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Development and validation of an isotope-dilution electrospray ionization tandem mass spectrometry method with an on-line sample clean-up device for the quantitative analysis of the benzene exposure biomarker S-phenylmercapturic acid in human urine
Abstract
An isotope-dilution electrospray ionization tandem mass spectrometry (ESI-MS/MS) method with an on-line sample clean-up device, for the quantitative analysis of human urine for the benzene exposure biomarker S-phenylmercapturic acid (SPMA), was developed and validated. The sample clean-up system was constructed from an autosampler, a reversed-phase C18 trap cartridge, a two-position switching valve, and controlling computer software and hardware. The sample clean-up system was interfaced via 1/20 splitting to the ESI source of a triple-quadrupole mass spectrometer using negative ion mode and multiple reaction monitoring for SPMA and the isotope-labeled internal standard. A strategy was adopted to acquire pooled blank urine matrix and quality control samples spiked with standards. Validated procedures and data on method specificity, detection limits, standard curves, precision and recovery, sample storage stability, and inter-laboratory comparison are presented. The analytical system was fully automated. No tedious manual sample clean-up procedures are required. With the selectivity and the sensitivity provided by ESI-MS/MS detection, the analytical system can be used for high-throughput and accurate determination of SPMA levels in human urine samples, as a biomarker for environmental as well as occupational benzene exposure.
... On the other hand, measuring PMA in urine is an expensive task due to the necessity of extensive sample pretreatment involving extraction and derivatization independently of the instrumental technique available for detection. In addition, highly selective chromatographic techniques are required (HPLC and GC) coupled to sensitive detectors such as the fluorimeter (Maestri et al. 1993), mass spectrometer (Angerer et al. 1998) or tandem mass spectrometer (Melikian et al. 1999, Lin et al. 2004a. These requirements enhance the costs of analyses and present disadvantages when planning the measurement of PMA in large-scale screening programmes. ...
... At present, PMA is determined in urine by HPLC methods requiring SPE extraction, derivatization and fluorimetric detection (Maestri et al. 1993, Inoue et al. 2001). Determination of PMA by GC-MS (Van Sittert et al. 1993, Einig et al. 1996, Angerer et al. 1998, Waidyanatha et al. 2004), GC-MS-MS (Van den Berg 2003), LC-MS (Maestri et al. 2005) and LC-MS-MS (Melikian et al. 1999, Liao et al. 2002, Pieri et al. 2003, Barbieri et al. 2004, Lin et al. 2004a were also reported. Ball et al. (1997) and Aston et al. (2002) reported the development and validation of a competitive enzyme-linked immunosorbent assay (ELISA) specific for PMA which was designed to simplify the procedures used until recently for routine biomonitoring of low levels of benzene exposure. ...
... Its use drastically reduces the number of the samples needed to be processed by complex and expensive techniques such as those based on MS and MS-MS detection like those recently reported, which require a full method validation, trained personnel and expensive laboratory facilities. Hyphenated chromatographic-mass spectrometric techniques adopted for PMA measurements like GC-MS, LC-MS (Maestri et al. 2005), LC-MS-MS (Melikian et al. 1999, Liao et al. 2002, Pieri et al. 2003, Barbieri et al. 2004, Lin et al., 2004a and GC-MS-MS (Van den Berg 2003) in the selected reaction monitoring afford undoubtedly outstanding sensitivity and selectivity as compared with chemiluminescence-ELISA and conventional GC-MS. However, the former methods appear more suitable for research purposes than for routine measurements on large numbers of samples. ...
S-phenylmercapturic acid (PMA) is a specific urinary biomarker of benzene at exposure levels lower than 1 ppm. However, measuring PMA in urine is an expensive task by either GC or HPLC due to the necessity of extensive sample pretreatment. In the present study, a commercial chemiluminescence enzyme-linked immunosorbent assay (ELISA) test for PMA and GC-MS were used for screening urine samples of 60 workers employed in petrochemical settings. The ELISA results were evaluated by comparison with the GC-MS. Overall, the ELISA test proved sensitive (limit of detection=0.1 microg l(-1)), rapid, robust and reliable, affording results in good agreement with the GC-MS (54% of measurements) and no false-negatives. On the other hand, 46% of the ELISA assays were assigned as false-positives (arbitrarily established when ELISA >5 microg l(-1), GC-MS <5 microg l(-1) and a correlation coefficient of 0.687 was calculated between the two methods. It appears that urinary PMA routine biomonitoring on large numbers of samples is carried out in a cost-effective and rapid approach by preliminary screening with the ELISA assay followed by GC-MS confirmation of concentrations exceeding the biological exposure index for PMA.
... The low conversion rate for this metabolic pathway results in low levels of urinary PMA, even among smokers. Other less specific urinary markers such as trans,trans-muconic acid are often used to estimate benzene exposure (22,24,33) despite the new approaches in analytical determination that have lowered the LOD of PMA to the sub-µg/L range (34)(35)(36). Paci et al. indicate that precursor PMA (pre-PMA) is a significant portion of total PMA in urine and that quantitative conversion of pre-PMA to PMA requires treatment with strong acid (35). Thus, our method, which uses 0.5% acetic acid, is unlikely to quantitatively convert pre-PMA to PMA. ...
... gBarr et al. (44). h Lin et al.(34). ...
The widespread exposure to potentially harmful volatile organic compounds (VOCs) merits the development of practical and accurate exposure assessment methods. Measuring the urinary concentrations of VOC mercapturic acid (MA) metabolites provides noninvasive and selective information about recent exposure to certain VOCs. We developed a liquid chromatography-tandem mass spectrometry method for quantifying urinary levels of six MAs: N-acetyl-S-(2-carboxyethyl)-L-cysteine (CEMA), N-acetyl-S-(3-hydroxypropyl)-L-cysteine (HPMA), N-acetyl-S-(2-hydroxy-3-butenyl)-L-cysteine (MHBMA), N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine (DHBMA), N-acetyl-S-(2-hydroxyethyl)-L-cysteine (HEMA), and N-acetyl-S-(phenyl)-L-cysteine (PMA). The method provides good accuracy (102% mean accuracy) and high precision (3.5% mean precision). The sensitivity (limits of detection of 0.01-0.20 microg/L) and wide dynamic detection range (0.025-500 microg/L) make this method suitable for assessing VOC exposure of minimally exposed populations and those with significant exposures, such as cigarette smokers. We used this method to quantify MA levels in urine collected from smokers and nonsmokers. Median levels of creatinine-corrected CEMA, HPMA, MHBMA, DHBMA, HEMA, and PMA among nonsmokers (n = 59) were 38.1, 24.3, 21.3, 104.7, 0.9, and 0.5 microg/g creatinine, respectively. Among smokers (n = 61), median levels of CEMA, HPMA, MHBMA, DHBMA, HEMA, and PMA were 214.4, 839.7, 10.2, 509.7, 2.2, and 0.9 microg/g creatinine, respectively. All VOC MAs measured were higher among smokers than among nonsmokers, with the exception of MHBMA.
... However, most of these methods determine only one metabolite 18,[20][21][22][23][24][25][26] . Over the past decades, the LC-MS/MS gained widespread use for quantitation of drugs and their metabolites in biological matrices 5,[27][28][29][30][31][32][33][34] . Several methods with isotopic dilutions have been developed on LC-MS/ MS for the determination of t,t-MA and SPMA in urine samples of workers exposed to low levels of benzene 28,35 . ...
