Analysis and evaluation of trans,trans-muconic acid as a biomarker for benzene exposure

Benzene is an important industrial chemical and, due to its occurrence in mineral oil and its formation in many combustion processes, a widespread environmental pollutant. Since benzene is hematoxic and has been classified as a human carcinogen, monitoring and control of benzene exposure is of importance. Although trans,trans-muconic acid (ttMA) was identified as a urinary metabolite of benzene at the beginning of this century, only recently has its application as a biomarker for occupational and environmental benzene exposure been investigated. The range of metabolic conversion of benzene to ttMA is about 2-25% and dependent on the benzene exposure level, simultaneous exposure to toluene, and probably also to genetic factors. For the quantitation of ttMA in urine, HPLC methods using UV and diode array detection as well as GC methods combined with MS or FID detection have been described. Sample pretreatment for both HPLC and GC analysis comprises centrifugation and enrichment by solid-phase extraction on anion-exchange sorbents. Described derivatization procedures prior to GC analysis include reaction with N,O-bis(trimethysilyl)acetamide, N,O-bis(trimethylsilyl)trifluoroacetamide, pentafluorobenzyl bromide and borontrifluoride-methanol. Reported limits of detection for HPLC methods range from 0.1 to 0.003 mg l(-1), whereas those reported for GC methods are 0.03-0.01 mg l(-1). Due to its higher specificity, GC methods appear to be more suitable for determination of low urinary ttMA levels caused by environmental exposure to benzene. In studies with occupational exposure to benzene (>0.1 ppm), good correlations between urinary ttMA excretion and benzene levels in breathing air are observed. From the reported regressions for these variables, mean excretion rates of ttMA of 1.9 mg g(-1) creatinine or 2.5 mg l(-1) at an exposure dose of 1 ppm over 8 h can be calculated. The smoking-related increase in urinary ttMA excretion reported in twelve studies ranged from 0.022 to 0.2 mg g(-1) creatinine. Only a few studies have investigated the effect of exposure to environmental levels of benzene (<0.01 ppm) on urinary ttMA excretion. A trend for slightly increased ttMA levels in subjects living in areas with high automobile traffic density was observed, whereas exposure to environmental tobacco smoke did not significantly increase the urinary ttMA excretion. It is concluded that urinary ttMA is a suitable biomarker for benzene exposure at occupational levels as low as 0.1 ppm. Biomonitoring of exposure to environmental benzene levels (<0.01 ppm) using urinary ttMA appears to be possible only if the ingestion of dietary sorbic acid, another precursor to urinary ttMA, is taken into account.     

Methods for the measurement of benzodiazepines in biological samples

A review of methods for the measurement of benzodiazepines in biological specimens published over the last five years is presented. A range of immunoassay procedures using EIA, ELISA, FPIA, agglutination or kinetic interaction of microparticles, or RIA methods are now available. Cross reactivities to benzodiazepines are variable such that no one kit will recognise all benzodiazepines and their relevant metabolites at concentrations likely to be encountered during therapeutic use. Prior hydrolysis of urine to convert glucuronide metabolites to immunoreactive substances improves detection limits for many benzodiazepines. Several radioreceptor assays have now been published and show good sensitivity and specificity to benzodiazepines and offer the advantage (over immunoassay) of being able to detect these drugs with equal sensitivity. Solvent extraction techniques using a variety of solvents were still popular and offer acceptable recoveries and lack of significant interference from other substances. A number of papers describing solid phase extraction procedures were also published. Direct injection of specimens into a HPLC column with back flushing were also successfully described. Seventy two chromatographic methods using HPLC, LC-MS, GC and GC-MS methods were reviewed. HPLC was able to achieve detection limits for many benzodiazepines using UV or DAD detection down to 1-2 ng/ml using 1-2 ml of urine or serum (blood). ECD detectors gave detection limits better than 1 ng/ml from 1 ml of specimen, which was an order of magnitude lower than for NPD. EI-MS offered similar sensitivity, whilst NCI-MS was capable of detection down to 0.1 ng/ml. Methods suitable for the separation of enantiomers of benzodiazepines have been described using HPLC. Electrokinetic micellar chromatography has also been shown to be capable of the analysis of benzodiazepines in urine.     

