Rosa Mercadante

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.

The role of salivary cortisol measured by liquid chromatography–tandem mass spectrometry in the diagnosis of subclinical hypercortisolism

OBJECTIVE The use of late-night salivary cortisol (LNSalC) for diagnosing subclinical hypercortisolism (SH) is debated. No data are available regarding the role of LNSalC as measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in SH diagnosis. The aim of this study was to evaluate the diagnostic accuracy of LNSalC measured by LC-MS/MS in SH. DESIGN Cross-sectional prospective study of outpatients. METHODS In 70 consecutive patients with adrenal incidentalomas (AI), without signs and symptoms of hypercortisolism, we diagnosed SH in the presence of at least two of the following: cortisol after 1 mg overnight dexamethasone suppression test (1  mg DST) >83  nmol/l, 24-h urinary free cortisol (UFC) >193  nmol/24  h, and morning ACTH <2.2  pmol/l. The LNSalC levels by LC-MS/MS at 2300  h (normal values <2.8  nmol/l) and the presence of hypertension, type 2 diabetes mellitus (T2DM), and osteoporosis (OP) were assessed. RESULTS The increased LNSalC levels (>2.8  nmol/l) had an 83.3% specificity (SP) and a 31.3% sensitivity (SN) for predicting the biochemical diagnosis of SH. The increased LNSalC had an 85.2% SP and a 55.6% SN for predicting the presence of hypertension, T2DM, and OP, while the combination of LNSalC >1.4  nmol/l (cutoff with 100% SN) plus 1 mg DST >50  nmol/l had an 88.9% SN and an 85.2% SP (similar to SH criterion at enrollment). CONCLUSIONS In AI patients, LNSalC measured by LC-MS/MS appears to be useful in combination with 1 mg DST for diagnosing SH, while it is not useful as a single criterion.

Comparison between urinary o-cresol and toluene as biomarkers of toluene exposure

The characteristics of urinary o-cresol (o-C) and urinary toluene (TOL-U) as biomarkers of occupational exposure to toluene were comparatively evaluated. One hundred healthy male rotogravure printing workers and 161 male and female control subjects were studied. Personal exposure to airborne toluene (TOL-A) during the shift was determined as a time-weighted average. Simple analytical procedures based on solid phase microextraction followed by gas chromatography/mass spectometry analysis were applied to the determination of end-shift o-C and TOL-U. Median TOL-A was 48 (6.0–162.0) mg/m3 in printers and 0.021 (<0.003–0.137) mg/m3 in controls. o-C was 0.185 (0.032–0.948) mg/g creatinine in printers and 0.027 (<0.006–0.330) mg/g creatinine in the controls. TOL-U was 7.6 (1.8–23.9) μ g/L in printers and 0.140 (0.094–0.593) μ g/L in the controls. According to all indices, exposure to toluene was higher in printers than in the controls. Nevertheless, the distribution of o-C in the two groups partially overlapped, whereas such behavior was not found in TOL-U. Both o-C and TOL-U in printers were correlated with TOL-A (Pearsons on log10-transformed variables r = 0.704 and 0.844, respectively) and with each other (r = 0.683). Smoking habits significantly increased the excretion of o-C but not of TOL-U. From the point of view of sampling conditions and analytical requirements, TOL-U and o-C showed similar properties, but comparison of their intrinsic characteristics showed that TOL-U had higher specificity and sensitivity, lower background values, was better correlated with airborne exposure, and was not influenced by cigarette smoking. Therefore TOL-U may be considered superior to o-C as a biomarker of occupational exposure to toluene.

High‐throughput determination of cortisol, cortisone, and melatonin in oral fluid by on‐line turbulent flow liquid chromatography interfaced with liquid chromatography/tandem mass spectrometry