Benzene is an important industrial solvent and a ubiquitous environmental pollutant. Urinary S-phenyl mercapturic acid (SPMA) and trans, trans - muconic acid (t,t-MA) are specific and sensitive biomarkers for the determination of low levels of benzene exposure. This paper describes a specific and sensitive LC-MS/ MS method for simultaneous determination of t,t-MA and SPMA from human urine samples using dual columns of two different internal diameters to reduce the matrix effect on ionization. This method was applied for the measurements of t,t-MA and SPMA from the random urine samples of exposed subjects of the footwear industry and comparable control subjects. The recoveries (Mean ± SD) of spiked standards of t,t-MA and SPMA in urine were 95.4 ± 12.3% (n = 6) ranging from 79.4 - 114% and 69.7 ± 9.5% (n=6) ranging from 60.1 - 89% respectively. The Limit of Detection (LOD) was found to be 1 ng/ml and 0.03 ng/ml and the Limit of Quantitation (LOQ) was found to be 5 ng/ml and 0.1 ng/ml for t,t-MA and SPMA at the S/N ratio of 3 and 10 respectively. The observed values of t, t-MA and SPMA in the exposed subjects of footwear industry were below the values of biological exposure indices (BEI) as described by ACGIH for benzene exposure.
... However, most of these methods determine only one metabolite 18,[20][21][22][23][24][25][26] . Over the past decades, the LC-MS/MS gained widespread use for quantitation of drugs and their metabolites in biological matrices 5,[27][28][29][30][31][32][33][34] . Several methods with isotopic dilutions have been developed on LC-MS/ MS for the determination of t,t-MA and SPMA in urine samples of workers exposed to low levels of benzene 28,35 . ...
... b-Glucuronidase enzymatic deconjugation was used to change the glucuronid-conjugated forms of seven phthalate metabolites into their free forms for quantification of the total amounts in urine samples. As previously reported, on-line solid phase extraction (SPE) was used to extract the seven urinary phthalate metabolites (Lin et al., 2004 ). Briefly, this analytical approach involved the enzymatic deconjugation of phthalate metabolites followed by on-line SPE and quantification using liquid chromatography/electrospray tandem mass spectrometry (LC– ESI-MS/MS). ...
... Deionized water was acquired from a Millipore system (Milford, MA, USA). Seven urinary phthalate monoesters were analyzed using on-line solid-phase extraction (SPE) coupled with Phthalate and GSTM1 polymorphism in estrogen-dependent diseases liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS), which was adapted from a previous study (Lin et al., 2004; Huang et al., 2007). Briefly, 1 ml aliquots of the sample containing 750 ml urine, 50 ml of 2000 ppb 13 C 4 -labeled phthalate monoesters as internal standards, 200 ml of 100 mM ammonium acetate buffer (pH 6.5) and 10 ml of b-glucuronidase were incubated at 378C for 90 min for deconjugation. ...
Phthalates are known to have estrogenic effects in cell models and experimental animals. However, the evidence regarding the effects of phthalates on human reproduction is still limited. We conducted a case-control study to determine whether estrogen-dependent diseases are associated with phthalate exposure and how the glutathione S-transferase M1 (GSTM1; a major detoxification enzyme) genotype modulates the risk.
We recruited subjects who underwent laparotomy and had pathologic confirmation of endometriosis (EN) (n = 28), adenomyosis (AD) (n = 16) and leiomyoma (LEI) (n = 36) from the Department of Obstetrics and Gynecology at a medical center in Taiwan between 2005 and 2007. Controls (n = 29) were patients without any of the three aforementioned gynecologic conditions. Urine samples were collected before surgery and analyzed for seven phthalate metabolites using liquid chromatography-tandem mass spectrometry. Peripheral lymphocytes were used for GSTM1 genotype determination.
Patients with LEIs had significantly higher levels of total urinary mono-ethylhexyl phthalate (SigmaMEHP; 52.1 versus 18.9 microg/g creatinine, P < 0.05) than the controls, whereas patients with EN had an increased level of urinary mono-n-butyl phthalate (94.1 versus 58.0 microg/g creatinine, P < 0.05). Subjects with GSTM1 null genotype had significantly increased odds for AD relative to those with GSTM1 wild genotype [odds ratio (OR) = 5.30; 95% CI, 1.22-23.1], even after adjustment for age and phthalate exposure. Subjects who carried the GSTM1 null genotype and had a high urinary level of SigmaMEHP showed a significantly increased risk for AD (OR = 10.4; 95% CI, 1.26-85.0) and LEIs (OR = 5.93; 95% CI, 1.10-31.9) after adjustment for age, compared with those with GSTM1 wild-type and low urinary level of SigmaMEHP.
These results suggest that both GSTM1 null and phthalate exposure are associated with AD and LEI. Larger studies are warranted to investigate potential interaction between GSTM1 null and phthalate exposure in the etiology of estrogen-dependent gynecologic conditions.
... The capture of benzene oxide by glutathione at pH 7 is inefficient compared to its rearrangement to phenol, accounting for the fact that SPMA is a minor metabolite of benzene (45). Nevertheless, SPMA has proven to be a useful and specific biomarker of benzene exposure (10,(46)(47)(48)(49)(50), with LC-MS/MS being used extensively for quantitation (4,(51)(52)(53)(54)(55)(56). Levels of SPMA are consistently higher in smokers than in non-smokers (8,10,55,56). ...
We determined the persistence at various times (3, 7, 14, 21, 28, 42, and 56 days) of eight tobacco smoke carcinogen and toxicant biomarkers in the urine of 17 smokers who stopped smoking. The biomarkers were 1-hydroxy-2-(N-acetylcysteinyl)-3-butene (1) and 1-(N-acetylcysteinyl)-2-hydroxy-3-butene (2) [collectively called MHBMA for monohydroxybutyl mercapturic acid] and 1,2-dihydroxy-4-(N-acetylcysteinyl)butane (3) [DHBMA for dihydroxybutyl mercapturic acid], metabolites of 1,3-butadiene; 1-(N-acetylcysteinyl)-propan-3-ol (4, HPMA for 3-hydroxypropyl mercapturic acid), a metabolite of acrolein; 2-(N-acetylcysteinyl)butan-4-ol (5, HBMA for 4-hydroxybut-2-yl mercapturic acid), a metabolite of crotonaldehyde; (N-acetylcysteinyl)benzene (6, SPMA for S-phenyl mercapturic acid), a metabolite of benzene; (N-acetylcysteinyl)ethanol (7, HEMA for 2-hydroxyethyl mercapturic acid), a metabolite of ethylene oxide; 1-hydroxypyrene (8) and its glucuronides (1-HOP), metabolites of pyrene; and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (9) and its glucuronides (total NNAL), a biomarker of exposure to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These biomarkers represent some of the major carcinogens and toxicants in cigarette smoke: 1,3-butadiene, acrolein, crotonaldehyde, benzene, ethylene oxide, polycyclic aromatic hydrocarbons (PAH), and NNK. With the exception of DHBMA, levels of which did not change after cessation of smoking, all other biomarkers decreased significantly after 3 days of cessation (P < 0.001). The decreases in MHBMA, HPMA, HBMA, SPMA, and HEMA were rapid, nearly reaching their ultimate levels (81-91% reduction) after 3 days. The decrease in total NNAL was gradual, reaching 92% after 42 days, while reduction in 1-HOP was variable among subjects to about 50% of baseline. Since DHBMA did not change upon smoking cessation, there appear to be sources of this metabolite other than 1,3-butadiene. The results of this study demonstrate that the tobacco smoke carcinogen/toxicant biomarkers MHBMA, HPMA, HBMA, SPMA, HEMA, 1-HOP, and NNAL are related to smoking and are good indicators of the impact of smoking on human exposure to 1,3-butadiene, acrolein, crotonaldehyde, benzene, ethylene oxide, PAH, and NNK.