Sensitive and selective detection of urinary 1-nitropyrene metabolites following administration of a single intragastric dose of diesel exhaust particles (SRM 2975) to rats

1-Nitropyrene (1-NP) has been proposed as a marker for exposure to diesel exhaust particles (DEP). Since the extent of the actual intake of 1-NP adsorbed on DEP will be relatively low, sensitive and selective methods are needed regarding human exposure assessment. Two analytical methods are presented for the assessment of 1-NP metabolites in urine of male Sprague-Dawley rats administered a single intragastric dose of native DEP (SRM 2975, 20 mg, 35.7 microgram of 1-NP/g). Enzymatically hydrolyzed urine was extracted using Blue Rayon. The extracts were analyzed directly, using HPLC with postcolumn on-line reduction and fluorescence detection (HPLC-Flu), or were processed further for GC/MS/MS analysis. Although sensitive to several metabolites, the HPLC-Flu method lacked selectivity for quantitation of some important metabolites in rat urinary extracts, and therefore seems suitable for screening purposes only. With regard to GC/MS/MS analysis, derivatization with heptafluorobutyrylimidazole (HFBI) yielded low limits of determination for hydroxy-1-aminopyrenes, hydroxy-N-acetyl-1-aminopyrenes (converted to derivatized hydroxy-1-aminopyrenes by the reagent), and 1-aminopyrene (1.8-9.2 fmol on the column). Derivatization of hydroxy-1-nitropyrenes yielded relatively high limits of determination, and therefore, hydroxy-1-nitropyrenes were reduced to hydroxy-1-aminopyrenes prior to derivatization with HFBI. Intragastric administration of DEP to rats resulted in urinary excretion of 6-hydroxy-N-acetyl-1-aminopyrene, 8-hydroxy-N-acetyl-1-aminopyrene, 6-hydroxy-1-nitropyrene, 8-hydroxy-1-nitropyrene, and 3-hydroxy-1-nitropyrene (7, 1.2, 1.6, 0.3, and 0.5% of the dose within 12 h, respectively). 1-Nitropyrene, N-acetyl-1-aminopyrene, and 3-, 6-, and 8-hydroxy-1-aminopyrene were not observed as urinary metabolites following administration of a single dose of DEP. The observed excretion pattern and urinary metabolite concentrations suggest that 1-NP present on unmodified DEP becomes bioavailable to a large extent and is metabolized in the same way as was previously observed following administration of pure 1-NP. The presented methods are promising for assessment of human exposure to 1-NP, e.g., following exposure to DEP, because of the possibility of analyzing large volumes of urine, the conversion of three types of metabolites to one (the amino metabolites), and the low detection limits that are achieved.     

