chromatographic data is presented. The compositions of various admixtures of 1- and 2-naphthylamine or their salts were determined by using SPF with calculations based on simultaneous equations.