Metrological traceability for benzo[a]pyrene quantification in airborne particulate matter

Airborne particulate matter (PM) represents one of the most important sources of urban pollution due to its physical and chemical properties. There is a great concern for PM dangerous effects on human health because particles can deeply penetrate into the respiratory system, carrying the contaminants adsorbed onto their surface. Polycyclic aromatic hydrocarbons (PAHs) are a class of organic contaminants that can be adsorbed onto PM and can have harmful effects on health, due to their particular chemical structure. The International Agency for Research on Cancer (IARC) classified PAHs as potential carcinogenic agents and benzo[a]pyrene (BaP) as carcinogenic to humans. In this paper, the development of a metrologically traceable procedure for the quantification of BaP in airborne PM, performed at the Italian National Institute of Metrological Research (Istituto Nazionale di Ricerca Metrologica—I.N.Ri.M.), is presented. The identification and quantification of BaP in PM samples were carried out by gas chromatography coupled with mass spectrometry (GC–MS). Metrological traceability was established in all the procedure steps, after performing the method validation. Suitable certified reference materials (CRMs) were used both to validate the analytical method and to calibrate the GC–MS. The measurement uncertainty was evaluated by identifying and taking into account all the relevant sources deriving from the whole procedure steps.     

ASE-SPE/GC-MS测定土壤中16种PAHs质量控制研究

优化了土壤中16种优控多环芳烃( PAHs)的分析方法,建立了一套完备的质量控制体系,解决了PAHs分析中常见的技术难点,如苯并(a)芘(BaP)回收率低,基质复杂的样品净化效果不理想,萘(Nap)和菲(Phe)挥发损失和环境本底影响等.样品经加速溶剂提取(ASE),固相萃取(SPE)净化,逐级减压浓缩,气相色谱质谱( GC - MS)测定,并以氘代苯并a芘(BaP - d12)作回收率指示物.实验比较了3种正相SPE吸附剂的效果,发现弗罗里硅土对BaP存在明显的降解现象,BaP的定量应使用同位素稀释法,以降低其分析不确定度;氧化铝对PAHs的吸附性过强,不利于样品净化;硅胶最为理想.PAHs的仪器检出限为0.26～5.7 pg,方法检出限为0.067 ～0.97 ng/g(干重),土壤基质加标回收率为71％～ 122％,相对标准偏差为1.6％～8.3％.将该法用于7个电子废物焚烧区域农田土壤样品的测定,PAHs含量在28～ 283 ng/g(干重)之间,样品中BaP-d12的回收率为90％～124％,各项质控指标符合检测要求.     

Estimation of measurement uncertainty of pesticides,polychlorinated biphenyls and polyaromatic hydrocarbons in sediments by using gas chromatography–mass spectrometry

The evaluation of the uncertainty associated to analytical methods is essential in order to demonstrate quality of a result. However, there is often lack of information about uncertainty of methods to estimate persistent organic pollutants concentration in complex matrix. Current work has thoroughly evaluated uncertainty associated to quantification of several organochloride pesticides, PCBs and PAHs in sediments. A discussion of the main contributions to the overall uncertainty is reported, allowing authors to establish the accuracy of results and plan future improvements. Combined uncertainties ranged between 5–9% (pesticides), 4–7% (PCBs) and 5–10% (PAHs), being uncertainty derived of calibration the main contribution. Also, the analytical procedure was validated analysing a standard reference material (IAEA-408).     

Photodynamic action of benzo[a]pyrene in K562 cells

Benzo[a]pyrene (BaP) is ubiquitously distributed in the environment, being considered the most phototoxic element among polycyclic aromatic hydrocarbon (PAHs). In presence of oxygen, PAHs can act as a photosensitizer by means of promoting photo-oxidation of biological molecules (photodynamic action, PDA). Thus, the present study analyzed the photodynamic action of BaP under UVA irradiation (BaP + UVA) and its oxidative effects on K562 cells. The evaluation of BaP kinetics showed that the highest intracellular concentration occurred after 18 h of incubation. Cell viability, reactive oxygen species (ROS) generation, lipid peroxidation, DNA damage (breaks and DNA-protein cross-link [DNAPC]) were assessed after exposure to BaP, UVA and BaP plus UVA irradiation (BaP + UVA). Cell viability was decreased just after exposure to BaP + UVA. Lipid peroxidation and DNA breaks increased after BaP + UVA exposure, while the DNAPC increased after BaP, UVA and BaP + UVA exposure, suggesting that BaP + UVA effects were a consequence of both type II (producing mainly singlet oxygen) and type I (producing others ROS) mechanisms of PDA.     