Allylmercapturic acid as urinary biomarker of human exposure to allyl chloride

OBJECTIVE: To evaluate the use of urinary mercapturic acids as a biomarker of human exposure to allyl chloride (3-chloropropene) (AC). During three regular shut down periods in a production factory for AC, both types of variables were measured in 136 workers involved in maintenance operations. METHODS: Potential airborne exposure to AC was measured by personal air monitoring in the breathing zone. In total 205 workshifts were evaluated. During 99 workshifts no respiratory protection equipment was used. Mercapturic acid metabolites were measured in urinary extracts by gas chromatography-mass spectrometry (GC-MS). RESULTS: During 86 work shifts when no respiratory protection was used the air concentrations of AC were below the Dutch eight hour time weighted average (8 h-TWA) occupational exposure limit (OEL) of AC (3 mg/m3), whereas in 13 workshifts the potential exposure, as measured by personal air monitoring, exceeded the OEL (3.3 to 17 mg/m3). With the aid of GC-MS, 3-hydroxypropylmercapturic acid (HPMA) was identified as a minor and allylmercapturic acid (ALMA) as a major metabolite of AC in urine samples from the maintenance workers exposed to AC. The concentrations of ALMA excreted were in a range from < 25 micrograms/l (detection limit) to 3550 micrograms/l. The increases in urinary ALMA concentrations during the workshifts correlated well with the 8h-TWA air concentrations of AC (r = 0.816, P = 0.0001, n = 39). Based on this correlation, for AC a biological exposure index (BEI) of 352 micrograms ALMA/g creatinine during an eight hour workshift is proposed. In some urine samples unexpectedly high concentrations of ALMA were found. Some of these could definitely be attributed to dermal exposure to AC. In other cases garlic consumption was identified as a confounding factor. CONCLUSION: The mercapturic acid ALMA was identified in urine of workers occupationally exposed to airborne AC and the increase in ALMA concentrations in urine during a workshift correlated well with the 8 h-TWA exposure to AC. Garlic consumption, but not smoking, is a potential confounding factor for this biomarker of human exposure to AC.     

A fatal case of benzene poisoning

Chronic effects following repeated exposure to low doses of benzene have been well assessed, whereas few data are available about acute exposure to benzene. We report a case of fatal acute intoxication which occurred aboard a chemical cargo ship. Autopsy findings included blood clots inside the heart and main vessels, multi-organ congestion, pulmonary edema and the presence of many vibices in the hypostatic areas. Toxicological analysis of blood and urine showed a benzene concentration of 31.67 and 2.26 micrograms/mL, respectively; high concentrations of benzene (microgram/g) were also found in the lungs (22.23), liver (378.60), brain (178.66), heart (182.57) and kidneys (75.15). The above data provide evidence for benzene distribution in various organs.     

Identification of urinary benzodiazepines and their metabolites: comparison of automated HPLC and GC-MS after immunoassay screening of clinical specimens

An automated high-performance liquid chromatographic method, benzodiazepines by REMEDi HS, was used to analyze benzodiazepines and their metabolites after beta-glucuronidase hydrolysis of 1-mL urine specimens from the following: 924 clinic and hospital patients whose specimens had previously been found to be presumptively positive using either EMIT or Triage immunoassay methodologies and 128 individuals whose specimens had screened negative by EMIT d.a.u.TM. REMEDi analyses did not correlate with the immunoassay results in 136 of the positive and three of the negative urine specimens. Gas chromatographic-mass spectrometric (GC-MS) confirmatory analyses were performed on these discordant specimens using 3 mL beta-glucuronidase-hydrolyzed urine followed by extraction with chloroform-isopropanol (9:1) and derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide. Two benzodiazepines, flunitrazepam and clonazepam, and their 7-amino metabolites were analyzed without prior derivatization. The analyses established 87% concordance between REMEDi and GC-MS versus 13% concordance with immunoassay for the subset. GC-MS analysis of these 142 specimens demonstrated two reasons for the nonconcurrence between REMEDi and EMIT: EMIT had given either false-negative or false-positive results and EMIT had given a positive result even though the determined metabolites were below the 200-ng/mL cutoff for the immunoassay and the 80-ng/mL cutoff for REMEDi. A total of 23 specimens were found to contain only lorazepam by REMEDi and GC-MS, 15 of which had been screened by Triage. A reevaluation of these 23 specimens by EMIT d.a.u. demonstrated that 11 were positive. This finding was in contrast to previous reports that EMIT will not detect lorazepam glucuronide in urine. An unexpected finding was the REMEDi identification and subsequent GC-MS confirmation of 7-aminoflunitrazepam, a urinary metabolite of flunitrazepam that is not available in the United States and that represented illicit use by four patients. A distinct advantage of REMEDi proved to be its capability in identifying demoxepam, a major metabolite of chlordiazepoxide; GC-MS analysis could not detect this metabolite because of its thermal decomposition to nordiazepam. To further evaluate the specificity of REMEDi, we conducted GC-MS analyses in a random fashion on 55 additional nondiscordant urine specimens that were identified as either positive or negative, as well as 22 specimens identified as containing 7-aminoclonazepam by REMEDi. Concurrence was observed between the two methods for all specimens, with the exception of one apparent false positive for alpha-hydroxyalprazolam by REMEDi. The reproducibility of the REMEDi method was found to be excellent; it was assessed by comparing results of 266 specimens that were reprocessed in different batches and for known calibrators and controls also processed with each batch. Study results demonstrated that the automated REMEDi assay for urinary benzodiazepines and their metabolites was comparable with GC-MS but had distinct advantages over GC-MS because of the following reasons: simplicity of the assay, less time required for analyses, and provision of additional information concerning the parent benzodiazepine.     