RATIONALE Cortisol, cortisone, and melatonin (CORTol, CORTone, and MELA, respectively) are hormones related to stress and sleep disorders. Their detection is relevant to epidemiological studies aimed at investigating the effects of circadian cycle disruption. The aim of this study was to develop and evaluate a high-throughput assay for the detection of CORTol, CORTone, and MELA concentrations in non-invasively collected oral fluid samples. METHODS A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method to measure levels of CORTol, CORTone, and MELA in oral fluid samples in the presence of deuterated analogs was optimized and validated. A 50 μL aliquot of oral fluid sample, obtained by centrifugation of a chewed swab, was purified using on-line turbulent flow liquid chromatography. Analytes were then separated using C18 reversed-phase chromatography, subjected to positive ionization using an electrospray source, then quantitated using a triple quadrupole mass detector in the selected reaction monitoring mode. RESULTS Limits of quantification and linear dynamic ranges were found to be 0.55 nmol/L, 5.5 nmol/L, and 0.004 nmol/L, and up to 28 nmol/L, 277 nmol/L, and 0.43 nmol/L for CORTol, CORTone, and MELA, respectively. Inter- and intra-run precisions as relative standard deviation values were <5%, and accuracies were within 95-106% of theoretical concentrations. An evaluation of matrix effects showed that the use of deuterated analogs controlled sources of bias. Furthermore, the total analysis time per sample was 13 min, resulting in a throughput of approximately 100 samples/day. CONCLUSIONS To our knowledge, this is the first automated, high-throughput assay for the simultaneous quantification of CORTol, CORTone, and MELA in oral fluid specimens.

Methodological issues in the biological monitoring of urinary benzene and S-phenylmercapturic acid at low exposure levels.

Biological monitoring of low level exposure to pollutants is a very challenging analytical activity, and the quality of results is difficult to assess, especially when a certified reference material is unavailable. The aim of this work was to evaluate the reliability of the assays used to measure urinary benzene (Benz-U) and S-phenylmercapturic acid (SPMA), by applying an internal quality control protocol. Urine spot samples from 705 subjects who were either members of the general urban population, gasoline station attendants, or refinery plant workers were assayed for Benz-U and SPMA, using GC/MS and LC/MS/MS, with quantification limits of 15 ng/L and 0.10 μg/L. The median Benz-U concentration was 263 ng/L (60-2789 ng/L, 5th-95th percentile), and the median SPMA concentration was 0.19 μg/L (<0.1-2.5 μg/L, 5th-95th percentile). Linearity of both assays was good, but a less-than-proportional response was found for SPMA concentrations below 1 μg/L. Between-run precision and accuracy for Benz-U concentration determination were assessed using quality controls at 120 ng/L and 1000 ng/L and were 10.3% and 4.8%, and 104.8% and 98.9%, respectively; while the precision and accuracy for SPMA concentration determination at 0.3 μg/L, 2.5 μg/L, and 20 μg/L were 40.3%, 6.2%, and 6.2%, and 48.3%, 96.3%, and 98.8%, respectively. Precision, estimated using duplicates of unknown samples, was 13.4% for Benz-U and 26.5% for SPMA analyses. Control charts for the means of the slope of the linear calibration curve of Benz-U showed good stability of the means over a five-year period. For SPMA, a two-laboratory comparison revealed acceptable agreement between ln-transformed data pairs, with a slope of the linear regression of 0.863 (confidence interval 0.774-0.952), null intercept, and a Pearsons r value of 0.844. Reliable results were obtained for Benz-U analyses over the entire concentration range, and for high and medium SPMA levels. However, the determination of SPMA concentrations at levels close to the limit of quantification was less reliable.

A quantitative approach to evaluate urinary benzene and S-phenylmercapturic acid as biomarkers of low benzene exposure.

Context: Benzene is a ubiquitous pollutant; smoking habit, genetic polymorphisms, and analytical difficulties impact the identification of the best biomarker. Objective: To apply a systematic quantitative approach to evaluate urinary benzene (BEN-U) and S-phenylmercapturic acid (SPMA) as biomarkers of low benzene exposures. Methods: Seventy-one blue collar refinery workers, 97 white collar refinery workers and 108 general population subjects were included. Intrinsic characteristics, sampling and analytical issues were compared. Results: BEN-U and SPMA were detected in 99% and 78% of samples, which correlated with benzene exposure (r = 0.456 and r = 0.636, respectively) and with urinary cotinine (r = 0.630 and r = 0.570, respectively). Intrinsic characteristics were similar for the two biomarkers: specificity (0.64 and 0.69 for BEN-U and SPMA), sensitivity (0.74 and 0.83), as well as intra- and inter-individual variability (150% and >14 for both). Conclusion: BEN-U and SPMA show similar intrinsic characteristics; analytical issues in detecting SPMA suggest that BEN-U is more convenient for investigating low exposure levels.