Uncertainty due to the quantification step in analytical methods

The quantification step is an important source of uncertainty in analytical methods, but it is frequently misunderstood and disregarded. In this paper, it is shown how this uncertainty is closely related to the linear response range of a method, and to the Pearson correlation coefficient of the calibration line. So, if there is a need for a pre-fixed quantification uncertainty, the linear response range will be affected. Some practical cases are given showing the quantification uncertainty significance. The theoretical equation giving the value of the quantification uncertainty is deduced from which new conclusions can be taken out. Because of that, the quantification uncertainty can easily be calculated and the parameters that really affect its value are shown along the paper. Some final considerations about detection limits and two-point calibration lines are also given. The paper can also be considered a reflection on uncertainty owed to calibration and on their consequences on the analytical methodology.     

Improved analysis of Polycyclic Aromatic Hydrocarbons in atmospheric particulate matter by HPLC-fluorescence

An improved method is reported for determination of Polycyclic Aromatic Hydrocarbons (PAHs) in atmospheric particulate matter by HPLC-FLD. The sampling step (air volume collected during each sampling period varies in the range 10/13 m3) is carried out by means of a medium-flow pumping system (15 L min(-1)) on a glass fiber filter (47 mm diameter) placed as collecting substrate in the sampling-cassette. After sampling, the filter is extracted with 3 ml of acetonitrile in an ultrasonic bath for 30 minutes. As for extraction of PAHs from loaded filters a new criterion here is proposed to evaluate the recovery efficiency of PAHs from the sample, instead of the usual spiking method of standard solution. The extract was then reduced to 100 microL and analysed by HPLC-FLD on line spectra system. The method is rapid (about one hour for extraction and analysis), reproducible and enables to measure with good accuracy the atmospheric concentration of benzo(b)fluoranthene (BbF), benzo(k)fluoranthene (BkF), benzo(a)pyrene (BaP), benzo(ghi)perylene (BghiP), carcinogenic compounds always present in the urban airborne particulate matter. So it is useful for routine pollution studies and suitable to substitute the official method used now. Monthly average air concentrations, for the four PAHs above mentioned, measured in Rome from July 2001 to June 2002, are reported.     

Determination of pyrene and benzo(a)pyrene residues in water by derivative synchronous solid-phase spectrofluorimetry

Benzo(a)pyrene (BaP) and pyrene (Pyr) are two polycyclic aromatic hydrocarbons (PAHs) showing native fluorescence in solution. Both compounds have been determined in water at trace levels by solid-phase spectrofluorimetry, in which BaP and Pyr are fixed on Sephadex G-25 gel and the relative fluorescence intensity is measured after the system is packed in a 1-mm silica cell. First-derivative synchronous spectra obtained at =38 nm were used to determine BaP and Pyr in the presence of other potentially interferent PAHs. The spectral characteristics of the PAHs-gel system are described, the applicable concentration ranges being 0.4–2.5 ng/ml for BaP and 0.7–4.5 ng/ml for Pyr. The relative standard deviations were 1.1% and 1.4% for BaP and Pyr respectively. The detection limits were 0.04 ng/ml for BaP and 0.1 ng/ml for Pyr. The method was applied to the analysis of both compounds in water at trace levels and a recovery study on tap, natural and sea waters was carried out.     

A metrological approach for the assessment of selenium status in human serum

This work addresses a metrological approach for the assessment of Se status in humans in terms of serum selenomethionine (SeMet). The quantification of SeMet was carried out using a primary method of chemical analysis, namely species-specific isotope dilution (SSID) in combination with HPLC coupled to collision/reaction cell inductively coupled plasma-mass spectrometry. SeMet was released from the serum selenoalbumin (a seleno-containing protein where SeMet is randomly incorporated) by enzymatic hydrolysis of the whole serum. This study is a follow-up of the analytical method development reported previously, and it focuses primarily on the evaluation of the uncertainty budget and the main uncertainty sources for SeMet determination in three commercial serums, namely BCR-637 (certified for total Se) and two serum standards, SERONORM level 1 (SERO-L1) and 2 (SERO-L2) (with indicative concentrations of total Se). The metrological approach reported here could be considered as a pilot study in terms of metrological determination of SeMet in human serum, hence being suitable for method validation and inter-laboratory comparison.     