Diffusion sampling of benzene present in the urine

Benzene is a widely diffuse solvent; in the industrial environment benzene is currently present at concentrations of ppm. A valid method of biological monitoring that is easy to perform is need for assessing occupational and non-occupational exposures. A new method has been developed to evaluate low concentrations of benzene in urine samples by means of a diffusion sampling. The solvent is absorbed from the urine surface and concentrated on an absorbent substrate (Tenax) that is placed inside the vial. The solvent is thermically desorbed from Tenax and injected into a column (Thermal Tube Desorber-Supelco; 250 degrees C thermal flash; borosilicate capillary glass-column SPB-I 60 m length, 0.75 mm I.D., 1 micron film thickness; GC Dani 8580-FID). The method, which had not been previously employed for the determination of volatile organic substances in biological fluids, has a linear range which extends up to 40 micrograms/l, and gives results in excellent agreement with the conventional Head Space method, except in the low concentration region: the new method permits the quantitative determination of benzene quantities smaller than the detection limit of Head Space method connected with mass spectrometer (approximately 1 microgram/l). The detection limit was not exactly determined, but is estimated to be of 100 ng/l with 25 ml of urine sample.     

Effect of phenobarbital pretreatment on in vitro enzyme kinetics and in vivo biotransformation of benzene in the rat

Phenobarbital pretreatment (50 mg/kg/day for 3 days orally) of male Wistar rats increased Vmax of benzene in vitro hepatic microsomal biotransformation about 6-fold without changing Km. However, benzene blood levels after oral, intraperitoneal, or subcutaneous benzene administration (3-3.5 mmoles/kg) were not influenced by phenobarbital pretreatment. The phenol blood levels after oral or intraperitoneal benzene were increased by phenobarbital pretreatment, but less than expected from in vitro data and only 3 h after benzene administration. Phenol elimination in urine after subcutaneous benzene was not affected by phenobarbital. After oral or intraperitoneal benzene administration, phenol urine excretion closely followed the levels of phenol in blood, i.e., rate of phenol urine excretion was significantly, but shortly increased, and the cumulative urine excretion of phenol increased very little or remained unchanged. Differences between the in vitro and in vivo observations of the effect of phenolbarbital on benzene biotransformation may partly be explained by distribution of benzene, which apparently limited benzene availability for biotransformation (Vd = 5.5) and caused rapid decrease of benzene concentrations in blood. Conditions for enzyme activity may have been substantially different in vitro vs. in vivo: in vitro concentrations of benzene were at least by an order of magnitude higher than phenol concentrations, while in vivo, an opposite relation prevailed making a competition for microsomal monooxygenase possible. Cofactor availability may be another rate-limiting step or factor of in vivo benzene biotransformation, as benzene ring hydroxylation requires high energy. The rate of in vitro hepatic microsomal benzene biotransformation proved to be of limited value when predicting benzene quantitative biotransformation in vivo in contradistinction to various substrates where the in vitro and in vivo biotransformation data are in good agreement.     

