Urinary levels of metal elements in the non-smoking general population in Italy: SIVR study 2012-2015.

The purpose of this study of the Italian Society of Reference Values (SIVR) was to provide the reference values for metals in the urine of the Italian general population. Thirteen sampling centres situated in the north, centre and south of Italy took part in this project. Each sampling centre selected 20 adult subjects. The sample was made up of 120 male and 140 female non-smokers or smokers who had abandoned the habit at least 5 years previously, aged between 18 and 60 years. Urine samples were obtained from the same subjects in June and again in November of the same year. During collection of these two samples, we administered an ad hoc questionnaire designed to assess factors that might influence exposure and the results of the study such as personal characteristics, occupational or extra-occupational activities, dietary habits, the presence of dental fillings containing amalgam, the frequency and mode of exposure to passive smoking and exhaust gases of motor vehicles (traffic). Therefore, the urinary levels of thirteen trace elements (Be, Cd, Co, Cr, Cu, In, Mn, Ni, Pb, Pt, Sb, Tl and V) were determined by Inductively Coupled Plasma Mass Spectrometry. The concentrations obtained (95th percentile) are in µg/L: 0.034, 0.900, 2.24, 0.600, 24.0, 0.013, 1.53, 4.44, 2.64, 0.022, 0.095, 0.759 and 0.855 for Be, Cd, Co, Cr, Cu, In, Mn, Ni, Pb, Pt, Sb, Tl and V, respectively. Further studies with a larger number of subjects are needed in order to continue the biomonitoring of the Italian general population and to monitor the modified levels over time.

Comprehensive approach to the validation of the standard method for total reflection X-ray fluorescence analysis of water.

In this work, we present the validation of the chemical method for total reflection X-ray fluorescence (TXRF) analysis of water, proposed as a standard to the International Standard Organization. The complete experimental procedure to define the linear calibration range, elements sensitivities, limits of detection and quantification, precision and accuracy is presented for a commercial TXRF spectrometer equipped with Mo X-ray tube. Least squares linear regression, including all statistical tests is performed separately for each element of interest to extract sensitivities. Relative sensitivities with respect to Ga, as internal standard, are calculated. Accuracy and precision of the quantification procedure using Ga as internal standard is evaluated with reference water samples. A detailed discussion on the calibration procedure and the limitation of the use of this method for quantitative analysis of water is presented.

Methodology to define biological reference values in the environmental and occupational fields: the contribution of the Italian Society for Reference Values (SIVR).

BACKGROUND: Biological reference values (RVs) explore the relationships between humans and their environment and habits. RVs are fundamental in the environmental field for assessing illnesses possibly associated with environmental pollution, and also in the occupational field, especially in the absence of established biological or environmental limits. OBJECTIVES: The Italian Society for Reference Values (SIVR) determined to test criteria and procedures for the definition of RVs to be used in the environmental and occupational fields. METHODS: The paper describes the SIVR methodology for defining RVs of xenobiotics and their metabolites. Aspects regarding the choice of population sample, the quality of analytical data, statistical analysis and control of variability factors are considered. The simultaneous interlaboratory circuits involved can be expected to increasingly improve the quality of the analytical data. RESULTS: Examples of RVs produced by SIVR are presented. In particular, levels of chromium, mercury, ethylenethiourea, 3,5,6-trichloro-2-pyridinol, 2,5-hexanedione, 1-hydroxypyrene and t,t-muconic acid measured in urine and expressed in micrograms/g creatinine (µg/g creat) or micrograms/L (µg/L) are reported. CONCLUSIONS: With the proposed procedure, SIVR intends to make its activities known to the scientific community in order to increase the number of laboratories involved in the definition of RVs for the Italian population. More research is needed to obtain further RVs in different biological matrices, such as hair, nails and exhaled breath. It is also necessary to update and improve the present reference values and broaden the portfolio of chemicals for which RVs are available. In the near future, SIVR intends to expand its scientific activity by using a multivariate approach for xenobiotics that may have a common origin, and to define RVs separately for children who may be exposed more than adults and be more vulnerable.

Trace determination of anthracyclines in urine: a new high-performance liquid chromatography/tandem mass spectrometry method for assessing exposure of hospital personnel.