Terbuthylazine in hair as a biomarker of exposure

Terbuthylazine (TBA) is an herbicide widely used in corn cultivation. Herein we evaluate the measurement of hair TBA as biomarkers of exposure. Five Sprague Dawley rats were gavaged with TBA for 3 days, and then the back hair was shaved and analyzed for TBA. In addition, head hair samples from 10 corn farmers, 9 rural residents, and 6 urban residents were collected at the end of the application season. Hair TBA was detected by liquid chromatography triple quadrupole mass spectrometry after solvent extraction. TBA was quantifiable in all rat samples with a mean concentration of 0.92 (±0.26)ng/mg, which corresponds to a 0.12% incorporation rate. TBA was quantifiable in all farmer samples (median: 0.67ng/mg), in 75% of rural resident samples (0.01ng/mg) and in none of the urban resident samples (<0.01ng/mg), with a statistical difference among groups (P<0.01). Our results suggest that TBA is incorporated in hair and prompt further investigation on the use of hair TBA as a potential biomarker of cumulative exposure.

Identification and quantification of metabolites of the fungicide tebuconazole in human urine.

Tebuconazole (TEB) is a fungicide used in agriculture; the objective of this work was to identify and quantify TEB metabolites in human urine. Samples from seven vineyard workers exposed to TEB were submitted to liquid chromatography interfaced with a triple quadrupole mass spectrometer, equipped with an electron spray source, and a linear ion trap to gain a profile of candidate metabolites. Based on the presence of the ion m/z 70 in the MS/MS spectra, which corresponds to protonated triazole (a specific moiety of TEB), and the isotopic pattern of the molecular ions, typical of molecules with one chlorine atom, hydroxyl and carboxyl derivatives of TEB, that is, TEB-OH and TEB-COOH, were identified as major metabolites, both as free molecules and as glucuronide (Glc) conjugates. The mean molar fractions were 0.67, 0.13, 0.13, and 0.07 for TEB-O-Glc, TEB-OH, TEB-COO-Glc, and TEB-COOH. Urine samples were submitted to hydrolysis with β-glucuronidase, and the free compounds were quantified in the presence of deuterated TEB (TEB-d6) as the internal standard (IS), by multiple reaction monitoring (MRM) mode. The assay was linear in the ranges of 0.2-600 μg/L and 0.1-240 μg/L for TEB-OH and TEB-COOH, respectively; precision, accuracy, and the limit of quantification (LOQ) were <3.1%, 98-103%, and 0.3 μg/L for both analytes. An evaluation of matrix effects showed that the use of TEB-d6 controlled these sources of bias. The urinary levels of TEB-OH and TEB-COOH in specimens collected from farmers exposed to TEB ranged from 10 to 473 and from 3 to 159 μg/L, respectively.

Biological monitoring of exposure to tebuconazole in winegrowers

Tebuconazole (TEB) is a fungicide widely used in vineyards and is a suspected teratogen for humans. The aim of this research was to identify urinary biomarkers and the best sampling time for the biological monitoring of exposure to TEB in agricultural workers. Seven vineyard workers of the Monferrato region, Piedemont, Italy, were investigated for a total of 12 workdays. They treated the vineyards with TEB for 1–2 consecutive days, one of them for 3 days. During each application coveralls, underwears, hand washing liquids and head coverings were used to estimate dermal exposure. For biomonitoring, spot samples of urine from each individual were collected starting from 24 h before the first application, continuing during the application, and again after the application for about 48 h. TEB and its metabolites TEB-OH and TEB-COOH were measured by liquid chromatography/triple quadrupole mass spectrometry. TEB contamination of coveralls and total dermal exposure showed median levels of 6180 and 1020 μg. Urinary TEB-OH was the most abundant metabolite; its excretion rate peaked within 24 h after product application (post 24 h). In this time frame, median levels of TEB-OH and TEB-COOH ranged from 8.0 to 387.8 μg/l and from 5.7 to 102.9 μg/l, respectively, with a ratio between the two metabolites of about 3.5. The total amount of urinary metabolites (U-TEBeq) post 24 h was significantly correlated with both TEB on coveralls and total dermal exposure (Pearson’s r=0.756 and 0.577). The amount of metabolites excreted in urine represented about 17% of total dermal TEB exposure. Our results suggest that TEB-OH and TEB-COOH in post-exposure urine samples are promising candidates for biomonitoring TEB exposure in agricultural workers.