... Unexposed Chinese workers 1.87 g/g creatinine [14] LOD 5 g/l [6] Automated sample clean-up system [27] Immunoassay ELISA LOD 8 g/l urine [7] Non-smokers 0.9 mol/mol creatinine [8] 2.4. Current study For a molecular epidemiology study of benzene exposure, part of which is reported below, three analytical approaches were used for S-PMA analysis, LC-MS/MS, immunoassay using the kit for laboratory use, and the immunoassay service supplied by AB Biomonitoring Ltd. ...
S-Phenylmercapturic acid (S-PMA), is a urinary metabolite of benzene, thought to be derived from the condensation product of benzene oxide with glutathione. S-PMA may be determined by GC, HPLC (UV or fluorescence detection), GC-MS, LC-MS/MS or immunoassays. The limit of sensitivities of most of these techniques is 1 microg/l urine or below. It has been suggested that S-PMA may have value as a biomarker for low level human exposure to benzene, in view of the facts that urinary excretion of S-PMA has been found to be related to airborne benzene in occupationally exposed workers, and that only low background levels of S-PMA have been found in control subjects. We have evaluated the use of S-PMA as a biomarker, using a commercially available analytical service, in a multicentre European study of populations exposed to varying levels of benzene, in Italy (Milan, Genoa) and in Bulgaria (Sofia). These were filling station attendants, urban policemen, bus drivers, petrochemical workers and referents (a total of 623 subjects). S-PMA was measured at the end of the work shift by an immunoassay procedure. Urinary benzene (in Milan only) and the benzene metabolite trans,trans-muconic acid (t,t-MA) were measured before and after the work shift. Air-borne benzene was measured as a monitor of exposure. Urinary benzene was the most discriminatory biomarker and showed a relationship with airborne benzene at all levels of exposure studied (including groups exposed to <0.1 ppm benzene), whereas t,t-MA and S-PMA, as determined by immunoassay, were suitable only in the highest exposed workers (petrochemical industry, geometric mean 1765 microg/m3 (0.55 ppm) benzene). All three biomarkers were positively correlated with smoking as measured by urinary cotinine).
... Effects of smoking cessation on levels of trans, trans-muconic acid in urine have not been reported. S-Phenylmercapturic acid (S-PMA) is another biomarker of benzene exposure, which is elevated in smokers and appears to have higher specificity than trans, trans-muconic acid (Hecht, 2002;Lin, Tyan, Shih, Chang, & Liao, 2004;Maestri, Negri, Ferrari, Ghittori, & Imbriani, 2005;Melikian et al., 2002;Tharnpoophasiam, Kongtip, Wongwit, Fungladda, & Kitayaporn, 2004). Hb adducts of the carcinogens ethylene oxide, acrylamide, acrylonitrile, and of an unknown ethylating agent are higher in smokers than in nonsmokers but are not specific to tobacco use (Bergmark, 1997;Carmella et al., 2002;Fennell et al., 2000). ...
To date, we have no valid biomarkers that serve as proxies for tobacco-related disease to test potential reduced exposure products. This paper represents the deliberations of four workgroups that focused on four tobacco-related heath outcomes: Cancer, nonmalignant pulmonary disease, cardiovascular disease, and fetal toxicity. The goal of these workgroups was to identify biomarkers that offer some promise as measures of exposure or toxicity and ultimately may serve as indicators for future disease risk. Recommendations were based on the relationship of the biomarker to what is known about mechanisms of tobacco-related pathogenesis, the extent to which the biomarker differs among smokers and nonsmokers, and the sensitivity of the biomarker to changes in smoking status. Other promising biomarkers were discussed. No existing biomarkers have been demonstrated to be predictive of tobacco-related disease, which highlights the importance and urgency of conducting research in this area.
β-Adrenergic agonist compounds are medicines that open up the lung's medium and large airways. β-Adrenergic agonist compounds have been illegally or legally used to increase lean muscle mass in meat animals, bodybuilding, weight-loss programs, and athletes. Developing a rapid analytical approach for determining β-adrenergic agonist compounds in biological samples is crucial for individual exposure assessment. This study established an analytical method for simultaneously measuring eight β-adrenergic agonist compounds in human urine, including clenbuterol, terbutaline, salbutamol, ractopamine, zilpaterol, cimaterol, tulobuterol, and fenoterol. Two hundred microliters of a urine sample were added to eight deuterium-labeled internal standard mixtures and glucuronidase/arylsulfatase for enzymatic hydrolysis, and were then analyzed using an online clean-up system coupled with a liquid chromatography-tandem mass spectrometry system (LC–MS/MS). The limit of quantification ranged from 0.03 to 0.12 ng/mL urine for the eight β-adrenergic agonist compounds. The relative standard deviations (RSD) of the within-run and between-run precisions were less than 10%, and the relative accuracy errors were less than 17% in the three-level spiked artificial urine samples. Two hundred eighty human urine samples collected from the general population in Taiwan were assessed to demonstrate the capability and feasibility of this method. The detection frequencies were 33% for clenbuterol, 5% for ractopamine, and less than 5% for the others. We concluded that the isotope dilution-online clean-up system coupled with LC–MS/MS method is a valuable analytical method for investigating urinary β-adrenergic agonist compounds in humans and is valuable for human biomonitoring studies.
Stand-alone electrospray ionization mass spectrometry (ESI-MS) has been advancing through enhancements in throughput, selectivity and sensitivity of mass spectrometers. Unlike traditional MS techniques which usually require extensive offline sample preparation and chromatographic separation, many sample preparation techniques are now directly coupled with stand-alone MS to enable outstanding throughput for bioanalysis. In this review, we summarize the different sample clean-up and/or analyte enrichment strategies that can be directly coupled with ESI-MS and nano-ESI-MS for the analysis of biological fluids. The overview covers the hyphenation of different sample preparation techniques including solid phase extraction (SPE), solid phase micro-extraction (SPME), slug flow micro-extraction/nano-extraction (SFME/SFNE), liquid extraction surface analysis (LESA), extraction electrospray, extraction using digital microfluidics (DMF), and electrokinetic extraction (EkE) with ESI-MS and nano-ESI-MS.
The indisputable relationship between the world's most widespread exogenous chemical carcinogen exposure - tobacco smoke - and cancer, provides a framework for a better understanding of cancer mechanisms in general, particularly the interaction between carcinogen exposure and host factors, and for the development of novel preventive and therapeutic strategies. Furthermore, the biomarkers that are developed in this research can be applied in the approaching era of tobacco product regulation. This chapter discusses the mechanisms of tumour induction by tobacco carcinogens and the status of tobacco carcinogen biomarkers.
A simple, rapid and low environmental toxicity ionic liquid-based ultrasonic-assisted dispersive liquid-liquid microextraction (DLLME) procedure was developed and validated for quantifying urinary levels of seven mercapturic acids (MAs) in human urine from nonsmokers and smokers. The ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate ([C8MIM][PF6]) and acetonitrile were used as extraction and disperser solvent, respectively, for the DLLME procedure, instead of using toxic chlorinated solvent. The factors affecting the extraction efficiency, such as the type and the amount of ionic liquid, type and volume of disperser solvent, extraction time and sample pH were optimized. In the proposed method, N-acetyl-S-(2-carboxyethyl)-l-cysteine (CEMA), N-acetyl-S-(3-hydroxypropyl)-l-cysteine (3-HPMA), N-acetyl-S-(2-hydroxy-3-butenyl)-l-cysteine + N-acetyl-S-(1-hydroxymethyl-2-propenyl)-l-cysteine (MHBMA isomers), N-acetyl-S-(phenyl)-l-cysteine (PMA), N-acetyl-S-(3,4-dihydroxybutyl)-l-cysteine (DHBMA), N-acetyl-S-(3-hydroxypropyl-1-methyl)-l-cysteine (HPMMA) and N-acetyl-S-(2-phenyl-2-hydroxyethyl)-l-cysteine + N-acetyl-S-(1-phenyl-2-hydroxyethyl)-l-cysteine (PHEMA isomers) can be determined. The optimized method showed a good precision level, with relative standard deviation values below 7.74 %. The method detection limits (MDL) between 0.05 and 2.50 µg L−1 and method quantification limits (MDQ) in the range of 0.20-8.25 µg L−1 were obtained for the proposed method. The recoveries for the analytes ranged from 85.32 to 127.61 %. Due to the good precision and the low limits of detection, the developed method is well suited for the determination of the analytes in human urine.
