Gas Chromatography-negative Ion Chemical Ionization-mass Research Articles

Investigation of NG-hydroxy-l-arginine interference in the quantitative determination of nitrite and nitrate in human plasma and urine by GC-NICI-MS

NG-Hydroxy-l-arginine (NOHA) is the intermediate product of the conversion of l-arginine to l-citrulline and nitric oxide (NO) by nitric oxide synthase (NOS). NO is further oxidized to nitrite and nitrate which circulate in the blood and are excreted in the urine. Nitrite and nitrate may therefore serve as surrogates of NO synthesis. NOHA has been reported to occur in various cells and in blood of animals and humans. The concentration of nitrite in the circulation is comparable to the concentration of NOHA in plasma and serum of humans and laboratory animals. NOHA is a relatively unstable compound and the interaction of its NG-hydroxy group with redox active species or during sample treatment such as derivatization in the heat may yield N-containing compounds including nitrite and nitrate. In theory, NOHA may interfere with the analysis of nitrite and nitrate. In the present study, we investigated a possible interference of synthetic NOHA (0–400 μM) with the gas chromatography-negative ion chemical ionization–mass spectrometry (GC-NICI-MS) method of analysis of circulating and urinary nitrite and nitrate involving derivatization with pentafluorobenzyl (PFB) bromide in aqueous acetone at 50 °C for 5 min (nitrite) or for 60 min (nitrite and nitrate). Our results show that NOHA does not interfere with the measurement of nitrite and nitrate in human plasma and urine by this method at concentrations up to 400 μM.

Determination of 16 polychlorinated biphenyls in fish oil by gas chromatography-negative ion chemical ionization-mass spectrometry

An analytical method for the simultaneous determination of 16 polychlorinated biphenyls (PCBs) in fish oil was developed. PCBs were extracted from fish oil with n-hexane, purified by sulfuric acid and determined by using gas chromatography-negative ion chemical ionization-mass spectrometry (GC-NCI-MS) in selected ion-monitoring (SIM) mode. A good linear relationship (r > 0.99) was observed with the PCBs concentrations from 0.01 µg/L to 10 µg/L, and the limits of quantification (LOQ, S/N = 10) were between 3 pg/g and 67 pg/g for different kinds of PCBs. The average recoveries ranged from 62.3% to 121.8% with the relative standard deviations ( RSDs, n = 3) smaller than 12%. Compared with the traditional pre-treatment of multiple material solid phase extraction, this new method is simple, rapid and less organic solvent usage. Meanwhile the method has good selectivity and sensitivity, and it is suitable for the determination of multiple trace PCBs in fish oil.

Pentafluorobenzyl esterification of haloacetic acids in tap water for simple and sensitive analysis by gas chromatography/mass spectrometry with negative chemical ionization

Chlorine is the most widely used disinfectant for control of waterborne diseases in drinking water treatment. It can react with natural organic matter in water and form haloacetic acids (HAAs). For analysis of HAA levels, derivatization with diazomethane is commonly recommended as the standard methodology in Japan. However, diazomethane is a carcinogenic alkylating agent. Therefore, in this study, a safe, simple, and sensitive quantification method was developed to monitor HAAs in drinking water. Pentafluorobenzyl esterification was used for pretreatment. The pentafluorobenzyl-ester derivative was detected by gas chromatography–negative ion chemical ionization-mass spectrometry analysis with very high sensitivity for HAAs analysis. The method has low detection limits (8–94ngL−1) and good recovery rates (89–99%) for HAAs. The method was applied to 30 tap water samples from 15 cities in the Kansai region of Japan. The levels of HAAs detected were in the range 0.54–7.83μgL−1. Dichloroacetic acid, trichloroacetic acid, and bromochloroacetic acid were the major HAAs detected in most of the tap water, and accounted for 29%, 20% and 19% of the total HAAs, respectively. This method could be used for routine monitoring of HAAs in drinking water without exposure of workers to occupational hazards.