Health-care workers handling antineoplastic agents may be exposed to extremely low doses of these drugs. Very sensitive and specific analytical methods are therefore needed for biological monitoring. The aim of this study was to develop and validate a method for trace level determination of doxorubicin, epirubicin, daunorubicin and idarubicin in human urine, using epi-daunorubicin as an internal standard. Solid-phase extraction (SPE) was used for sample preparation. Urine samples were loaded onto Bond Elut C18 cartridges. The analytes were eluted in methylene chloride/2-propanol (1:1, v/v) and then evaporated to dryness. The residue was reconstituted with the mobile phase prior to high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) analysis. Quantitation of each analyte was performed using the multiple reaction monitoring (MRM) method. The urine assay was linear over the range 0.1-2.0 microg/L, with a lower limit of quantification (LLOQ) of 0.10 microg/L for doxorubicin and epirubicin, and 0.03 microg/L for daunorubicin and idarubicin. The respective limits of detection (LODs) were 0.04 and 0.01 microg/L. The precision and accuracy of the assay were determined on three different days. The within-series precision was found to be always less than 13.9% for all the analytes. The overall precision expressed as relative standard deviation (RSD) was always less than 10.6%. The recovery of anthracyclines was assessed at two concentrations of the range tested (0.1 and 2.0 microg/L) and it ranged from 87.7% (daunorubicin) to 102.0% (doxorubicin) and from 79.1% (daunorubicin) to 90.7% (idarubicin) for the lower and the higher level, respectively, with a RSD always less than 9.1%. The uncertainty of the present assay was also evaluated and the combined uncertainty was always less than 20% over all the days of the validation study. This is the first method that makes use of LC/MS/MS for the biological monitoring of occupational exposure to anthracyclines.

Validated procedure for simultaneous trace level determination of the anti-cancer agent gemcitabine and its metabolite in human urine by high-performance liquid chromatography with tandem mass spectrometry.

A sensitive, accurate and reproducible procedure has been developed for the quantitative determination of gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and its metabolite 2',2'-difluorodeoxyuridine (2dFdU) in human urine. The samples (2 mL) were extracted by solid-phase extraction (SPE) and analyzed by reversed-phase high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC/MS/MS), operating in multiple reaction monitoring (MRM mode). This procedure was validated using 2'-deoxycytidine as internal standard (IS). The urine assay was linear over the range 0-50 microg/L, with a limit of quantification (LLOQ) of 0.2 microg/L for gemcitabine and 1.0 microg/L for the metabolite. The respective limits of detection (LODs) for dFdC and 2dFdU were 0.05 and 0.3 microg/L. The precision and accuracy of the assay were determined on three different days. The within-series precision was found to be always less than 8.5 and 12.7% for gemcitabine and 2dFdU, respectively. The overall precision expressed as relative standard deviation (CVr) was always less than 7.1% for both analytes. The recovery of gemcitabine was always greater than 90% with a CVr <6.3%. The measurement uncertainty determined from the validation data assessed the possibility of determining this drug and its metabolite at trace levels in urine, considering that the combined uncertainty of the whole procedure was always less than 30%.

Critical evaluation of a high-throughput analytical method for polychlorinated biphenyls in human serum: which detector for the establishment of the reference values?

A rapid screening method, previously developed and validated for the determination of 60 PCB congeners in human serum by using high-resolution gas chromatography coupled with low-resolution mass spectrometry, was applied to the establishment of the reference values in the general population. The reliability of this method was further confirmed by analyzing ten serum samples, obtained from the same population group involved in the original study, by electron impact ionization with either low- or high-resolution mass spectrometry. Analyses in electron capture negative ionization mode were also performed for the determination of 'dioxin-like' isomers. The data were found to be in good accordance with one another, especially for the congeners of highest environmental significance. Then, 162 real serum samples were analyzed for the presence of PCBs. Comparison with the results from other studies reported in the literature showed that a similar profile was observed; the most abundant congeners were #153, #180, #138 and #170. A statistical analysis was performed to determine whether there were significant correlations between PCB concentrations and specific variables such as age, gender, and dietary habits.

Analysis of uranium and isotopic ratio measurement in fish and marine invertebrates from the North Adriatic Sea by inductively coupled plasma mass spectrometry.