Abstract A framework of "Common Criteria" (i.e. a series of questions) has been developed to inform the use and evaluation of biomonitoring data in the context of human exposure and risk assessment. The data-rich chemical benzene was selected for use in a case study to assess whether refinement of the Common Criteria framework was necessary, and to gain additional perspective on approaches for integrating biomonitoring data into a risk-based context. The available data for benzene satisfied most of the Common Criteria and allowed for a risk-based evaluation of the benzene biomonitoring data. In general, biomarker (blood benzene, urinary benzene and urinary S-phenylmercapturic acid) central tendency (i.e. mean, median and geometric mean) concentrations for non-smokers are at or below the predicted blood or urine concentrations that would correspond to exposure at the US Environmental Protection Agency reference concentration (30 µg/m(3)), but greater than blood or urine concentrations relating to the air concentration at the 1 × 10(-5) excess cancer risk (2.9 µg/m(3)). Smokers clearly have higher levels of benzene exposure, and biomarker levels of benzene for non-smokers are generally consistent with ambient air monitoring results. While some biomarkers of benzene are specific indicators of exposure, the interpretation of benzene biomonitoring levels in a health-risk context are complicated by issues associated with short half-lives and gaps in knowledge regarding the relationship between the biomarkers and subsequent toxic effects.
The aim of this study is to validate isotope-dilution electrospray ionization tandem mass spectrometry (ESI–MS–MS) method with a dual-loop cleanup device for simultaneous quantitation of two benzene metabolites, trans, trans-muconic acid (ttMA) and S-phenylmercapturic acid (SPMA), in human urine. In this study, a pooled blank urine matrix from rural residents was adopted for validation of the analytical method. The calibration curve, detection limit, recovery, precision, accuracy and the stability of sample storage for the system have been characterized. Calibration plots of ttMA and SPMA standards spiked into two kinds of urine matrixes over a wide concentration range, 1/32–8-fold biological exposure indices (BEIs) values, showed good linearity (R > 0.9992). The detection limits in pooled urine matrix for ttMA and SPMA were 1.27 and 0.042 μg g−1 creatinine, respectively. For both of ttMA and SPMA, the intra- and inter-day precision values were considered acceptable well below 25% at the various spiked concentrations. The intra- and inter-day apparent recovery values were also considered acceptable (apparent recovery >90%). The ttMA accuracy was estimated by urinary standard reference material (SRM). The accuracy reported in terms of relative error (RE) was 5.0 ± 2.0% (n = 3). The stability of sample storage at 4 or −20 °C were assessed. Urinary ttMA and SPMA were found to be stable for at least 8 weeks when stored at 4 or −20 °C. In addition, urine samples from different benzene exposure groups were collected and measured in this system. Without tedious manual sample preparation procedure, the analytical system was able to quantify simultaneously ttMA and SPMA in less than 20 min.
An electrospray ionization tandem mass spectrometry (ESI-MS/MS) system with an online dual-loop cleanup device was developed for simultaneous quantitation of the urinary benzene exposure biomarkers trans,trans-muconic acid (ttMA) and S-phenylmercapturic acid (SPMA). The cleanup device was constructed from an autosampler, two electrically operated two-position switching valves, a reversed-phase C18 trap cartridge, a 200-microL loop, and two solvent-delivery pumps. The device was interfaced directly with a triple-quadrupole mass spectrometer and fully controlled by computer software and hardware. Because isotope dilution by introducing 13C-labeled ttMA and SPMA as internal standards was employed, the precision of the analytical system was high (for ttMA, intra- and inter-day CV values ranged from 3.82-4.53%; for SPMA, 2.13-7.06%). The calibration curves obtained using human urine spiked with ttMA were linear from 15.6-4000 microg/L (R = 0.9998) and SPMA at concentrations from 0.78-200 microg/L (R = 0.9993). The method detection limit (MDL) for SPMA was 0.23 microg/L. The MDL of ttMA could not be determined accurately because of unavailability of an appropriate blank urine matrix, but was estimated to be lower than 7.43 microg/L. Without tedious manual sample cleanup procedures the analytical system is fully automated and is therefore useful for high-throughput simultaneous determination of urinary ttMA and SPMA. The sample throughput is roughly 100 samples per day. With the selectivity and the sensitivity provided by MS/MS detection, the analytical system can be used for large-scale monitoring of environmental or occupational exposure of humans to benzene.
A high-performance liquid chromatography/single quadrupole mass spectrometry (LC/MS) method is described for the determination of urinary S-phenylmercapturic acid (S-PMA), a specific metabolite of benzene. Urine samples were spiked with [13C6]S-PMA (used as the internal standard) and acidified; then they were purified by solid-phase extraction (SPE) on C18 cartridges. Analyses were conducted on a reversed-phase column by gradient runs with 1% aqueous acetic acid/methanol mixtures at different proportions as the mobile phase. The detector was used in electrospray negative ion mode (ESI-), the ions m/z 238 for S-PMA and 244 for [13C6]S-PMA being recorded simultaneously. The detection limit (for a signal-to-noise ratio = 3) was 0.2 microg/L, thus allowing for the measurement of background excretion of S-PMA in the general population. The use of the internal standard allowed us to obtain good precision (CV% values < 3%) and a linear calibration curve within the range of interest for monitoring occupational exposure to benzene (up to 500 microg/L). The method was applied to assay the metabolite concentration in a group of 299 workers (68 smokers and 231 non-smokers) occupationally exposed to relatively low levels of benzene (environmental concentration = 0.4-220 microg/m3, mean 11.4 microg/m3 and 236 non-exposed subjects (134 smokers and 102 non-smokers). The results clearly showed that smoking must be taken into account for the correct interpretation of the results of S-PMA measurements for the assessment of work-related benzene exposure. When only non-smokers were selected, the mean excretion of S-PMA was significantly higher in workers exposed to benzene (1.2 +/- 0.9 microg/g creatinine) than in the control group (0.7 +/- 0.6 microg/g creatinine) (p < 0.001), thus confirming the role of S-PMA as a biomarker of benzene on a group basis, even for relatively low exposure degrees.
S-phenylmercapturic acid (PMA) is one specific urinary biomarker of low-level benzene exposure. It is used for biological monitoring of benzene-exposed workers in the petrochemical industry and normally ranges from non-measurable to 10 microg/l levels in non-exposed non-smoking subjects. Benzene-exposure caused by workplace or lifestyle sources is frequently accompanied by toluene exposure, which can cause the occurrence of high levels (from 10 mg/l to more than 2000 mg/l) of hippuric acid (HA) in urine. Both solvents are toxic, and benzene is classified as a human carcinogen. The biological monitoring of benzene and toluene is therefore required for preventive care of exposed workers health. In this study a GC-MS method was adopted for measuring urinary PMA, which involved liquid-liquid extraction (LLE) with ethyl acetate from acidified urine and esterification with 0.5 N hydrochloric acid in methanol. The method evidenced a GC effect in a conventional HP-5 (30 m x 0.25 mm i.d., 0.25 microm film-thickness) methyl-phenylsilicone capillary column produced by HA on PMA. The results demonstrate that HA at concentrations as low as 250 mg/l can delay the elution of PMA and labelled internal standard from the column. The recognition and discussion of this particular GC phase soaking effect may be of help for those who are occupied in the determination of PMA and of urinary acidic metabolites by GC.