Simultaneous dislocation of both interphalangeal joints and flexor tendon tear in a finger

A case in which the death of a 2-year-old male child was the result of an acute intoxication with chloral hydrate, lidocaine, and nitrous oxide is presented. Trichloroethanol (TCE), the primary metabolite of chloral hydrate, was qualitatively detected by the Fujiwara reaction. Quantitation of TCE was carried out by gas chromatography-mass spectrometry (GC-MS) with the following results: plasma, 79.0 mg/L; urine, 31.0 mg/L; gastric contents, 454.0 mg/L; bile, 111.0 mg/L; vitreous, 40.2 mg/L; cerebrospinal fluid (CSF), 68.3 mg/L; and liver, 164 mg/kg. Lidocaine was quantitated by GC analysis using nitrogen-phosphorus detection with the following results: plasma, 11.9 mg/L; urine, 3.7 mg/L; gastric contents, 15.3 mg/L; bile, 19.0 mg/L; vitreous, 17.8 mg/L; CSF, 9.4 mg/L; and liver, 19.0 mg/kg. Nitrous oxide was quantitated in the blood with a value of 4.4 mL/L.     

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.     

Rat fetal ventral mesencephalon grown as solid tissue cultures: influence of culture time and BDNF treatment on dopamine neuron survival and function

The method of analysis described permits the determination of 2,4-dinitrobenzoic acid down to the lower microg l(-1) range in the urine of persons exposed to dinitrotoluene. 2,4-Dinitrobenzoic acid is the main metabolite of 2,4-dinitrotoluene and technical dinitrotoluene. After acidic hydrolysis, which served to release the conjugated part of the 2,4-dinitrobenzoic acid, the analyte was selectively separated from the urine matrix via various extraction steps and then derivatised to the methyl ester. Quantitative analysis was carried out using capillary gas chromatography and mass selective detection. 3,5-Dinitrobenzoic acid was used as an internal standard. The detection limit was 1 microg l(-1) urine. The relative standard deviations of within-series imprecision were between 5 and 6%. The relative recoveries were between 91 and 110% depending on the concentration. The analytical method developed as part of this study was used to investigate a collective consisting of 82 urine samples from persons working in the area of explosives disposal. The concentrations of 2,4-dinitrobenzoic acid determined ranged from the detection limit to 95 microg l(-1) urine. The method allowed the quantification of low-level internal exposure to dinitrotoluene.     

Metabolism of Ro 21-5998 (mefloquine) in the rat (author's transl)

After administration of 15 mg/kg 14C-Ro 21-5998/001 i.p. to rats, the metabolite patterns in feces, urine, bile and blood were compared. Metabolites from feces were identified by GC/MS, in all other cases by TLC. The main component in the feces consists of mefloquine (Ro 21-5998). In addition the acid Ro 21-5104, a derivative of mefloquine with a hydroxy group in the piperidine moiety (M 12), the alcohol Ro 14-0518 and a metabolite (M 4a), which is supposed to be a lactam, were shown to be present. The acid Ro 21-5104 is the main metabolite in the urine. The bile contains the parent compound Ro 21-5998, the acid Ro 21-5104 and the alcohol Ro 14-0518, partially as conjugates. The structurally investigated components account for about 40% of the administered dose. The blood contains the parent compound Ro 21-5998 and the acid Ro 21-5104 as main components. In comparing the various metabolite patterns, it can be summarized that that in urine is slightly different from those in bile (after hydrolysis of the conjugates), feces and blood which show more similarities between each other.     

Medical surveillance studies of workers exposed to low level benzene

The paper presents th results of an investigation of haematotoxicity in workers exposed to low benzene concentrations. Forty-seven female workers in the shoemaking industry, exposed to solvent mixture and twenty-seven non-exposed controls were examined. Benzene concentrations in the working atmosphere ranged from 1.9 to 14.8 ppm. Significant differences in the levels of benzene in blood and phenols in pre- and post-shift urine between the exposed and control groups confirmed benzene exposure. Haemoglobin level and mean corpuscular haemoglobin concentration were significantly lower, and mean corpuscular volume was higher in the shoemaking workers than in controls. In the subgroup of shoemaking workers exposed to benzene concentrations of 5 ppm or lower, no differences in haematological parameters were found. In conclusion, exposure to a benzene concentration lower than 5 ppm does not appear to produce an increased level of abnormal haematological outcomes detectable in routine medical surveillance. The results of the study corroborate the present maximum permissible concentrations (5 ppm) as a protective limit preventing the onset of haematotoxic non-leukemogenic effects of chronic benzene exposure.     

