A Gas Liquid Chromatographic Fluorescent Procedure for the Analysis of Benzo(A)Pyrene in 24 Hour Atmospheric Particulate Samples

Abstract

This paper describes a selective method for the assay of benzo(a)pyrene (BaP) in 24 hour particulate samples. Benzo(a)pyrene is partially separated from the other polynuclear aromatic hydrocarbons via gas chromatography and then is completely resolved optically from traditionally difficult to resolve compounds (benzo(e)pyrene, perylene, benzo(k)fluoranthene) by means of a gas phase fluorescent detector. Ambient air concentrations as low as 20 picograms per cubic meter of air can be assayed with this gas chromatographic - gas phase fluorescent detection system.     

Quantification of monohydroxy-PAH metabolites in urine by solid-phase extraction with isotope dilution-GC–MS

For measurement of biomarkers from polycyclic aromatic hydrocarbon (PAH) exposure, an analytical method is described quantifying hydroxylated PAH (OH-PAH) in urine samples. This method determined monohydroxy metabolites of naphthalene, fluorene, phenanthrene, fluoranthene, pyrene, chrysene, benzo[c]phenanthrene, and benz[a]anthracene. The sample preparation consisted of enzymatic hydrolysis, solid-phase extraction and derivatization with a silylating reagent. Five carbon-13 labeled standards were used for isotope dilution. Analytes were separated by gas chromatography (GC) and quantified with high-resolution mass spectrometry (HRMS). This method produced good recoveries (41-70%), linearity, and specificity. Data were corrected for blank levels from the naphthalene, fluorene, and phenanthrene metabolites. Method detection limits ranged from 2 ng L(-1) for 1-hydroxypyrene to 43.5 ng L(-1) for 1-hydroxynaphthalene. Using quality control charts from two urine pools, the method can be readily applied to biomonitoring PAH exposure.     

Quantification of several monohydroxylated metabolites of polycyclic aromatic hydrocarbons in urine by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method with fluorescence detection has been developed to determine the urinary polycyclic aromatic hydrocarbon metabolites 2-hydroxynaphthalene, 2-hydroxyfluorene, 9-hydroxyphenanthrene, 1-hydroxypyrene and 3-hydroxybenz[a]pyrene. Solid phase extraction (SPE) was used to clean up the samples, and washing with 30% methanol was found to be the best way to remove interferences in the matrix. The method detection limits ranged from 0.044 μg/L for 1-hydroxypyrene to 1.615 μg/L for 3-hydroxybenz[a]pyrene, and the recoveries ranged between 40% for 3-hydroxybenz[a]pyrene and 99% for 2-hydroxynaphthalene. The within-day relative standard deviation was lowest for 2-hydroxynaphthalene at 0.67% and went up to 2.42% for 3-hydroxybenz[a]pyrene, and the between-day relative standard deviation ranged from 3.84% for 9-hydroxyphenanthrene to 10.42% for 2-hydroxyfluorene. The correlation coefficients were between 0.9962 and 0.9998. The developed method was successfully used to analyze samples from student volunteers in a high school.     

Immunochemical detection of metabolites of parent and nitro polycyclic aromatic hydrocarbons in urine samples from persons occupationally exposed to diesel exhaust

An immunochemical assay was developed for the detection of metabolites excreted in urine as a result of occupational exposure to PAHs and nitro-PAHs. These metabolites were analyzed in a competitive ELISA, using an existing antibody (4D5) that has been developed against 6-aminobenzo[a]pyrene coupled to bovine serum albumine (B[a]P-BSA). A solid phase extraction (SPE) work-up procedure was optimized by dilution of pooled urine samples from rats exposed to 1-NP, in human urine. The application was validated by comparison of test results with urinary excreted 1-hydroxypyrene (1-OH-P) levels in a study among 28 railroad workers. Excretion of urinary metabolites was determined over two consecutive workdays. The 24 h average excretion of metabolites as determined in the immunoassay was not related to levels of particulate matter in the breathing zones of workers, not to 1-OH-P excretion levels of the day of urine collection. However, it was significantly associated with the personal dust exposure of the day before (P<0.0001), suggesting slow excretion of urinary metabolites. Smoking habits caused minor interference (P<0.1).     

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).     