S-phenylmercapturic acid is widely accepted as a specific biomarker for the evaluation of benzene exposure. Here, we describe a fast, specific and sensitive high-performance liquid achromatography coupled with tandem mass spectrometry (LC-MS/MS) method that has been developed and validated for the determination of S-phenylmercapturic acid in human urine. Isotope-labeled S-phenylmercapturic acid-d5 was used as internal standard to improve the method ruggedness. The fully automated solid-phase extraction method on a 96-well Oasis MAX (mix-mode anion exchange) plate was employed to clean up the urine samples before analysis. The rapid LC-MS/MS analysis of extracted samples was achieved on a Genesis C18 column with a run time of only 3 min. Negative electrospray ionization with multiple reaction monitoring (ESI-MRM) mode was used to detect S-phenylmercapturic acid (m/z 238 --> 109) and S-phenylmercapturic acid -d5 (m/z 243 --> 114). The method fulfils all the standard requirements of method validation. The calibration curve was linear within the concentration range 0.400-200 ng/mL. The method performed accurately and precisely in validation with <7.5% relative error and <6.5% relative standard deviation of quality control samples. The method efficacy was also verified by the analysis of urine samples from 12 smokers and 12 non-smokers. With the fully automated sample cleanup procedure and the fast LC-MS/MS analysis, a sample analysis throughput of 384 samples per day could be achieved.
A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and fully validated, according to U.S. Food and Drug Administration guidance, for the simultaneous determination of phenylmercapturic acid, benzylmercapturic acid and o-methylbenzyl mercapturic acid in human urine as biomarkers of exposure to benzene, toluene and xylenes (BTX). After solid phase extraction and LC separation, samples were analyzed by a triple-quadrupole mass spectrometer operated in negative ion mode, using isotope-labeled analogs as internal standards (ISs). The method meets all the validation criteria required. The limits of detection of the three analytes, ranging from 0.30 to 0.40microgl(-1), and the high throughput make the method suitable for the routine biological monitoring of co-exposure to BTX both in the occupational and environmental settings. The validated method was applied to assess exposure to BTX in a group of 354 urban traffic wardens.
It has been suggested that the threshold limit value (TLV) for the time-weighted average (TWA), of benzene be lowered because of its possible leukemogenic effect at low exposure concentrations. This requires the development of new methods of biological monitoring. In this cross-sectional study the diagnostic power of blood and breath benzene and of urinary phenol, catechol, hydroquinone, S-phenylmercapturic acid, and muconic acid were compared in a population of 410 male workers exposed to benzene in garages, in two coke plants, and in a by-product plant. Benzene exposure was assessed by personal air sampling (charcoal tube and passive dosimeter). In all, 95% of the workers were exposed to less than 0.5 ppm benzene. According to the multiple regression equation, the muconic acid and S-phenylmercapturic acid concentrations detected in nonsmokers exposed to 0.5 ppm benzene were 0.3 mg/g and 6 micrograms/g, respectively (range 0.2-0.6 mg/g and 1.2-8.5 micrograms/g, respectively). With muconic acid very few false-positive test results were found, and this determination remained reliable even around a cutoff level of 0.1 ppm benzene. Moreover, the diagnostic power of this test proved to be good even when diluted or concentrated urine samples were not excluded. S-Phenylmercapturic acid (S-PMA) also performed fairly well. Blood and breath benzene as well as urinary phenol (PH) and hydroquinone (HQ) were clearly less suitable biomarkers than muconic acid (MA). Catechol (CA) was not associated with occupational benzene exposure. According to the results of biological monitoring, the skin resorption of benzene from gasoline or other fuels seems negligible. Correlation, multiple regression, and likelihood ratios consistently showed that MA and S-PMA concentrations were fairly good indicators of benzene exposure in the 0.1- to 1-ppm range, even in a population comprising both smokers and nonsmokers. PH, HQ, CA, and blood and breath benzene were less suitable, if at all, in the same exposure range.
A hand-saving HPLC method to measure urinary phenylmercapturic acid (PMA) was developed which allows about 35 PMA determinations per day. The method involves conversion of pre-PMA to PMA by the addition of sulfuric acid to a urine sample, extraction into an ether-methanol mixture followed by condensation under a nitrogen stream. The condensate was introduced to a ODS-3 column in a HPLC system, and PMA in the column was eluted into a mobile phase of acetonitrile: methanol: perchloric acid: water. The elution of PMA was monitored at 205 nm. One determination will be completed in 40 min. The method was applied to analysis of end-of-shift urine samples from 152 workers exposed up to 210 ppm benzene, 66 workers exposed to a mixture of benzene (up to 116 ppm) and toluene + xylenes (up to 118 ppm), and 131 non-exposed controls of both sexes. A linear regression was established between time-weighted average intensity of exposure to benzene and urinary PMA. From the regression, it was calculated that urinary PMA level will be about 6.4 mg/l after 8-hour exposure to benzene at 100 ppm, and that PMA in urine accounted for about 0.1% of benzene absorbed. No effects of sex, age, and smoking habit of individuals were detected, and the effect of co-exposure to toluene + xylenes at the levels comparable to that of benzene was essentially nil, which indicates an advantage of PMA as a benzene exposure marker over monoto tri-phenolic metabolites or t,t-muconic acid.
The concentrations of environmental tobacco smoke (ETS) constituents including benzene were measured in the living rooms of 10 nonsmoking households and 20 households with at least one smoker situated in the city and suburbs of Munich. In the city, the median benzene levels during the evening, when all household members were at home, were 8.1 and 10.4 μg/m3 in nonsmoking and smoking homes, respectively. The corresponding levels of 3.5 and 4.6 μg/m3 were considerably lower in the suburbs. Median time-integrated 1-week benzene concentrations in the city were 10.6 μg/m3 in nonsmoking homes and 13.1 μg/m3 in smoking homes. In the suburbs, the corresponding values were 3.2 and 5.6 μg/m3. While the benzene concentrations in nonsmoking homes located in the city were significantly higher (p < 0.05) than in suburban nonsmoking households, no difference was found between smoking and nonsmoking households located either in the city or in the suburbs. Individual exposures to benzene and to specific markers for tobacco smoke of all household members (82 nonsmokers and 32 smokers) were determined by questionnaire, personal monitoring, and biomonitoring. Within the city, the benzene exposure determined by personal samplers was 11.8 μg/m3 for nonsmokers living in nonsmoking homes and 13.3 μg/m3 for nonsmokers in smoking homes. The corresponding values for nonsmokers living in the suburbs were 5.9 and 6.9 μg/m3, respectively. Neither difference was statistically significant. Nonsmokers living in nonsmoking households in the city had significantly higher exposure to benzene compared to their counterparts living in the suburbs (personal samplers: 11.8 vs 5.9 μg/m3, p < 0.001; benzene in exhalate: 2.4 vs. 1.1 μg/m3, p < 0.05; trans,trans-muconic acid excretion in urine: 92 vs. 54 μg/g creatinine, p < 0.05). Nonsmokers from all households with smokers were significantly more exposed to benzene than nonsmokers living in the nonsmoking households (personal samplers: 13.2 vs. 7.0 μg/m3, p < 0.05; benzene in exhalate: 2.6 vs. 1.8 μg/m3, p < 0.01; trans,trans-muconic acid excretion in urine: 73 vs. 62 μg/g creatinine), but the contribution of ETS to the total benzene exposure was relatively low compared to that from other sources. Analysis of variance showed that at most 15% of the benzene exposure of nonsmokers living in smoking homes was attributable to ETS. For nonsmokers living in nonsmoking households benzene exposure from ETS was insignificant.