Uranium analysis in fish, echinoderms and shellfish samples collected in the north part of the Adriatic Sea is presented. The aim of the work was to evaluate uranium concentrations in samples of this kind, and also to investigate possible contamination from depleted uranium (DU) in consequence of the war operations previously conducted in that area. DU contamination was checked by measuring the (235)U/(238)U isotope ratio. The samples were dissolved according to the EPA 3052 procedure, and the determinations were performed by inductively coupled plasma mass spectrometry (ICP-MS), optimized in order to perform isotope ratio measurements with good precision. The method was validated by evaluating the recovery from spiked samples; results in the range 93-107% were obtained. The isotope ratio measurement was evaluated in terms of accuracy and precision by analyzing the certified reference materials IAEA 326 and GBW 07305, and good agreement with the certified values was obtained here also. The concentration of uranium was higher in invertebrate samples than in fish (0.3-2 microg/g of U vs. 0.05-0.1 microg/g, respectively). The isotope ratio measurements for all the samples gave values very similar to the natural ratio, permitting exclusion of the presence of DU.

Analytical method for the quantitative determination of urinary ethylenethiourea by liquid chromatography/electrospray ionization tandem mass spectrometry.

A direct, rapid and selective method for the quantitative determination of the ethylenethiourea (ETU) in human urine has been validated and is reported in the present study. It allows the accurate quantification of ETU in this complex matrix without the use of any internal standard as the sample cleanup is effective enough for the removal of interferences that could lead to ion suppression in the electrospray ionization (ESI) source. This simple and rapid purification system, based on the use of a Fluorosil phase of a BondElut column followed by a liquid-liquid extraction procedure, achieves mean extracted recoveries, assessed at three different concentrations (2.5, 10.0, and 25.0 microg/L), always more than 85%. High-performance liquid chromatography (HPLC) with positive ion tandem mass spectrometry, operating in selected multiple reaction monitoring (MRM) mode, is used to quantify ETU in human urine. The assay is linear over the range 0-50 microg/L, with a lower limit of quantification (LOQ) of 1.5 microg/L and a coefficient of variation (CV) of 8.9%. The lower limit of detection (LOD) is assessed at 0.5 microg/L. The overall precision and accuracy were determined on three different days. The values for within- and between-day precision are < or = 8.3 and 10.1%, respectively, and the accuracy is in the range 97-118%. The relative uncertainties for the LOQ and QC concentrations have been estimated to be 18 and 8%, respectively. The assay was applied to quantify ETU in human urine from growers that regularly handle ethylenebisdithiocarbamate pesticides in large crop plantations. The biological samples were collected at the start and end of the working day, and the ETU urine levels were found to vary between 1.9 and 8.2 microg/L.

Inductively coupled plasma mass spectrometric determination of molybdenum in urine.

A method was developed for the determination of molybdenum (Mo) in human urine by direct dilution of the sample in doubly distilled water with 1% HNO3 (v/v) and inductively coupled mass spectrometry (ICP-MS). In and Y were used as internal standards. Since (98)Mo provides a higher sensitivity, it was chosen as the reference isotope. The influence of different factors, such as sample dilution, HNO3 concentration and the stability of the analyte were evaluated. The detection limit (LOD) was assessed at 0.2 microg/L Mo, while the lower limit of quantification (LOQ) was 0.6 microg/L. Recoveries ranged between 97.2 and 100.7% from solutions containing from 10 to 50 microg/L Mo. Linear calibration curves were generated from 2.1 and 52.1 microg/L with coefficients of variation (CV ) ranging from 1.62 to 3.56%. In order to establish reference values (RV) for molybdenum, the procedure presented here was used to determine Mo in the urine of a population group living in Tuscany, Italy.

Determination of rare earth elements in urine by electrothermal vaporization inductively coupled plasma mass spectrometry.

A method was developed for the determination of rare earth elements (REEs) in urine with electrothermal vaporization inductively coupled plasma mass spectrometry (ETV-ICPMS). The undiluted sample was directly injected into the graphite tube and trifluoromethane (Freon-23) was used as chemical modifier in order to reduce the vaporization temperature and the memory effect of most of the lanthanides. The detection limits were in the range 1-10 ng/L with relative standard deviation of 3-5% at concentration levels of 1microg/L, and less than 10-15% at 100 ng/L. Two different procedures, external calibration and a standard additions method, were evaluated to measure the concentration levels of lanthanides in the urine samples and the second procedure was considered to be the best choice for calibration in this work. The level of REEs in urine of 50 healthy volunteers was in the range 5-20 ng/L, above the detection limit of ETV-ICPMS.