Comparison of behaviour and fate of tetrabromobisphenol A (TBBPA) in membrane bioreactors and conventional activated sludge process

Research was completed on the behaviour and fate of tetrabromobisphenol A (TBBPA) in pilot-scale membrane bioreactors and conventional activated sludge (CAS) process at the City of Guelph Wastewater Treatment Plant. Emphasis was on the physicochemical processes, biological transformation and sorption kinetics. Measurement of TBBPA at the environmental level can be challenging, so the main focus of this paper was the development of an analytical method for the nano-level determination of TBBPA. The approach used was gas chromatography-negative ion chemical ionization-mass spectrometry in select ion monitoring mode. For the wastewater matrix studied, the developed method had a low method detection level (MDL) of 2.4 ng/L. The matrix effects for influent and activated sludge mixed liquor were determined to be 36.5 ± 5.9% (n = 10) and 79.2 ± 4.5% (n = 10), respectively. Preliminary results show that the CAS system demonstrates better performance for TBBPA removal, with removal at 83%. This was corroborated by the batch experiments that showed that the synergistic effects of biodegradation and biosorption potentially provide more removal of the TBBPA through the activated sludge process.

Improvements in the Methodology of Monitoring Sulfur Mustard Exposure by Gas Chromatography-Mass Spectrometry Analysis of Cleaved and Derivatized Blood Protein Adducts

An analytical method for determining exposure to 2,2'-dichlorodiethyl sulfide (sulfur mustard, HD) has been enhanced. The method is based on the cleavage of adducted HD (protein-hydroxyethylthioethyl esters) to produce thiodiglycol. Following cleavage, a deuterated internal standard is added, and the analytes are extracted, derivatized, and analyzed by gas chromatography-negative ion chemical ionization-mass spectrometry. Inclusion of a concentration step, addition of solid sodium bicarbonate to neutralize excess derivatization reagent, and optimization of method and instrument conditions provided dramatic increases in signal-to-noise ratio. A five-day precision and accuracy study was conducted, including interday and intraday unknown analysis. Linearity was verified by a R(2) > 0.9995 for all five curves evaluated. The precision and accuracy of the assay were demonstrated to be excellent by evaluation of the interday and intraday unknown samples (< 10% relative standard deviation and relative error in most cases). Statistical treatment of the method blanks and calibration results demonstrated a reduction in the limit of quantitation from 25 nM (HD, human plasma, in vitro) to 1.56 nM. Sample and calibration stability through the analytical sequence was established by the inclusion of continuing calibration verification standards (< 5% error). Short-term sample stability was verified by reinjection of a calibration set after 18 days (R(2) = 0.9997). Quantitative agreement with the previous method was supported by the analysis of a 50 nM standard protein sample (HD, rat plasma) with both methodologies (< 1% error).

4-Hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts in lung, lower esophagus and cardia of sudden death victims

4-Hydroxy-l-(3-pyridyl)-l-butanone (HPB)-releasing adducts are formed by metabolic activation of N′-nitrosonornicotine and 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone and have been proposed as specific biomarkers for exposure to tobacco smoke. However, in several studies hemoglobin adducts releasing HPB were on average less than threefold higher in smokers compared to nonsmokers. Using an improved analytical method we have recently found a sevenfold difference in DNA adduct levels in the lung from smoking and nonsmoking lung cancer patients. In the present study we extended the determination of HPB-releasing DNA adducts by gas chromatography–negative ion chemical ionization-mass spectrometry (GC–NICI-MS) to samples of peripheral lung, lower esophagus and cardia from tumor-free sudden death victims (primarily road traffic accidents, suicide and sudden cardiac arrest). The donors were classified as either current smokers or nonsmokers based on cotinine in either blood or urine (cut-off values for active smoking: >15 ng cotinine/ml blood or >100 ng cotinine/ml urine). Contrary to our expectation, DNA adduct levels (fmol HPB/mg DNA) in lung tissue from tumor-free smokers ( N = 32, 92 ± 148) were not significantly different from values in nonsmokers ( N = 56, 61 ± 66). The values in tumor-free smokers were on average more than fourfold lower compared to smoking lung cancer patients in our previous study. Adduct levels in the mucosa of esophagus ( N = 82; 133 ± 160) and cardia ( N = 30; 108 ± 102) of sudden death victims did not show any difference according to the current smoking status. HPB-releasing DNA adduct levels in cardia and esophagus were significantly correlated ( N = 29; Spearman r = 0.609; p < 0.001). In contrast, adduct levels in lung did not correlate with either esophagus (77 cases) or cardia (28 cases). Further studies are necessary to elucidate the discrepancies in lung DNA adduct levels in smokers with or without lung cancer and to identify obvious additional sources other than tobacco for these adducts.