Desorptivity versus chemical reactivity of polycyclic aromatic hydrocarbons (PAHs) in atmospheric aerosols collected on quartz fiber filters

Nine polycyclic aromatic hydrocarbons (PAHs) contained in air samples collected on quartz fiber filters inside an urban tunnel and in a nearby mixed commercial residential area in the city of Rio de Janeiro, Brazil, were exposed to scrubbed air (to measure desorption loss) and to particle-free ambient air (to measure chemical reaction losses in the absence of desorption). The exposures were conducted for 5.5 to 9 hour periods at ambient temperature (22-26 degrees C) at face velocities typical of high volume sampling. Under prevailing atmospheric conditions all nine PAHs experienced filter losses which (for most of them) followed first order kinetics. For the ambient samples, in a 6 hour exposure period, the following five PAHs showed filter losses (% in parantheses) attributed exclusively to chemical reaction: benzo(b)fluoranthene (43), benzo(k)fluoranthene (39), benzo(a)pyrene (70), benzo(ghi)perylene (44), and indeno (1,2,3-cd)pyrene (41). The other four showed the following unassigned losses: pyrene (100), fluoranthene (65), crysene (72), and benzo(a)anthracene (71). The results are discussed in the light of possible filter artifacts in PAH sampling and the use of PAH profile signatures for source identification of atmospheric particulate matter in receptor modeling.     

Solvents and Techniques For The Extraction Of Polynuclear Aromatic Hydrocarbons From Filter Samples Of Diesel Exhaust

Abstract

The amounts of five polynuclear aromatic hydrocarbons-fluoranthene, pyrene, benzo(a)anthracene, benzo(k)fluoranthene, and benzo(a)pyrene-extracted by six solvents with four extraction techniques have been determined for diesel exhaust particulate collected on Teflon-coated glass fiber filters. Samples were analyzed by high pressure liquid chromatography with fluorescence detection. Toluene and methylene chloride gave higher recoveries than methanol, isopropanol, acetonitrile, and acetone for benzo(a)anthracene, benzo(k)fluoranthene, and benzo(a)pyrene. Soxhlet extraction for two hours (approximately 48 cycles) with toluene or acetonitrile was more effective than simple mechanical agitation, ultrasonic agitation for 15 minutes or immersion in refluxing ‘solvent’ for two hours.     

Determination of Polycyclic Aromatic Hydrocarbons in Airborne Particulate by High Performance Liquid Chromatography

This paper presents a trisolvent ultrasonic extraction and HPLC analysis method for the determination of 11 polycyclic aromatic hydrocarbons in air particulate collected on an air filter by a commercial high volume air sampler. A reverse phase column, Vydac 201 TP, and a gradient mobile phase, acetonitrile/water, were used. The 11 PAHs, fluoranthene, pyrene, benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a, h]anthracene, benzo[ghi]perylene, indeno[1,2,3-cd]pyrene, and coronene were completely resolved under experimental conditions. All the PAHs except coronene were monitored by fluorescence with λ_ex=270 nm, λ_em>389 nm. Coronene was monitored by UV with λ=300 nm. The methodology was evaluated by spiking SRM 1649 with a PAH standard and then going through different extraction procedures and analyzing the PAH concentrations without clean-up. An external standard method was used for quantitation. The recovery yields for fluoranthene, benz[a]anthracene, benzo[a]pyrene, benzo[ghi]perylene and indeno[l,2,3-cd]pyrene were above 90%. The detection limits of PAH with fluorescence at λ_ex=270 nm, λ_em>389 nm ranged from 5.7 pg to 69.5 pg.     

Photochemical degradation of polycyclic aromatic hydrocarbons (PAH) in real and laboratory conditions

Studies were carried out on the effects induced by the main components of airborne particulate matter (APM) as soot, inorganic and cyclohexane soluble substances and by glass fiber filters on photodegradation of pyrene, benz(a)anthracene and benzo(a)pyrene exposed to UV and solar radiation. In both experimental models tested hydrocarbons showed a higher photochemical stability when absorbed on APM. Inorganic components of APM slightly enhance UV photodegradation. In real condition (outdoor exposure to solar radiation) PAH half lives generally showed a good linear correlation with mean solar radiation intensity; only degradation rate of benzo(a)pyrene on APM, exposed to sunlight was practically constant. Pyrene, in particular, showed a higher degradation rate when high ozone concentrations (0.2 ppm) occurred.     