Benzene is a carcinogenic compound, which is emitted from petrol-fuelled cars and thus is found ubiquitous in all cities. As part of the project Monitoring of Atmospheric Concentrations of Benzene in European Towns and Homes (MACBETH) six campaigns were carried out in the Municipality of Copenhagen, Denmark. The campaigns were distributed over 1 year. In each campaign, the personal exposure to benzene of 50 volunteers (non-smokers living in non-smoking families) living and working in Copenhagen was measured. Simultaneously, benzene was measured in their homes and in an urban network distributed over the municipality. The Radiello diffusive sampler was applied to sample 5 days averages of benzene and other hydrocarbons. Comparison of the results with those from a BTX-monitor showed excellent agreement. The exposure and the concentrations in homes and in the urban area were found to be close to log-normal distribution. The annual averages of the geometrical mean values were 5.22, 4.30 and 2.90 μg m−3 for personal exposure, home concentrations and urban concentrations, respectively. Two main parameters are controlling the general level of benzene in Copenhagen: firstly, the emission from traffic and secondly, dispersion due to wind speed. The general level of exposure to benzene and home concentrations of benzene were strongly correlated with the outdoor level of benzene, which indicated that traffic is an important source for indoor concentrations of benzene and for the exposure to benzene.
Benzene, a common industrial chemical and a component of gasoline, is radiomimetic and exposure may lead progressively to aplastic anaemia, leukaemia, and multiple myeloma. Although benzene has been shown to cause many types of genetic damage, it has consistently been classified as a non-mutagen in the Ames test, possibly because of the inadequacy of the S9 microsomal activation system. The metabolism of benzene is complex, yielding glucuronide and sulphate conjugates of phenol, quinol, and catechol, L-phenylmercapturic acid, and muconaldehyde and trans, trans-muconic acid by ring scission. Quinol is oxidised to p-benzoquinone, which binds to vital cellular components or undergoes redox cycling to generate oxygen radicals; muconaldehyde, like p-benzoquinone, is toxic through depletion of intracellular glutathione. Exposure to benzene may also induce the microsomal mixed function oxidase, cytochrome P450 IIE1, which is probably responsible for the oxygenation of benzene, but also has a propensity to generate oxygen radicals. The radiomimetic nature of benzene and its ability to induce different sites of neoplasia indicate that formation of oxygen radicals is a major cause of benzene toxicity, which involves multiple mechanisms including synergism between arylating and glutathione-depleting reactive metabolites and oxygen radicals. The occupational exposure limit in the United Kingdom (MEL) and the United States (PEL) was 10 ppm based on the association of benzene exposure with aplastic anaemia, but recently was lowered to 5 ppm and 1 ppm respectively, reflecting a concern for the risk of neoplasia. The American Conference of Governmental Industrial Hygienists (ACGIH) has even more recently recommended that, as benzene is considered an A1 carcinogen, the threshold limit value (TLV) should be decreased to 0.1 ppm. Only one study in man, based on nine cases of benzene associated fatal neoplasia, has been considered suitable for risk assessment. Recent re-evaluation of these data indicated that past assessments may have overestimated the risk, and different authors have considered that lifetime exposure to benzene at 1 ppm would result in an excess of leukaemia deaths of 9.5 to 1.0 per 1000. Although in this study, deaths at low levels of benzene exposure were associated with multiple myeloma and a long latency period, instead of leukaemia, which might justify further lowering of the exposure limit, the risk assessment model has been found to be non-significant for response at low levels of exposure. The paucity of data for man, the complexity of the metabolic activation of benzene, the interactive and synergistic mechanisms of benzene toxicity and carcinogenicity, the different disease endpoints (aplastic anaemia, leukaemia, and multiple myeloma), and different individual susceptibilities, all indicate that in such a complex scenario, regulators should proceed with caution before making further changes to the exposure limit for this chemical.
In an inhalation study rats were exposed to different doses of benzene, ranging from 1 to 500 p.p.m. The urine was sampled
during the inhalation period of 8 h and for 24 h after exposure. S-Phenylmercapturic acid (S-PMA) in the urine was determined
by amino acid analysis. Phenol was measured by gas chromatography/mass spectrometry. In both cases the correlation between
benzene uptake and the excretion of the urinary metabolites was significant at the level of P = 0.01. The same significant correlation (P = 0.01) was demonstrable after i.p. administration of benzene at doses between 0.7 and 140.0 μI/kg body weight. In the case
of two collectives of workers who were exposed to air concentrations of up to 0.15 p.p.m. for 8 h and of up to 1.13 p.p.m.
for 12 h respectively, the amount of S-PMA in the first urine samples after the shift was significantly higher than in samples
collected at the beginning of the shift (P = 0.01). In the first collective the mean values and the standard deviations of the S-PMA concentrations in the samples at
the beginning of the shift were 12.0 ± 16.7 compared with 48.5 ± 64.5 μg/g creatinine at shift end. In the second collective
they were 25.1 ± 25.1 compared with 70.9 ± 109.2 μg/g creatinine. The level of significance of the difference between the
concentration values of S-PMA at the beginning and end of the shift was P = 0.01. The phenol concentration did not differ significantly. These results suggest that S-PMA can be regarded as a useful
indicator for monitoring individuals and collectives exposed to benzene at levels even <1 p.p.m.
An HPLC method for the determination of S-phenyl-N-acetylcysteine in urine is described. The sensitivity is 6 mumol/L (CV = 9%) urine. Exposure of rats to six different concentrations of benzene, ranging from 0-30 ppm, was highly associated with urinary excretion of S-phenyl-N-acetylcysteine (r = 0.86) and with total phenol (r = 0.81). A background level of phenol was found in urine of both non-exposed rats and of non-exposed referents. However, no background excretion of S-phenyl-N-acetylcysteine was found, either in rats or in humans. In urine of exposed rats, the level of S-phenyl-N-acetylcysteine was approximately five times lower than the phenol level. Workers occupationally exposed to benzene, showing high levels of urinary phenol, revealed low concentrations of urinary S-phenyl-N-acetylcysteine. The biological monitoring of industrial exposure to benzene by determination of S-phenyl-N-acetylcysteine in urine is not better than the determination of phenol in urine.
Different parameters of biological monitoring were applied to 26 benzene-exposed car mechanics. Twenty car mechanics worked in a work environment with probably high benzene exposures (exposed workers); six car mechanics primarily involved in work organization were classified as non-exposed. The maximum air benzene concentration at the work places of exposed mechanics was 13 mg/m3 (mean 2.6 mg/m3). Elevated benzene exposure was associated with job tasks involving work on fuel injections, petrol tanks, cylinder blocks, gasoline pipes, fuel filters, fuel pumps and valves. The mean blood benzene level in the exposed workers was 3.3 micrograms/l (range 0.7-13.6 micrograms/l). Phenol proved to be an inadequate monitoring parameter within the exposure ranges investigated. The muconic and S-phenylmercapturic acid concentrations in urine showed a marked increase during the work shift. Both also showed significant correlations with benzene concentrations in air or in blood. The best correlations between the benzene air level and the mercapturic and muconic acid concentrations in urine were found at the end of the work shift (phenylmercapturic acid concentration: r = 0.81, P < 0.0001; muconic acid concentration: r = 0.54, P < 0.05). In conclusion, the concentrations of benzene in blood and mercapturic and muconic acid in urine proved to be good parameters for monitoring benzene exposure at the workplace even at benzene air levels below the current exposure limits. Today working as a car mechanic seems to be one of the occupations typically associated with benzene exposure.