"Vast sums of money ... to keep the controversy alive"--the 1988 BAT memo

A young man (22 years old) died of a cardiorespiratory arrest a few hours following admission to the emergency department of a hospital. He was found lying seriously ill in the parking lot of a dance club. Screening of postmortem blood and urine with enzyme multiplied immunoassay (EMIT) detected only amphetamines, caffeine, and cotinine. Further screening of blood, urine, and stomach contents with thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) was negative for all three matrices. Specific conditions for amphetamines were used for the gas chromatographic (GC) screening (GC-mass spectrometric [MS] and GC-nitrogen-phosphorus detection). This resulted in the preliminary identification of amphetamine in both blood and urine. Confirmation of the presence of amphetamine in all available postmortem specimens was provided by mass and infrared spectral data (GC-MS and GC-Fourier transform infrared spectrometry) after derivatization. Quantitative results and differentiation between the enantiomers of amphetamine were obtained after chiral derivatization. The calculated concentrations disclosed amphetamine ingestion as the cause of this fatality.     

Determination of drugs of abuse in blood

The detection and quantitation of drugs of abuse in blood is of growing interest in forensic and clinical toxicology. With the development of highly sensitive chromatographic methods, such as high-performance liquid chromatography (HPLC) with sensitive detectors and gas chromatography-mass spectrometry (GC-MS), more and more substances can be determined in blood. This review includes methods for the determination of the most commonly occurring illicit drugs and their metabolites, which are important for the assessment of drug abuse: Methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), N-ethyl-3,4-methylenedioxyamphetamine (MDEA), 3,4-methylenedioxy-amphetamine (MDA), cannabinoids (delta-9-tetrahydrocannabinol, 11-hydroxy-delta-9-tetrahydrocannabinol, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol), cocaine, benzoylecgonine, ecgonine methyl ester, cocaethylene and the opiates (heroin, 6-monoacetylmorphine, morphine, codeine and dihydrocodeine). A number of drugs/drug metabolites that are structurally close to these substances are included in the tables. Basic information about the biosample assayed, work-up, GC column or LC column and mobile phase, detection mode, reference data and validation data of each procedure is summarized in the tables. Examples of typical applications are presented.     

Hemoglobin and albumin adducts of benzene oxide among workers exposed to high levels of benzene

Benzene oxide (BO) reacts with cysteinyl residues in hemoglobin (Hb) and albumin (Alb) to form protein adducts (BO-Hb and BO-Alb), which are presumed to be specific biomarkers of exposure to benzene. We analyzed BO-Hb in 43 exposed workers and 42 unexposed controls, and BO-Alb in a subsample consisting of 19 workers and 19 controls from Shanghai, China, as part of a larger cross-sectional study of benzene biomarkers. The adducts were analyzed by gas chromatography-mass spectrometry following reaction of the protein with trifluoroacetic anhydride and methanesulfonic acid. When subjects were divided into controls (n = 42) and workers exposed to < or =31 (n = 21) and >31 p.p.m. (n = 22) benzene, median BO-Hb levels were 32.0, 46.7 and 129 pmol/g globin, respectively (correlation with exposure: Spearman r = 0.67, P < 0.0001). To our knowledge, these results represent the first observation in humans that BO-Hb levels are significantly correlated with benzene exposure. Median BO-Alb levels in these 3 groups were 103 (n = 19), 351 (n = 7) and 2010 (n = 12) pmol/g Alb, respectively, also reflecting a significant correlation with exposure (Spearman r = 0.90, P < 0.0001). The blood dose of BO predicted from both Hb and Alb adducts was very similar. These results clearly affirm the use of both Hb and Alb adducts of BO as biomarkers of exposure to high levels of benzene. As part of our investigation of the background levels of BO-Hb and BO-Alb found in unexposed persons, we analyzed recombinant human Hb and Alb for BO adducts. Significant levels of both BO-Hb (19.7 pmol/g) and BO-Alb (41.9 pmol/g) were detected, suggesting that portions of the observed background adducts reflect an artifact of the assay, while other portions are indicative of either unknown exposures or endogenous production of adducts.     