Ultrasound-assisted extraction and on-line LC–GC–MS for determination of polycyclic aromatic hydrocarbons (PAH) in urban dust and diesel particulate matter

A method has been developed for determination of polycyclic aromatic hydrocarbons (PAH) in particulate matter from ambient air and diesel exhaust emissions. It is reproducible and accurate and, compared with similar methods for analysis of individual PAH components in complex matrices, it is relatively fast and simple. Single PAH components can be determined in samples of particulate matter from ambient air and diesel exhaust emissions with LOD of approximately 1 pg/sample. Further, sample throughput is high, because more than 20 samples can be extracted and prepared for analysis in one working (8-h) day. The particulate matter is subjected to ultrasound-assisted extraction, a technique that is shown to extract PAH from particulate material with efficiencies fully comparable with those of Soxhlet extraction. An aliphatic/PAH-enriched fraction is obtained by solid-phase extraction before isolation, separation, and identification/quantification of PAH by on-line liquid chromatography–gas chromatography–mass spectrometry. The method was validated by analysis of US National Institute of Standards and Technology (NIST) Standard Reference Materials (SRM) 1649a, Urban Dust, and 2975, Diesel Particulate Matter. Results from the method are in good agreement with the NIST-certified PAH concentrations and with NIST reference PAH concentrations.     

The determination of benzo[a]pyrene in the total particulate matter of cigarette smoke

A procedure for the isolation and determination of benzo[a]pyrene in the total particulate matter of cigarette smoke is described. Two high-pressure liquid chromatographic (HPLC) techniques are employed: a normal-phase, mu Bondapak-NH2, amino column is used for isolation of the benzo[a]pyrene fraction and a reversed-phase, Vydac 201TP54, polymeric octadecyl silane column is used for quantitation. Fluorescence detection is used in both modes of chromatography. The wavelengths of excitation and emission are evaluated for analytical detection. Extraction media and various isolation techniques are compared for their extraction efficiency and isolation from interferences, respectively. The procedure is efficient, reproducible, sensitive (3 pg), and gives results that compare favorably with other techniques reported in the literature for the B[a]P content of reference cigarettes, 1R1 and 1R4F.     

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.     

Identification and quantification of urinary benzo[a]pyrene and its metabolites from asphalt fume exposed mice by microflow LC coupled to hybrid quadrupole time-of-flight mass spectrometry

Prolonged, extensive exposure to asphalt fume has been associated with several adverse health effects. Inhaled polycyclic aromatic hydrocarbons (PAHs) from asphalt fume exposure have been suspected of inducing such effects. In this study, a bioanalytical method was proposed and evaluated to identify and quantify benzo[a]pyrene and its hydroxy-metabolites. This method is based on coupling a microflow liquid chromatography (LC) to a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometry (Q-TOFMS). In the experiment, thirty-two B6C3FI mice were exposed to asphalt fume in a whole body inhalation chamber for 10 days (4 h day(-1)) and twelve other mice were used as controls. The asphalt fume was generated at 180 degrees C and the concentrations in the animal exposure chamber ranged 175-182 mg m(-3). Benzo[a]pyrene and its metabolites of 3-hydroxybenzo[a]pyrene, benzo[a]pyrene-7,8-dihydrodiol(+/-), benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide(+/-), and benzo[a]pyrene-7,8,9,10-tetrahydrotetrol(+/-) in the urine of asphalt fume exposed mice were identified and found at 3.18 ng 100 mL(-1), 31.36 ng 100 mL(-1), 11.56 ng 100 mL(-1), 54.92 ng 100 mL(-1), and 45.23 ng 100 mL(-1) respectively. The results revealed that the urinary benzo[a]pyrene and its hydroxy-metabolites from exposed mice were at significantly higher levels (p < 0.001) than those from the control groups. Compared with several other technologies such as HPLC-UV and HPLC-fluorescence, the new method is more sensitive and selective, and it can also provide additional useful information on the structures of the metabolites. Hence, this method can be used to perform the assessment and to study the mechanisms of the adverse health effects. The fragmentation patterns established in this study can also be used to identify and quantify PAH metabolites in other biological fluids.     