Comparison of the suitability of two minor urinary metabolites of benzene, trans,trans-muconic acid (tt-MA) and S-phenylmercapturic acid (S-PMA), as biomarkers for low levels of benzene exposure.
The sensitivity of analytical methods of measuring tt-MA and S-PMA were improved and applied to 434 urine samples collected from 188 workers in 12 studies in different petrochemical industries and from 52 control workers with no occupational exposure to benzene. In nine studies airborne benzene concentrations were assessed by personal air monitoring.
Strong correlations were found between tt-MA and S-PMA concentrations in samples from the end of the shift and between either of these variables and airborne benzene concentrations. It was calculated that exposure to 1 ppm (8 hour time weighted average (TWA)) benzene leads to an average concentration of 1.7 mg tt-MA and 47 micrograms S-PMA/g creatinine in samples from the end of the shift. It was estimated that, on average, 3.9% (range 1.9%-7.3%) of an inhaled dose of benzene was excreted as tt-MA with an apparent elimination half life of 5.0 (SD 2.3) hours and 0.11% (range 0.05%-0.26%) as S-PMA with a half life of 9.1 (SD 3.7) hours. The mean urinary S-PMA in 14 moderate smokers and 38 non-smokers was 3.61 and 1.99 micrograms/g creatinine, respectively and the mean urinary tt-MA was 0.058 and 0.037 mg/g creatinine, respectively. S-PMA proved to be more specific and more sensitive (P = 0.030, Fisher's exact test) than tt-MA. S-PMA, but not tt-MA, was always detectable in the urine of smokers who were not occupationally exposed. S-PMA was also detectable in 20 of the 38 non-smokers from the control group whereas tt-MA was detectable in only nine of these samples. The inferior specificity of tt-MA is due to relatively high background values (up to 0.71 mg/g creatinine in this study) that may be found in non-occupationally exposed people.
Although both tt-MA and S-PMA are sensitive biomarkers, only S-PMA allows reliable determination of benzene exposures down to 0.3 ppm (8 h TWA) due to its superior specificity. Because it has a longer elimination half life S-PMA is also a more reliable biomarker than tt-MA for benzene exposures during 12 hour shifts. For biological monitoring of exposure to benzene concentrations higher than 1 ppm (8 h TWA) tt-MA is also suitable and may even be preferred due to its greater ease of measurement.
Urinary phenol determinations have traditionally been used to monitor high levels of occupational benzene exposure. However, urinary phenol cannot be used to monitor low-level exposures. New biological indexes for exposure to low levels of benzene are thus needed. The aim of this study was to investigate the relations between exposure to benzene (A-benzene, ppm), as measured by personal air sampling, and the excretion of benzene (U-benzene, ng/l), trans,trans-muconic acid (MA, mg/g creatinine), and S-phenylmercapturic acid (PMA, micrograms/g creatinine) in urine. The subjects of the study were 145 workers exposed to benzene in a chemical plant. The geometric mean exposure level was 0.1 ppm (geometric standard deviation = 4.16). After logarithmic transformation of the data the following linear regressions were found: log (U-benzene, ng/l) = 0.681 log (A-benzene ppm) + 4.018; log (MA, mg/g creatinine) = 0.429 log (A-benzen ppm) - 0.304; and log (PMA, micrograms/g creatinine) = 0.712 log (A-benzene ppm) + 1.664. The correlation coefficients were, respectively, 0.66, 0.58, and 0.74. On the basis of the equations it was possible to establish tentative biological limit values corresponding to the respective occupational exposure limit values. In conclusion, the concentrations of benzene, mercapturic acid, and muconic acid in urine proved to be good parameters for monitoring low benzene exposure at the workplace.
A method was developed for the determination of the specific benzene metabolite S-phenylmercapturic acid in urine. The analyte is determined by HPLC with fluorescence detection after solid-phase extraction of urine with C18 material and hydrolysis followed by precolumn derivatization. The samples are separate by a column-switching method with a dual column system. As the method is highly sensitive (detection limit ca. 1 microgram/l), urinary S-phenylmercapturic acid concentrations for non-exposed persons (e.g., non-smokers) can also be measured precisely.
Methods for the biological monitoring of benzene and its metabolites in exhaled air, blood and urine are reviewed. Analysis of benzene in breath can be carried out by using an exhaled-air collection tube and direct analysis by GC or GC-MS; however, this technique is less reliable when compared to analysis using blood or urine. For the determination of non-metabolized benzene in blood and urine, GC head-space analysis is recommended. Phenol, the major metabolite of benzene can be monitored by either HPLC or GC methods. However, urinary phenol has proved to be a poor biomarker for low-level benzene exposure. Recent studies have shown that trans,trans-muconic acid, a minor metabolite of benzene can be determined using HPLC with UV detection. This biomarker can be used for detection of low-level benzene exposure. Urinary S-phenylmercapturic acid is another sensitive biomarker for benzene, but it can be detected only by GC-MS. Hydroquinone, catechol and 1,2,4-benzenetriol can be measured using HPLC with either ultraviolet or fluorimetric detection. Nevertheless, their use for low-level assessment requires further studies. Eventually, for the assessment of health risks caused by benzene, biological-exposure reference values need to be established before they can be widely used in a field setting.
This paper describes a procedure for the identification of phenylmercapturic acid in urine of benzene-exposed mice. Collected urine of benzene exposed mice was adjusted to pH 7 and applied to an anion exchanger. After extraction with diethyl ether and evaporation to dryness, the sample was dissolved in aqueous phosphoric acid and injected into the HPLC. HPLC conditions included an ODS column and an eluent consisting of tetrabutylammoniumhydrogensulfate-methanol (75:25, v/v), the absorbance wavelength was 255 nm. The detection limit of phenylmercapturic acid was 3 mg/l in mouse urine.
We examined the urinary excretion of S-benzyl-N-acetylcysteine (SBAC) of toluene sniffers using capillary gas chromatography. SBAC was extracted from 10 ml urine with chloroform and backextracted into 1 M sodium bicarbonate solution. After acidification, the aqueous solution was reextracted with ethyl acetate, and then derivatized to its methyl ester (ME). The peak appearing in the gas chromatogram was identified as SBAC-ME by mass spectrometry. The calibration curve was constructed by plotting the peak height ratio of SBAC-ME and internal standard (S-phenethyl-N-acetylcysteine)-ME against analyte concentration using 10 ml toluene unexposed urine. It showed good linearity over the range of 0.05-3.0 mg/l (r = 0.99). We have applied this technique to urine samples from toluene sniffers. SBAC was detected in all urinary samples of sniffers (n = 30, 0.11-47.13 mg/l), but not at all in the urine of toluene unexposed subjects (n = 60). These results prove that SBAC is also formed from toluene by human metabolism, and detection of SBAC is considered a useful marker for inhalation of toluene.
Collision-induced dissociation (CID) methods are described for the quantification of nanogram per millilitre (ppb) concentrations of 2-acetamido-3-(3'-hydroxypropylthio)-propanoic acid (I) and 2-acetamido-3-phenylthiopropanoic acid (II) in human urine extracts. I and II are potential detoxification products of acrolein and benzene in conjugation with N-acetyl(-L-)cysteine derived from glutathione. We have studied the potential of tandem mass spectrometry (MS/MS) under electron impact (EI) and chemical ionization (CI) conditions as a confirmatory screening technique for these compounds. Our main goals were high selectivity and low detection limits along with little or no sample clean-up. The effects of the mode of ionization and of collision conditions on the CID spectra have been investigated. Direct insertion probe without any derivatization or short-column gas chromatographic separation techniques are used. Total instrument and data analysis time is about 15 min for direct insertion probe MS/MS and about 30 min for short-column gas chromatography (GC)/MS/MS. Detection limits are: direct insertion probe MS/MS (EI mode), 50 ppb (100 pg) for compound I; short-column GC/MS/MS (EI mode), 1.5 ppb (5 pg) for compound II; and short-column GC/MS/MS (CI mode), 0.6 ppb (2 pg) for the methyl ester of compound II. Results are compared with non-mass spectrometric methods. The MS/MS methods were applied for the determination of I (EI mode) and II (CI mode) in urinary samples of a smoker and eight non-smokers. After smoking, the urinary levels of I and II were elevated, whereas no increase was observed after experimental passive smoking.