MEASURING TOXICITY, INCORPORATION, AND REPAIR OF 5-HYDROXYMETHYL-2′-DEOXYURIDINE IN CELLS.

5-Hydroxymethyl-2′-deoxyuridine (HmdU) is among the most common endogenous oxidative DNA lesions and is repaired by more than one DNA glycosylase in human cells. This DNA-repair pathway may be exploited for use in cancer chemotherapy. Exogenous delivery of HmdU to cells in culture leads to its incorporation into the DNA and subsequent repair, resulting in death by apoptosis if the HmdU load is too heavy for the cell. Both incorporation into the DNA and repair of HmdU out of the DNA are necessary for cell death to occur. To measure the incorporation into and subsequent repair of HmdU out of the DNA of cells in culture, we have developed a highly sensitive and specific method using stable isotope dilution and gas chromatography-negative ion chemical ionization-mass spectrometry (GC-NICI-MS) for measuring HmdU levels found in DNA. Using the osteosarcoma cell line U2OS as a model system, we treat cells with HmdU, harvest cells, and isolate DNA. Hydrolysis of the DNA with formic acid releases the free bases, and we convert the base 5-hydroxymethyluracil (HmU) into its methyl ester, 5-methoxymethyluracil, subsequently derivatizing with 3,5-bis(trifluoromethyl)benzyl bromide to yield a stable compound. When analyzing trace levels of HmdU deoxynucleoside standard, this derivatization process yields a detection limit of approximately 500 attomoles (5 × 10−16 mol) on column. We demonstrate in U2OS cells that toxicity of exogenously delivered HmdU is proportional to its incorporation into the DNA, and we show that repair of HmdU out of the DNA is occurring. Application of our method in cell lines with varying sensitivities to HmdU treatment will allow us to gain further insight into the mechanism of and resistance to this potential chemotherapeutic agent.

Combined Analysis of r-1,t-2,3,c-4-Tetrahydroxy-1,2,3,4-Tetrahydrophenanthrene and 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanol in Smokers' Plasma

Polycyclic aromatic hydrocarbons (PAH) and tobacco-specific nitrosamines, such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are widely accepted to be two important types of lung carcinogens in cigarette smoke. In this study, we have developed a method to estimate individual uptake of these compounds by quantifying r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in 1 mL of smokers' plasma. PheT and NNAL are biomarkers of PAH and NNK uptake, respectively. [D10]PheT and [pyridine-D4]NNAL were added to plasma as internal standards. The plasma was treated with beta-glucuronidase to release any conjugated PheT and NNAL. The analytes were enriched by solid-phase extraction on a mixed mode cation exchange cartridge and the PheT fraction was further purified by high-performance liquid chromatography. The appropriate fractions were analyzed by gas chromatography-negative ion chemical ionization-mass spectrometry for PheT and liquid chromatography-electrospray ionization-mass spectrometry for NNAL. The method was sensitive (limits of quantitation: PheT, 13 fmol/mL; NNAL, 3 fmol/mL), accurate, and precise. Levels of PheT and NNAL in plasma from 16 smokers averaged 95 +/- 71 and 36 +/- 21 fmol/mL, respectively, which are approximately 1% to 2% of the amounts found in urine. This method should be useful in molecular epidemiology studies of carcinogen uptake and lung cancer in smokers.

Free 3-nitrotyrosine in exhaled breath condensates of children fails as a marker for oxidative stress in stable cystic fibrosis and asthma