Highly sensitive routine method for urinary 3-hydroxybenzo[a]pyrene quantitation using liquid chromatography-fluorescence detection and automated off-line solid phase extraction

Many workers and also the general population are exposed to polycyclic aromatic hydrocarbons (PAHs), and benzo[a]pyrene (BaP) was recently classified as carcinogenic for humans (group 1) by the International Agency for Research on Cancer. Biomonitoring of PAHs exposure is usually performed by urinary 1-hydroxypyrene (1-OHP) analysis. 1-OHP is a metabolite of pyrene, a non-carcinogenic PAH. In this work, we developed a very simple but highly sensitive analytical method of quantifying one urinary metabolite of BaP, 3-hydroxybenzo[a]pyrene (3-OHBaP), to evaluate carcinogenic PAHs exposure. After hydrolysis of 10 mL urine for two hours and concentration by automated off-line solid phase extraction, the sample was injected in a column-switching high-performance liquid chromatography fluorescence detection system. The limit of quantification was 0.2 pmol L(-1) (0.05 ng L(-1)) and the limit of detection was estimated at 0.07 pmol L(-1) (0.02 ng L(-1)). Linearity was established for 3-OHBaP concentrations ranging from 0.4 to 74.5 pmol L(-1) (0.1 to 20 ng L(-1)). Relative within-day standard deviation was less than 3% and relative between-day standard deviation was less than 4%. In non-occupationally exposed subjects, median concentrations for smokers compared with non-smokers were 3.5 times higher for 1-OHP (p<0.001) and 2 times higher for 3-OHBaP (p<0.05). The two urinary biomarkers were correlated in smokers (ρ=0.636; p<0.05; n=10) but not in non-smokers (ρ=0.09; p>0.05; n=21).     

Assessment uncertainty associated to the analysis of organic compounds linked to particulate matter of atmospheric aerosols

Current work has evaluated uncertainty associated to quantification of several organic compounds present in particulate matter of atmospheric aerosols, setting out the stages of analytical procedure that contribute most to the global uncertainty. Several sources of uncertainty have been identified, which were clustered into five main contributions: sampling, extraction, clean-up, derivatization and analysis. A discussion of the main contributions to the overall uncertainty is reported, allowing authors to locate the largest ones and plan future improvements. Combined uncertainties ranged between 10-18% (alkanes), 12-16% (PAHs), 10-18% (alcohols) and 9-21% (acids). The analytical procedure was validated by analysing a standard reference material (SRM1649a, urban dust). Also, the proposed method was applied to the analysis of four samples of particulate matter.     

Method validation for determination of the 15 European-priority polycyclic aromatic hydrocarbons in primary smoke condensates by gas chromatography/mass spectrometry: interlaboratory study

A collaborative study was conducted to validate an analytical method for quantification of the 15 polycyclic aromatic hydrocarbons (PAHs) regarded in 2002 as a health concern by the former Scientific Committee on Food of the European Commission (SCF) in primary smoke condensates. The method is based on gas chromatography/mass spectrometry of a cyclohexane extract with solid-phase cleanup through silica gel. The analytes were detected in the selected-ion monitoring mode and quantified by using 3 isotopically labeled internal standard compounds. Seventeen laboratories participated in the collaborative validation study, of which 12 reported valid results. The data were subjected to Cochran, single Grubbs, and double Grubbs tests for statistical outliers. A maximum of 2 outliers was eliminated before further statistical evaluation of the method performance characteristics. Depending on the analyte, the results showed relative standard deviations for repeatability between 4.2 and 30% and for reproducibility from 9.9 to 60%. The recoveries varied between about 50 and 85%, except those for cyclopenta[cd]pyrene, dibenzo[a,l]pyrene, and dibenzo[a,h]pyrene. Nevertheless, because Commission Directive 2005/10/EC allows for a recovery range of 50-120% for (BaP) benzo[a]pyrene in various foods, it can be concluded that the method performs appropriately within the analytical range between 5 and 25 microg/kg of primary smoke condensate. For BaP the validated analytical range covered 5-20 microg/kg, and for benzo[a]anthracene (BaA) 10-25 microg/kg. The method is suitable for monitoring BaP and BaA at their respective maximum permitted levels of 10 and 20 microg/kg. Three analytes, benzo[b]-, benzo[j]-, and benzo[k]-fluoranthene could not be separated by all of the participants and were therefore treated as the sum. Nevertheless, with this method the pattern of the respective concentrations of these 15 PAHs can be monitored in primary smoke condensate as suggested by the SCF.     