Protein phylogeny of translation elongation factor EF-1 alpha suggests microsporidians are extremely ancient eukaryotes

trans, trans-Muconic acid (1,3-butadiene-1, 4-dicarboxylic acid, MA), a minor urinary metabolite of benzene exposure, was determined, after clean-up by solid-phase anion-exchange chromatography, by reversed-phase HPLC on a C18 column (5 x 0.46 cm I.D., 3 microns particle size), using formic acid-tetrahydrofuran-water (14:17:969) as mobile phase and UV detection at 263 nm. The recovery of MA from spiked urine was > 95% in the 50-500 microgram/l range; the quantification limit was 6 micrograms/l; day-to-day precision, at 300 micrograms/l, was C.V. = 9.2%; the run time was less than 10 min. Urinary MA excretion was measured in two spot urine samples of 131 benzene environmentally exposed subjects: midday values obtained in non-smokers (mean +/- S.D. = 77 +/- 54 micrograms/l, n = 82) were statistically different from those of smokers (169 +/- 85 micrograms/l, n = 30) (P < 0.0001); each group showed a statistically significant increase between MA excretion in midday over morning samples. Moreover, in subjects grouped according to tobacco-smoke exposure level, median values of MA were positively associated with and increased with daily smoking habits.     

Oral platelet aggregation inhibitor Ro 48-3657: determination of the active metabolite and its prodrug in plasma and urine by high-performance liquid chromatography using automated column switching

A sensitive and highly automated high-performance liquid chromatography (HPLC) column-switching method has been developed for the simultaneous determination of the active metabolite III and its prodrug II, both derivatives of the oral platelet inhibitor Ro 48-3657 (I), in plasma and urine of man and dog. Plasma samples were deproteinated with perchloric acid (0.5 M), while urine samples could be processed directly after dilution with phosphate buffer. The prepared samples were injected onto a pre-column of a HPLC column switching system. Polar plasma or urine components were removed by flushing the precolumn with phosphate buffer (0.1 M, pH 3.5). Retained compounds (including II and III) were backflushed onto the analytical column, separated by gradient elution and detected by means of UV detection at 240 nm. The limit of quantification for both compounds was 1 ng/ml (500 microl of plasma) and 25 ng/ml (50 microl of urine) for plasma and urine, respectively. The practicability of the new method was demonstrated by the analysis of about 6000 plasma and 1300 urine samples from various toxicokinetic studies in dogs and phase 1 studies in man.     

Involvement of NK-1 and NK-2 tachykinin receptor mechanisms in jaw muscle activity reflexly evoked by inflammatory irritant application to the rat temporomandibular joint

The analysis of methaqualone (MTQ) in biological matrices by capillary electrophoresis (CE) is described. This methods uses liquid-liquid extraction and micellar electrokinetic capillary chromatography (MECC), an operation mode of CE. Separations are made using a 25 cm long capillary and a borate/phosphate buffer at pH 8.2. Using gas chromatography with mass spectrometry detection (GC-MS) as reference method, MTQ has been analyzed in urine, blood, gastric content and hair. For hair analysis, supercritical fluid extraction was compared with liquid-liquid extraction. Linearity was established in urine and blood between 0.25 and 10.0 micrograms/ml. MTQ recovery from blood was estimated at 60%. The limit of detection of this method in urine is about 0.10 microgram/ml. Drawbacks and advantages of MECC over GC-MS are discussed.