3-Nitrotyrosine (3-NT) is considered as a marker of oxidative stress, which occurs during inflammation. Since 3-NT levels in exhaled breath condensate (EBC) are very low, we applied a specific and sensitive gas chromatography–negative ion chemical ionization–mass spectrometry (GC–NICI–MS) method and high performance liquid chromatography (HPLC) with electrochemical detection for the analysis of free 3-NT in EBC. A total of 42 children (aged 5–17 years) were enrolled in this study, including children with asthma ( n = 12), cystic fibrosis ( n = 12), and healthy controls ( n = 18). Additionally, 14 healthy non-smoking adults (aged 18–59 years) were included. An EcoScreen system was used for the collection of EBC samples. Free 3-NT levels in EBC ranged from 0.54–6.8 nM. Median (interquartile range) concentrations (nM) were similar in all groups: 1.46 (0.97–2.49) in healthy adults, 2.51 (1.22–3.51) in healthy children, 1.46 (0.88–2.02) in children with asthma, and 1.97 (1.37–2.35) in CF children, respectively ( p = 0.24, Kruskall–Walis test). No difference was found between the children with airway disease and age-matched healthy controls. In healthy subjects, there was no effect of age on 3-NT concentrations. HPLC analyses provided similar concentration ranges for EBC 3-NT when compared with GC–NICI–MS. Our study has clearly demonstrated that free 3-NT in EBC fails as a marker for oxidative stress in children with stable CF and asthma.

Characterization and Quantification of Cysteinyl Adducts of Benzene Diol Epoxide

The production of macromolecular adducts of benzene diol epoxide (BDE), a toxic metabolite of benzene, has received little attention despite the demonstrated mutagenicity and carcinogenicity of BDE in rodents. Syn and anti enantiomers of BDE were relatively stable in 0.1 M ammonium acetate buffer, pH 7.6 (half times were greater than 5 h), and showed evidence of pseudo-first-order reactions with albumin (half times were about 4 h) and glutathione (GSH) (half times were about 0.3-0.4 h). Reaction products of BDE isomers with l-cysteine, N-acetyl-l-cysteine, N-acetyl-l-cysteine methyl ester, and GSH were characterized by a combination of electrospray ionization mass spectrometry and/or gas chromatography-mass spectrometry with electron impact ionization of trimethylsilyl derivatives of the adducts. Products corresponded to 1:1 addition of BDE isomers with each nucleophilic species, suggesting that adduction occurred primarily at the free sulfhydryl group. To investigate the disposition of the BDEs in vivo, we developed an assay for cysteinyl BDE-protein adducts. The assay involves enzymatic hydrolysis of the protein followed by derivatization of the released adducts and gas chromatography-negative ion chemical ionization-mass spectrometry. Preliminary applications of the assay showed linear increases in the formation of BDE-GSH adducts in samples of GSH incubated with increasing concentrations of BDE (10-300 microM) and showed the presence of BDE-albumin following incubation of albumin with 10 microM BDE.

Occurrence of polybrominated diphenylethers, polychlorinated dibenzo- p-dioxins, dibenzofurans and biphenyls in coastal sediments from Spain

Fifteen sediment samples were analysed in order to determine their content of polybrominated diphenylethers (PBDEs), polychlorinated dibenzo- p-dioxins (PCDDs), dibenzofurans (PCDFs) and biphenyls (PCBs). Samples were collected from several hot spots on the Spanish coast, such as the harbours of Almeria and Tarragona, and the mouths of the Besós and Llobregat rivers in Barcelona. A generic analytical procedure based on Soxhlet extraction followed by an automated cleanup system and gas chromatography-ion trap-mass spectrometry was employed for determining the toxic congeners of PCDDs and PCDFs, as well as dioxin-like PCBs. As regards PBDE determinations, a rapid method based on the use of selective pressurized liquid extraction followed by gas chromatography-negative ion chemical ionization-mass spectrometry was applied. Total toxicity equivalent (WHO-TEQ) values were calculated using the toxicity equivalent factors proposed by WHO for dioxin-like PCBs, PCDDs and PCDFs. WHO-TEQ values ranged from 0.3 to 75 pg/g dry weight (dw), with PCB contribution on the toxicity of the samples between 1 and 84%. Total PBDE levels ranged from 2.7 to 134 ng/g dw, with BDE-209 contribution on the total PBDE contamination between 50 and 99%.

Validation of a simple, sensitive method for the determination of beta-estradiol in bovine urine using gas-chromatography negative-ion chemical ionization mass spectrometry.