Innovative method for determination of 19 polycyclic aromatic hydrocarbons in food and oil samples using gas chromatography coupled to tandem mass spectrometry based on an isotope dilution approach

An efficient and selective analytical method for the determination and the quantification of 19 polycyclic aromatic hydrocarbons (PAHs) in food and oil has been developed. This method includes the monitoring of 15 PAHs stated as a priority by the EU in their 2005/108 recommendation. The samples were extracted according to a selective extraction step using pressurized liquid extraction followed by a purification with polystyrene-divinylbenzene SPE. Identification and quantification were performed using GC-MS/MS, with an isotope dilution approach using (13)C-labelled PAHs. The novel combination of selective extraction followed by purification provides highly purified analytes combined to a fast and automated method. The advantages of GC-MS/MS as compared to other detection methods are tremendous in terms of sensitivity, selectivity and interpretation facilities. Limits of detection varied between 0.008 and 0.15 microg kg(-1), limits of quantification between 0.025 and 0.915 microg kg(-1) for PAHs in food. The calibration curves showed a good linearity for all PAHs (R(2)>0.99) and precision and recovery were fit for purpose. Trueness of the method was carried out using the US National Institute of Standards and Technology SRM 2977 reference material.     

Validation of the analytical procedure for the determination of polyaromatic hydrocarbons in smoke flavourings using high performance liquid chromatography coupled to an ultraviolet,diode array or fluorescence detector

High performance liquid chromatography (HPLC) coupled to an ultraviolet (UV), diode array or fluorescence detector (UV/DAD/FLD) has been used to set up an analytical procedure for the quantification of 16 EU priority polyaromatic hydrocarbons (PAHs) in smoke flavourings. The following parameters have been determined for the 16 EU priority PAHs: limit of detection, limit of quantification, precision (repeatability and intermediate precision), recovery and measurement uncertainty, using the concept of accuracy profiles. They were in close agreement with quality criteria described in the Commission Regulation (EC) no. 627/2006 concerning PAHs in smoke flavourings. Presented at the AOAC Europe Workshop, November 2006, Limassol, Cyprus     

A new approach for measuring protein adducts from benzo[a]pyrene diolepoxide by high performance liquid chromatography/tandem mass spectrometry

The long-range goal of the present study is the development of a general approach for in vivo dosimetry of reactive metabolites of polycyclic aromatic hydrocarbons (PAHs), to be used as a tool in cancer risk assessment. With benzo[a]pyrene (BaP) chosen as indicator and a model of PAHs this study aims at the development of a method for the determination of adducts to histidine (His) in hemoglobin (Hb) and serum albumin (SA) of reactive metabolites of BaP. The predominantly mutagenic metabolite of BaP has been shown to be a diolepoxide isomer, +(anti)r-7, t-8-dihydroxy-t-9,10-epoxy-7,8, 9,10-tetrahydrobenzo[a]pyrene (+BPDE). In comparison with other methods for protein degradation, hydrazinolysis was found to be sufficiently effective and mild. The His adduct isolated after protein hydrazinolysis, with protection by tert-butyloxycarbonyl (Boc) of the hydrazide and alpha-amino groups, was shown to be N(im)- +/- (r-7, t-8, t-9-trihydroxy-7, 8, 9, 10-tetrahydrobenzo[a]pyren-c-10-yl)-N(alpha), N(2)-bis(tert-butyloxycarbonyl)-L-histidinehydrazide. Isomers of this compound, used as references, were synthesized and characterized by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Adducts in Hb and SA from in vitro treatment with BPDE were characterized after hydrazinolysis by HPLC-UV/MS, muHPLC/MS/MS and gas chromatography/mass spectrometry (GC/MS). Approximately 70 and 10% of the isolated BPDE adducts from SA and Hb, respectively, were His adducts. Other products were released as BaP tetrols and BaP triols. For the purpose of enrichment/purification of BPDE-His adducts, C(18) and cation exchange solid phase extraction (SPE) were utilized. The sensitivity obtained by this new approach, based on hydrazinolysis of protein, enrichment by SPE and analysis with muHPLC/MS/MS (APCI), is in the low-fmole range.     

