Federica Rossella

Development of a gas chromatography/mass spectrometry method to quantify several urinary monohydroxy metabolites of polycyclic aromatic hydrocarbons in occupationally exposed subjects

The aim of this study was the development of a method for the determination of 12 urinary monohydroxy metabolites of PAHs, namely 1-hydroxynaphthalene, 2-hydroxynaphthalene, 2-hydroxyfluorene, 9-hydroxyfluorene, 1-hydroxyphenanthrene, 2-hydroxyphenanthrene, 3-hydroxyphenanthrene, 4-hydroxyphenanthrene, 9-hydroxyphenanthrene, 1-hydroxypyrene, 6-hydroxychrysene, and 3-hydroxybenzo[a]pyrene. Analytes were determined in the presence of deuterated analogues as internal standards, by GC/MS operating in the electron impact mode. Sample preparation was performed by enzymatic hydrolysis of glucoronate and sulphate conjugates of hydroxy metabolites of PAHs, liquid-liquid extraction with n-hexane, and derivatization with a silylating reagent. The method is very specific, limits of quantification are in the range 0.1-1.4 microg/l, and range of linearity is from limit of detection to 208 microg/l. Within- and between-run precision, expressed as coefficient of variation, are <20%; accuracy for most analytes is within 20% of the theoretical value. An application of the proposed method to the analysis of 10 urine samples from coke-oven workers shows that 1-hydroxynaphthalene and 2-hydroxyfluorene were the most abundant compounds (median 61.4 and 69.7 microg/l, respectively), while 6-hydroxychrysene, and 3-hydroxybenzo[a]pyrene were always below the quantification limit.

Quantification of 13 priority polycyclic aromatic hydrocarbons in human urine by headspace solid-phase microextraction gas chromatography–isotope dilution mass spectrometry

Polycyclic aromatic hydrocarbons (PAHs) are common environmental pollutants in both living and working environments. The aim of this study was the development of a headspace solid-phase microextraction gas chromatography-isotope dilution mass spectrometry (HS-SPME/GC-IDMS) method for the simultaneous quantification of 13 PAHs in urine samples. Different parameters affecting PAHs extraction by HS-SPME were considered and optimized: type/thickness of fiber coatings, extraction temperature/time, desorption temperature/time, ionic strength and sample agitation. The stability of spiked PAHs solutions and of real urine samples stored up to 90 days in containers of different materials was evaluated. In the optimized method, analytes were absorbed for 60min at 80 degrees C in the sample headspace with a 100mum polydimethylsiloxane fiber. The method is very specific, with linear range from the limit of quantification to 8.67 x 10(3)ngL(-1), a within-run precision of <20% and a between-run precision of <20% for 2-, 3- and 4-ring compounds and of <30% for 5-ring compounds, trueness within 20% of the spiked concentration, and limit of quantification in the 2.28-2.28 x 10(1)ngL(-1) range. An application of the proposed method using 15 urine samples from subjects exposed to PAHs at different environmental levels is shown.

Urinary BTEX, MTBE and naphthalene as biomarkers to gain environmental exposure profiles of the general population

The aim of this work was to evaluate urinary benzene, toluene, ethylbenzene, m+p-xylene, o-xylene (BTEX), methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and naphthalene (NAP) as biomarkers of exposure to environmental pollutants. Personal air and urine samples from 108 subjects belonging to the Italian general population were compared. Urinary profiles were obtained by headspace gas chromatography-mass spectrometry. BTEX, MTBE, ETBE and NAP median airborne exposures during a 5-h sampling were 4.0, 25.3, 3.8, 9.3, 3.4, 3.4, <0.8, and 3.4 microg/m(3), respectively. Meanwhile, median urinary levels, as geometric means of three determinations were: 122, 397, 74, 127, 43, 49, <15, and 46 ng/L, respectively. Urinary benzene and toluene concentrations were 4.6- and 1.2-fold higher in smokers than in non-smokers. For most chemicals, significant positive correlations between airborne exposure (log-transformed) and the corresponding biological marker (log-transformed) were found, with Pearsons r values for correlation, ranging from 0.228 to 0.396. Multiple linear regression analysis showed that the urinary level of these chemicals was influenced by personal airborne exposure, urinary creatinine, and urinary cotinine, with R(2) 0.733 for benzene. Urinary chemicals are useful biomarkers of environmental exposure. Given the ease of rapidly obtaining urine samples, they represent a non-invasive alternative to blood chemical analysis. The possibility of obtaining urinary exposure profiles makes this method an appealing tool for environmental epidemiology.

Urinary polycyclic aromatic hydrocarbons and monohydroxy metabolites as biomarkers of exposure in Coke-Oven workers