A new method is presented for the analysis of 17[small beta]-estradiol in bovine urine. After deconjugation, the sample is cleaned up using an OASIS[trade mark sign] HLB disposable cartridge and extracted into 1-chlorobutane. The hormone is derivatized using pentafluorobenzoyl chloride. The derivatized estradiol is quantitated using gas-chromatography negative-ion chemical ionization mass spectrometry. Calibrations, obtained using spiked blank urine, are linear in the range of 100-1000 pg mL(-1) with CC[small alpha] approximately 170 pg mL(-1) and CC[small beta] of 287 pg mL(-1). Recoveries are in the range of 80 to 130%. The method is rugged, rapid and sensitive when compared to other hormone methods.

Determination of decabromodiphenyl ether in sediments using selective pressurized liquid extraction followed by GC-NCI-MS.

A method based on selective pressurized liquid extraction (SPLE) followed by gas chromatography-negative ion chemical ionization-mass spectrometry (GC-NCI-MS) has been evaluated for analysis of decabromodiphenyl ether (PBDE-209) in sediment samples. Instrumental operating conditions such as source temperature and system pressure were optimized in the NCI-MS system, giving an instrumental detection limit of 2 pg. The limit of determination of the entire SPLE-GC-NCI-MS procedure was around 50 pg g(-1) dry weight (dw), with repeatability of replicates between 4 and 21% relative standard deviation. Application of the method to 13 different river and marine sediment samples collected in Spain revealed that levels of decabromodiphenyl ether ranged between 2 and 132 ng g(-1) dry weight.

Effect of DNA repair gene polymorphisms on BPDE-DNA adducts in human lymphocytes.

To determine whether variations in DNA repair genes are related to host DNA damage, we investigated the association between polymorphism in the XPD gene (codon 199, 312, 751) and the XRCC1 gene (codon 194, 399) and the presence of benzo(a)pyrene diolepoxide adducts to lymphocyte DNA (BPDE-DNA) in a group of male patients with incident lung cancer, all current smokers. BPDE-DNA adducts were analyzed by high-resolution gas chromatography-negative ion chemical ionization-mass spectrometry. XPD and XRCC1 genotypes were identified by PCR-RFLP. XRCC1 and XPD genotypes did not affect the levels and proportion of detectable BPDE-DNA adducts. The patients were also genotyped for the GSTM1 polymorphism, given its role in the detoxification of BPDE. Individuals with the GSTM1 deletion had significantly higher levels of BPDE-DNA adducts when they were XPD-Asp312Asp+Lys751Lys than carriers of at least one variant allele. No such association was found with the XRCC1 genotypes. Because of the small study population (n = 60), further statistical analysis of possible gene-gene and gene-environment would not be informative. This is the first study analysing the specific BPDE-DNA adduct in vivo with regard to polymorphic repair genes (XPD, XRCC1) and xenobiotic metabolizing gene (GSTM1). Our results raise the possibility that the XPD-Asp312Asp+Lys751Lys genotype may increase BPDE-DNA damage; this effect might be evident in individuals who are especially likely to have accumulated damage, probably because of lower detoxification capacity and high environmental exposure.

Simultaneous determination of delta9-tetrahydrocannabinol and 11-nor-9-carboxy-delta9-tetrahydrocannabinol in human plasma by solid-phase extraction and gas chromatography-negative ion chemical ionization-mass spectrometry.