Recently, the determination of S-phenylmercapturic acid (S-PMA) in urine has been proposed as a suitable biomarker for the monitoring of low level exposures to benzene. In the study reported here, the test has been validated in 12 separate studies in chemical manufacturing plants, oil refineries, and natural gas production plants. Parameters studied were the urinary excretion characteristics of S-PMA, the specificity and the sensitivity of the assay, and the relations between exposures to airborne benzene and urinary S-PMA concentrations and between urinary phenol and S-PMA concentrations. The range of exposures to benzene was highest in workers in chemical manufacturing plants and in workers cleaning tanks or installations containing benzene as a component of natural gas condensate. Urinary S-PMA concentrations were measured up to 543 micrograms/g creatinine. Workers' exposures to benzene were lowest in oil refineries and S-PMA concentrations were comparable with those in smoking or nonsmoking control persons (most below the detection limit of 1 to 5 micrograms/g creatinine). In most workers S-PMA was excreted in a single phase and the highest S-PMA concentrations were at the end of an eight hour shift. The average half life of elimination was 9.0 (SD 4.5) hours (31 workers). Tentatively, in five workers a second phase of elimination was found with an average half life of 45 (SD 4) hours. A strong correlation was found between eight hour exposure to airborne benzene of 1 mg/m3 (0.3 ppm) and higher and urinary S-PMA concentrations in end of shift samples. It was calculated that an eight hour benzene exposure of 3.25 mg/m3 (1 ppm) corresponds to an average S-PMA concentration of 46 micrograms/g creatinine (95% confidence interval 41-50 micrograms/g creatinine). A strong correlation was also found between urinary phenol and S-PMA concentrations. At a urinary phenol concentration of 50 mg/g creatinine, corresponding to an eight hour benzene exposure of 32.5 mg/m3 (10 ppm), the average urinary S-PMA concentration was 383 micrograms/g creatinine. In conclusion, with the current sensitivity of the test, eight hour time weighted average benzene exposures of 1 mg/m3 (0.3 ppm) and higher can be measured.
A method was developed for sensitive determination of the specific benzene metabolite S-phenylmercapturic acid and the corresponding toluene metabolite S-benzylmercapturic acid in human urine for non-occupational and occupational exposure. The sample preparation procedure consists of liquid extraction of urine samples followed by precolumn derivatization and a clean-up by normal-phase HPLC. Determination of analytes occurs by gas chromatography with electron-capture detection. With this highly sensitive method (detection limits 60 and 65 ng/l, respectively) urinary S-phenylmercapturic and S-benzylmercapturic acid concentrations for non-occupationally exposed persons (e.g. non-smokers) can be measured precisely in one chromatographic run. Validation of the method occurred by comparison with a HPLC method we have published recently.
Exposure biomarkers, which have long been restricted to the framework of occupational hygiene, currently arouse increasing interest in the field of environmental pollution. To assess their validity, we propose here a conceptual framework that is based on their intrinsic characteristics and on properties related to the procedures for their analysis. The most important criteria are specificity for the toxic substance under consideration and sensitivity, that is, the ability to distinguish contrasted levels of exposure. Their analytic sensitivity and specificity are also important. Fulfilling these criteria is especially important in the context of environmental pollution, because the levels of exposure, and thus the contrasts, are low. This framework is used to assess the validity of some biomarkers for polycyclic aromatic hydrocarbons (1-hydroxypyrene and DNA adducts) and for benzene (urinary and serum benzene, trans,trans muconic acid, and S-phenylmercapturic acid). This evaluation shows that the most relevant biomarkers for estimating individual exposure to environmental pollution are 1-hydroxypyrene for polycyclic aromatic hydrocarbons and urinary benzene and S-phenylmercapturic for benzene.
The use of unleaded gasoline, together with an increase in the number of vehicles in Bangkok, has significantly influenced benzene and toluene concentrations in vehicular emissions and contributes to the air pollution problem. As a matter of practical necessity, a quick test program is done for the measurement of emission concentrations/rates for vehicles driven on the road. Exhaust emission measurement at idle mode was conducted in a fleet of 12 vehicles of different model years and manufacturers. The study revealed that the benzene and toluene concentrations in the exhaust effluent averaged 4.4-22.02 and 12.24-44.75 mg/m3, respectively for 1990-1992 cars and decreased to 0.76-4.14 and 0.89-6.26 mg/m3, respectively for 1994-1995 cars. In another study, exhaust emission measurement on a chassis dynamometer was carried out in a fleet of nine selected, in-use cars. It was observed that benzene and toluene emission rates were considerably higher-in the range of 70.84-85.82 and 354.15- 429.00 mg/km, respectively, for 1990-1991 model year cars. Lower benzene and toluene emission rates of 0.43-95.07 and 2. 15-475.35 mg/km, respectively, were represented by newer cars with model years 1994-1995. These results indicated that there was a significant increase in benzene and toluene emission concentrations and rates with increasing car mileage and model year. The finding also revealed that only 28% of the tested vehicles complied to the approved emission standard.
S-phenylmercapturic acid (S-PMA) was measured in urine from 145 subjects exposed to low benzene concentrations in the air (C(I), benzene). The 8-h, time-weighted exposure intensity of individual workers was monitored by means of charcoal tubes and subsequent gas-chromatographic analysis after desorption with CS2. S-PMA excretion level in urine was determined by high-performance liquid chromatography with fluorescence detection. The following linear correlation was found between S-PMA concentrations in urine and benzene concentrations in the breathing zone: log(S-PMA, microg/g creatinine) = 0.712 log (C(I)-benzene, ppm) + 1.644 (n = 145, r = 0.74, P < 0.001). The geometric mean (GSD) of S-PMA concentrations in urine from 45 subjects occupationally not exposed to benzene but smoking more than 20 cigarettes/day was 7.8 microg/g creatinine (2.11), the corresponding value among non-smokers being 1.0 microg/g creatinine (2.18). It is concluded that the urinary level of S-PMA can be regarded as a useful indicator of exposure to benzene.
An online automatic sample cleanup system was developed for use with electrospray ionization tandem mass spectrometry (ESI-MS-MS)
for the quantitative detection of the benzene exposure biomarker S-phenylmercapturic acid (S-PMA) in human urine. The sample clean-up system was constructed with an autosampling device, a
reversed-phase C18 trap cartridge, a two-position switching valve, and controlling computer software and hardware. The sample
cleanup system was interfaced directly with the ESI source of a triple-stage-quadrupole MS using multiple reaction monitoring
of negative product ions derived from S-PMA and the internal standard as the detection mode. The calibration curve was linear
using human urine spiked at concentrations from 0.25 to 100 mg/L S-PMA (R2 = 0.997). The detection limit of the analytical system for neat S-PMA standard solution was 0.04 µg/L, whereas the detection
limit was estimated to be lower than 0.35 µ g/L for a urine matrix containing trace amounts of S-PMA. Without tedious manual
sample cleanup procedures, the analytical system is fully automatic and therefore useful for high-throughput urinary S-PMA
determination. With the selectivity and the sensitivity provided by MS-MS detection, the analytical system can be used for
highthroughput and accurate determination of S-PMA levels in human urinary samples as a biomarker for benzene exposure.