Objectives: To assess exposure to polycyclic aromatic hydrocarbons (PAHs) using 13 unmetabolised PAHs (U-PAHs) and 12 monohydroxy metabolites (OHPAHs) in urine, and to compare the utility of these biomarkers. Methods: 55 male Polish coke oven workers collected urine spot samples after a workshift. U-PAHs (naphthalene, acenaphtylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzo[k]fluoranthene, benzo[b]fluoranthene, benzo[a]pyrene) were determined by automatic solid phase micro-extraction followed by gas chromatography/mass spectrometry (GC/MS). OHPAHs (1- and 2-hydroxynaphthalene, 2- and 9-hydroxyfluorene, 4-, 9-, 3-, 1- and 2-hydroxyphenanthrene, 1-hydroxypyrene, 6-hydroxychrysene, 3-hydroxybenzo[a]pyrene) were determined, after liquid/liquid extraction and derivatisation, by GC/MS. Results: U-PAHs from naphthalene to chrysene were found in 100% of samples, and heavier U-PAHs in 7–22% of samples. OHPAHs up to 1-hydroxypyrene were found in 100% of samples, while 6-hydroxychrysene and 3-hydroxybenzo[a]pyrene were always below the quantification limit. Median naphthalene, phenanthrene, pyrene, chrysene and benzo[a]anthracene levels were 0.806, 0.721, 0.020, 0.032 and 0.035 μg/l, while hydroxynaphthalenes, hydroxyphenanthrenes and 1-hydroxypyrene levels were 81.1, 18.9 and 15.4 μg/l. For each chemical, the ratio between U-PAH and the corresponding OHPAH ranged from 1:26 to 1:1000. Significant correlations between logged values of U-PAHs and OHPAHs, between U-PAHs, and between OHPAHs were found, with Pearson’s r ranging from 0.27 to 0.97. Conclusion: Current analytical techniques allow specific and simultaneous measurement of several urinary determinants of PAHs in humans. The results of these measurements support the use of U-PAHs as biomarkers of exposure and suggest the spectrum of chemicals to be investigated, including carcinogenic chrysene and benzo[a]anthracene, should be widened.

Development and validation of a gas chromatography/ mass spectrometry method for the assessment of genomic DNA methylation

A method for the determination of DNA global methylation, taken as the ratio (%) of 5-methylcytosine (5mCyt) versus the sum of cytosine (Cyt) and 5mCyt, via gas chromatography/mass spectrometry (GC/MS), was developed and validated. DNA (2.5 microg) was hydrolyzed with aqueous formic acid 88%, spiked with cytosine-2,4-(13)C(2),(15)N(3) and 5-methyl-(2)H(3)-cytosine-6-(2)H(1) as internal standards, and derivatized with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide and 1% tert-butyldimethylchlorosilane, in the presence of acetonitrile and pyridine. GC/MS, operating in single ion monitoring mode, separated and specifically detected all nucleobases as tert-butyldimethylsilyl derivatives, without interferences, with the exception of guanosine. The method was linear throughout the range of clinical interest and had good sensitivity, with a limit of quantification of 3.2 pmol for Cyt and 0.056 pmol for 5mCyt, the latter corresponding to a methylation level of 0.41%. Intra- and inter-day precision and accuracy were below 4.0% for both analytes and methylation. The matrix absolute effect, process efficiency and coefficient of variation ranged from 96.5 to 101.2%. The matrix relative effect was below 1%. The method was applied to the analysis of different human DNAs, including: nonmethylated DNA from PCR (methylation 0.00%), hypermethylated DNA prepared using M.SssI CpG methyltransferase (methylation 18.05%), DNA from peripheral blood leukocytes of healthy subjects (N = 6, median methylation 5.45%), DNA from bone marrow of leukemia patients (N = 5, 3.58%) and DNA from myeloma cell lines (N = 4, 2.74%).

Exposure to BTEX and Ethers in Petrol Station Attendants and Proposal of Biological Exposure Equivalents for Urinary Benzene and MTBE

OBJECTIVE To assess exposure to benzene (BEN) and other aromatic compounds (toluene, ethylbenzene, m+p-xylene, o-xylene) (BTEX), methyl tert-butyl ether (MTBE), and ethyl tert-butyl ether (ETBE) in petrol station workers using air sampling and biological monitoring and to propose biological equivalents to occupational limit values. METHODS Eighty-nine petrol station workers and 90 control subjects were investigated. Personal exposure to airborne BTEX and ethers was assessed during a mid-week shift; urine samples were collected at the beginning of the work week, prior to and at the end of air sampling. RESULTS Petrol station workers had median airborne exposures to benzene and MTBE of 59 and 408 µg m(-3), respectively, with urinary benzene (BEN-U) and MTBE (MTBE-U) of 339 and 780 ng l(-1), respectively. Concentrations in petrol station workers were higher than in control subjects. There were significant positive correlations between airborne exposure and the corresponding biological marker, with Pearsons correlation coefficient (r) values of 0.437 and 0.865 for benzene and MTBE, respectively. There was also a strong correlation between airborne benzene and urinary MTBE (r = 0.835). Multiple linear regression analysis showed that the urinary levels of benzene were influenced by personal airborne exposure, urinary creatinine, and tobacco smoking [determination coefficient (R(2)) 0.572], while MTBE-U was influenced only by personal exposure (R(2) = 0.741). CONCLUSIONS BEN-U and MTBE-U are sensitive and specific biomarkers of low occupational exposures. We propose using BEN-U as biomarker of exposure to benzene in nonsmokers and suggest 1457 ng l(-1) in end shift urine samples as biological exposure equivalent to the EU occupational limit value of 1 p.p.m.; for both smokers and nonsmokers, MTBE-U may be proposed as a surrogate biomarker of benzene exposure, with a biological exposure equivalent of 22 µg l(-1) in end shift samples. For MTBE exposure, we suggest the use of MTBE-U with a biological exposure equivalent of 22 µg l(-1) corresponding to the occupational limit value of 50 p.p.m.