Delta9-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-delta9-tetrahydrocannabinol (THCA) in human plasma can be simultaneously detected using solid-phase extraction with gas chromatography and negative ion chemical ionization mass spectrometry. THC-d3 and THCA-d3 are added as internal standards; protein is precipitated with acetonitrile and the resulting supernatants diluted with 0.1 M sodium acetate (pH 7.0) prior to application to the solid-phase extraction columns. THC and THCA were eluted separately and then pooled, dried under air, and derivatized with trifluoroacetic anhydride and hexafluoroisopropanol. The derivatized THC-d0 gives abundant molecular anions (m/z 410), and the derivatized THCA-d0 gives abundant fragment ions (m/z 422) formed by loss of (CF3)2CHOH from its molecular anion. The recoveries of THC and THCA were 74% and 17%, respectively. The lower and upper limits of quantitation were 0.5 and 100 ng/mL for THC and 2.5 ng/mL and 100 ng/mL for THCA. The within-run accuracy and precision for THC (measured at 0.5, 1, 10 and 75 ng/mL) ranged from 98 to 106% (% target) and 4.1 to 9.5 (%CV), respectively. For THCA, the within-run accuracy and precision (measured at 2.5, 5, 10, and 75 ng/mL) ranged from 89 to 101% and 4.3 to 7.5%, respectively. The between-run accuracy and precision for THC ranged from 92 to 110% and 0.4 to 12.4%, respectively. The between-run accuracy and precision for THCA ranged from 97 to 103% and 6.5 to 12.3%, respectively. In processed samples stored in reconstituted form at -20 degrees C, THC and THCA were stable for at least three days. THC and THCA stored in plasma were stable following three freeze/thaw cycles. THC and THCA in whole blood at room temperature for 6 h, or in plasma stored at room temperature for 24 h, did not show significant change. Storage in polypropylene containers for 7 days at -20 degrees C and the presence of 1% sodium fluoride or the cannabinoid receptor antagonist, SR141716, at 1 microg/mL did not interfere with the quantitation of THC and THCA. In three individuals who smoked marijuana under controlled dosing conditions, peak THC concentrations of 151, 266, and 99 ng/mL were seen in the first plasma samples drawn immediately after the end of smoking, and corresponding peak THCA concentrations of 41, 52, and 17 ng/mL occurred at 0.33 to 1 h after cessation of smoking.

Concentration of 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) in urine and alkali-hydrolyzed urine after consumption of charbroiled beef

Heterocyclic amines (HAs) and polycyclic aromatic hydrocarbons are carcinogenic products formed during the cooking of meat at moderate to high temperatures. We have previously shown that the urinary concentration of 1-hydroxypyrene-glucuronide, a metabolite of pyrene, increased significantly in ten subjects who had ingested charbroiled ground beef. We now report the time course and interindividual variation of 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) concentration in the urine samples from these ten subjects. PhIP concentration was determined in both untreated and alkali-hydrolyzed urine to obtain estimates of the proportion of conjugated PhIP metabolites in each subject. PhIP was measured by gas chromatography-negative ion chemical ionization-mass spectrometry after derivatization with pentafluorobenzyl bromide. Ten healthy non-smoking males consumed identical amounts of broiled beef on five consecutive days. The morning after the first day of broiled beef consumption, urinary concentration of PhIP increased 14–38 fold above mean pre-feed concentration of PhIP in individual alkali-hydrolyzed urine samples. Following cessation of broiled beef consumption, urinary PhIP concentration declined to near pre-feed levels within 48–72 hrs. The ratio of total alkali–labile PhIP metabolites to unmetabolized PhIP varied by about 2.7-fold among subjects, ranging from 18:1 to 48:1, suggesting that interindividual differences in PhIP metabolism occur and can be detected by this method. This study of urinary PhIP following ingestion of meat cooked by charbroiling, that contains both HAs and polycyclic aromatic hydrocarbons, extends previous studies of ingestion of pan-fried meat that contains primarily HAs. The results indicate that significant amounts of PhIP are bioavailable from ingestion of charbroiled ground beef and that measurement of proportions of alkali–labile PhIP metabolites and parent PhIP in human urine may yield information on individual metabolism of ingested PhIP.

Determination of estrogens in river water by gas chromatography–negative-ion chemical-ionization mass spectrometry

A method for the determination of estrogens (17α-estradiol, 17β-estradiol, estrone, ethynyl estradiol, and estriol) as pentafluorobenzyl-trimethylsilyl (PFB–TMS) derivatives by gas chromatography–mass spectrometry (GC–MS) with negative-ion chemical-ionization (NICI) is described. The NICI of all the derivatives produced an intense [M–PFB] − ion as the base peak. The reagent gas (methane) flow-rate and the ion source temperature were determined to be 2.0 ml/min and 240°C, respectively, for the optimized NICI-selected ion monitoring (SIM) conditions. The sensitivities of the PFB–TMS derivatives in the NICI mode were 8.0–130 times higher than those of the PFB–TMS derivatives in electron ionization (EI) mode, and 12–25 times higher than those of all the TMS derivatives in the EI mode. This method was applied to the analysis of estrogens in river water using a solid-phase extraction as the sample preparation. The recoveries of the target chemicals from a river-water sample spiked with standards at 2 ng/l level were 85.8–126.5% (RSD, 6.2–13.0%). The methodical detection limits ranged from 0.10 to 0.28 ng/l.