Solid-surface phosphorescence characterization of polycyclic aromatic hydrocarbons and selective determination of benzo(a)pyrene in water samples

This paper presents the phosphorescence characterization of polycyclic aromatic hydrocarbons (PAHs) on solid-surface for obtaining new flow-through phosphorescence optosensors for PAHs-based on-line, immobilized onto a non-ionic resin solid support coupled to a continuous flow system and the applications for the selective determination of benzo(a)pyrene (BaP). The phosphorescent characterization of 15 PAHs, described as major pollutants by the Environmental Protection Agency (EPA) (naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, chrysene, benzo(a)anthracene, benzo(k)fluoranthene, benzo(b)fluoranthene, benzo(a)pyrene, indeno(1,2,3-cd)pyrene, benzo(g,h,i)perylene and dibenzo(a,h)anthracene) has been carried out. The experimental variables (heavy atom, deoxygenation and organic solvent in samples) for obtaining different possibilities for developing mono and multi-parameter PAH sensors and the conditions for PAH screening have been carefully studied and the experimental conditions to determination of BaP in presence of other PAHs in water samples have been optimized.     

Multifactorial optimization approach for the determination of polycyclic aromatic hydrocarbons in river sediments by gas chromatography-quadrupole ion trap selected ion storage mass spectrometry

A procedure for the determination of very low polycyclic aromatic hydrocarbons (PAHs) concentrations in sediment samples has been developed by gas chromatography-quadrupole ion trap mass spectrometry (GC-QIT MS) after extraction with dichloromethane and purification by using silica gel cleanup. Identification and quantification of analytes were based on the selected ion storage (SIS) strategy using deuterated PAHs as internal standards. In order to search out the main factors affecting the SIS mass spectrometry efficiency, four MS parameters, including target total ion count (TTIC), waveform amplitude (WA), transfer line (XLT) and ion trap temperatures (ITT) were subjected to a complete multifactorial design. The most relevant parameters obtained (TTIC and WA) were optimized by a rotatable and orthogonal composite design. Optimum values for these parameters were selected for the development of the method involving PAH determination in sediment samples. The optimized method exhibited a range of 111-760% higher signal-to-noise (S/N) ratios for PAHs in comparison with the method operated by the default conditions, demonstrating that the multifactorial optimization contributed to substantially improve the sensitivity of the GC-QIT MS determination. The accuracy of the method was verified by analyzing NWRI EC-3 certified reference material (Lake Ontario sediment). The selectivity, sensitivity (limits of quantification were in the range of 0.02-11.0 ng g(-1)), accuracy (recoveries >or=77%) and precision (RSD相似文献   

Burial effects of organic coatings on the heterogeneous reactivity of particle-borne benzo[a]pyrene (BaP) toward ozone

With an aerosol flow tube coupled to an Aerodyne aerosol mass spectrometer (AMS), room temperature (296 ± 3 K) kinetics studies have been performed on the reaction of gas-phase ozone with benzo[a]pyrene (BaP) adsorbed in submonolayer amounts to dry ammonium sulfate (AS) particles. Three organic substances, i.e., bis(2-ethylhexyl)sebacate (BES, liquid), phenylsiloxane oil (PSO, liquid), and eicosane (EC, solid), were used to coat BaP-AS particles to investigate the effects of such organic coatings on the heterogeneous reactivity of PAHs toward ozone. All the reactions of particle-borne BaP with excess ozone exhibit pseudo-first-order kinetics in terms of BaP loss, and reactions with a liquid organic coating proceed by the Langmuir-Hinshelwood (L-H) mechanism. Liquid organic coatings did not significantly affect the kinetics, consistent with the ability of reactants to rapidly diffuse through the organic coating. In contrast, the heterogeneous reactivity of BaP was reduced substantially by a thin (4-8 nm), solid EC coating and entirely suppressed by thick (10-80 nm) coatings, presumably because of slow diffusion through the organic layer. Although the heterogeneous reactivity of surface-bound PAHs is extremely rapid in the atmosphere, this work is the first to experimentally demonstrate a mechanism by which the lifetime of PAHs may be significantly prolonged, permitting them to undergo long-range transport to remote locations.     