Benzo(a)pyrene diolepoxide-haemoglobin and albumin adducts at low levels of benzo(a)pyrene exposure

A biomonitoring study was conducted to simultaneously measure individual benzo(a)pyrene (BaP) exposure in 50 office employees, not occupationally exposed to polycyclic aromatic hydrocarbons (PAH), using personal samplers and the formation of (+) r-7, t-8-dihyroxy-t-9,t-10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) adducts to haemoglobin (BPDE–Hb) and serum albumin (BPDE–SA). The population enrolled was exposed to an average of 0.58 ± 0.46 ng BaP m−3 (mean ± SD). The concentration of BaP collected from smokers' samples was double that from non-smokers (P = 0.007). BPDE adducts to Hb and SA were quantified as BaP tetrols released from hydrolysis of macromolecules and measured by high-resolution gas chromatography–negative ion chemical ionization–mass spectrometry. BPDE–Hb adducts were detected in 16% of the population and BPDE–SA adducts in 28%. Smoking did not affect adduct formation. When BaP personal monitoring data were used as the criterion of exposure, no correlation was found with the presence and the levels of BPDE–Hb and BPDE–SA adducts. Undetected sources of PAH, such as the diet, might markedly alter the exposure profile depicted by individual air sampling and affect the frequency and levels of protein biomarkers. This is the first comparative analysis of BPDE–Hb and BPDE–SA adducts, providing reference values for these biomarkers in a general urban population. However it is difficult to establish which biomarkers would be the more relevant in assessing low BaP exposure, due to undetectable factors such as dietary PAHs, that might have influenced theresults to some degree.

Effects of taurine, glutamine and culture dishes on the in vitro development of bovine embryos

Prostanoids can he assayed by the highly sensitive and reproducible gas chromatography-negative ion chemical ionization-mass spectrometry (GC-NICI-MS) technique. This paper describes a simplified method applied to the quantification of prostaglandins (PG) in the surpernatants of limited amounts of cells, cultured in multiwell plates (30,000 cells/well). The culture medium was extracted on C2 columns and the derivatization was performed in three steps. Prostanoids were then analyzed by GC-NICI-MS within 10 min. The storage conditions at each step were tested in order to achieve maximal yields. This procedure allowed the determination and quantification of PG produced by cultured human synovial cells and chondrocytes at the basal level as well as after interleukin-1 stimulation. PGE2, PGF2α, and 6-keto-PGF1α, the stable form of PGI2, were the main PG detected. Derivatization and GC-MS analyses of 18 samples could easily be performed in 1 day. The simplified GC-NICI-MS method can thus be routinely applied for assaying PG in any cell line cultured in multiwells.

Screening for forensically relevant benzodiazepines in human hair by gas chromatography-negative ion chemical ionization-mass spectrometry

A procedure is presented for the detection in human hair of forensically relevant benzodiazepines, i.e. nordiazepam, oxazepem, bromazepam, diazepam, lorazepam, flunitrazepam, alprazolam and triazolam. The method involves decontamination of hair with methylene chloride, pulverization in a ball mill, incubation of 50 mg powdered hair in Soerensen buffer (pH 7.6) in the presence of prazepam-d 5 used as internal standard, liquid-liquid extraction with diethyl ether-chloroform (80:20, v/v) and gas chromatography-mass spectrometry using negative chemical ionization after derivatization with, N, O-bis(trimethylsilyl)trifluoroacetamide plus 1% trimethylchlorosilane. The limits of detection for all benzodiazepines ranged from 1 to 20 pg/mg using a 50-mg hair sample. Coefficients of variation and extraction recoveries, ranging from 7.4 to 25.4% and 47.6 to 90%, respectively, were found suitable for a screening procedure. One hundred and fifteen samples were submitted to this screening procedure, and specimens tested positive for nordiazepam (0.20-18.87 ng/mg, n = 42) and its major metabolite oxazepam (0.10-0.50 ng/mg, n = 14), flunitrazepam (19–148 pg/mg, n = 31), lorazepam (31–49 pg/mg, n = 4) and alprazolam (0.3-1.24 ng/mg, n = 2). Bromazepam, diazepam and triazolam were not detected.