Uncertainty in liquid chromatographic analysis of pharmaceutical product: influence of various uncertainty sources

An ISO GUM measurement uncertainty estimation procedure was developed for a liquid-chromatographic drug quality control method-assay of simvastatin in drug formulation. In quantification of uncertainty components several practical approaches for including difficult-to-estimate uncertainty sources (such as uncertainty due to peak integration, uncertainty due to nonlinearity of the calibration curve, etc.) have been presented. Detailed analysis of contributions of the various uncertainty sources was carried out. The results were calculated based on different definitions of the measurand and it was demonstrated that unequivocal definition of the measurand is essential in order to get rigorous uncertainty estimate. Two different calibration methods - single-point (1P) and five-point (5P) - were used and the obtained uncertainties and uncertainty budgets were compared. Results calculated using 1P and 5P calibrations agree very well. The uncertainty estimate for 1P is only slightly larger than with 5P calibration.     

Comparison of immunoassay and gas chromatography-mass spectrometry for measurement of polycyclic aromatic hydrocarbons in contaminated soil

Polycyclic aromatic hydrocarbons (PAHs) are frequently encountered in the environment and may pose health concerns due to their carcinogenicity. A commercial enzyme-linked immunosorbent assay (ELISA), was evaluated as a screening method for monitoring PAHs at contaminated sites. The ELISA was a carcinogenic PAH (C-PAH) RaPID assay testing kit that cross-reacts with several PAHs and utilizes benzo[a]pyrene (BaP) as a calibrator. Soil samples were extracted with 50% acetone in dichloromethane (DCM) for analysis by ELISA and gas chromatography-mass spectrometry (GC-MS). The overall method precision was within ±30% for ELISA and within ±20% for GC-MS. Recovery data for spiked soils ranged from 46 to 140% for BaP as determined by ELISA. Recoveries data of the GC-MS surrogate standards, 2-fluorobiphenyl and chrysene, were greater than 70%. The GC-MS procedure detected a total of 19 priority PAHs (2-6-ring PAHs) including seven probable human carcinogens (4-6-ring B2-PAHs). The ELISA results were compared to GC-MS summation results for the total 19 target PAHs as well as for the subset of the seven B2-PAH compounds. For all soil samples, the PAH concentrations derived from ELISA were greater than the sum of B2-PAH concentrations obtained by GC-MS. ELISA determinations were also frequently greater than the results obtained by GC-MS for the total 19 PAH compounds. This discrepancy can be expected, since the ELISA is a screening assay for the detection of several related PAHs while the GC-MS procedure detects priority PAH compounds. Thus, only a subset of PAHs (e.g. 19 PAHs) in the soil samples were measured by GC-MS while additional PAHs, including alkylated PAHs, and PAH derivatives have been demonstrated to be cross-reactive in the C-PAH ELISA. Results of paired tests show that the PAH data from ELISA and GC-MS methods are significantly different (P<0.001), but highly correlated. The ELISA data had a strong positive relationship with the GC-MS summation data for the B2-PAHs as well as for the 19 PAHs targeted by the GC-MS method. Results indicate that the ELISA may be useful as a broad screen for monitoring PAHs in environmental samples.     

分子吸收分光光度法测定辉钼矿中钼的不确定度评定

对分子吸收分光光度法测定辉钼矿中钼元素的不确定度进行了评定,分析了不确定度的来源,包括工作曲线拟合、标准溶液配制、样品重复测定、分析仪器、样品称量、样品溶液定容等引入的不确定度。计算了钼含量测定结果的合成标准不确定度和扩展不确定度。当辉钼矿中钼含量为0.108 1%时,扩展不确定度为0.002 1%(k=2)。