Determination of dibenzopyrenes in standard reference materials (SRM) 1649a, 1650, and 2975 using ultrasonically assisted extraction and LC–GC–MS

A method has been developed for analysis of the highly potent polycyclic aromatic hydrocarbon (PAH) carcinogens dibenzo(a,l)pyrene, dibenzo(a,h)pyrene, and dibenzo(a,i)pyrene (molecular weight 302) present in small amounts in diesel and air particulate material. The method can also be used for analysis of the PAH benzo(a)pyrene, coronene, and perylene, for which reference and certified values are available for the standard reference materials used for validation of the method—SRM 1649a (urban dust) and SRM 2975 (diesel particulate matter). The only NIST values that have been published for these dibenzopyrene isomers in the analyzed SRM are reference values for dibenzo(a,i)pyrene and dibenzo(a,h)pyrene in SRM 1649a. The concentrations determined in the SRM were in good agreement with reported NIST-certified and reference values and other concentrations reported in the literature. Standard reference material 1650 (diesel particulate matter) was also analyzed. The method could not, however, be validated using this material because certification of SRM 1650 had expired. The method is based on ultrasonically assisted extraction of the particulate material, then silica SPE pre-separation and isolation, and, separation and detection by hyphenated LC–GC–MS. The method is relatively rapid and requires only approximately 1–5 mg SRM particulate material to identify and quantify the analytes. Low extraction recoveries for the analytes, in particular the dibenzopyrenes, when extracting diesel SRM 2975 and 1650 resulted, however, in the dibenzopyrenes being present in amounts near their limits of quantifications in these samples. The method’s limit of quantification (LOQ), based on analyses of SRM 1649a, is in the range 10–77 pg. By use of this method more than 25 potential PAH isomers with a molecular weight of 302 could be separated.     

Possibilities and limitations of an enzyme-linked immunosorbent assay for the detection of 1-nitropyrene in air particulate matter

ELISAs (enzyme-linked immunosorbent assays) are generally used in environmental analytical chemistry for screening purposes. In this work the applicability of a polyclonal-based 1-nitropyrene-ELISA for the quantification of the target analyte in air particulate matter was investigated. Validation was performed using an HPLC method with fluorescence detection of the reduced form of 1-nitropyrene. It was found that the immunoassay is not only sensitive for the target analyte but to a certain extent also for other cross-reacting molecules present in the sample, such as 2-nitropyrene and 2-nitrofluoranthene, which were identified by GC-HRMS and are considered to be products of photochemically induced reactions of pyrene or fluoranthene, respectively. The degree of correlation between ELISA and HPLC results for collected samples of air particulate matter was inversely dependent on the distance between the sampling location and the source.     

Thermal desorption gas chromatography–mass spectrometry as an enhanced method for the quantification of polycyclic aromatic hydrocarbons from ambient air particulate matter

Polycyclic aromatic hydrocarbons (PAHs) from ambient air particulate matter (PM) were analysed by a two-step thermal desorption (TD) injection system integrated to a gas chromatograph–mass spectrometer (GC/MS). The operational variables of the TD method were optimised and the analytical expanded uncertainties were calculated to vary from 8% to 16% over the operative concentration range (40–4000 pg). The performance of the TD method was validated by the analysis of a standard reference material and by comparison of PAH concentrations in PM samples to those obtained by a conventional liquid extraction (LE) method. The TD method reported lower uncertainties than the LE method for the analysis of similar concentrations in air. The TD method also showed advantages for shorter sampling times in comparison to 24 h for source apportionment applications and for reducing losses of more reactive compounds such as benzo[a]pyrene.     

Collection and analytical characterisation of the atmospheric particulate in the city of Bari

In this paper an application of new procedures for atmospheric particulate analysis is illustrated. PM10, PAHs (benzo[a]anthracene (BaA), benzo[b]fluoranthene (BbF), benzo[j]fluoranthene (BjF), benzo[k]fluoranthene (BkF), benzo[a]pyrene (BaP), indeno[1, 2, 3-cd]pyrene (Ip), dibenzo[a, h]anthracene (DbA)) and heavy metals (Cu, Ni, Zn, Co, Mn, Cd, Fe and Pb) were investigated. PM10 determination was performed by gravimetric method, PAHs were measured by GC-MS, and heavy metals by HPIC. An air quality monitoring campaign on the territory of Bari municipality has been organised, and its results are shown.     

Possibilities and limitations of an enzyme-linked immunosorbent assay for the detection of 1-nitropyrene in air particulate matter

ELISAs (enzyme-linked immunosorbent assays) are generally used in environmental analytical chemistry for screening purposes. In this work the applicability of a polyclonal-based 1-nitropyrene-ELISA for the quantification of the target analyte in air particulate matter was investigated. Validation was performed using an HPLC method with fluorescence detection of the reduced form of 1-nitropyrene. It was found that the immunoassay is not only sensitive for the target analyte but to a certain extent also for other cross-reacting molecules present in the sample, such as 2-nitropyrene and 2-nitrofluoranthene, which were identified by GC-HRMS and are considered to be products of photochemically induced reactions of pyrene or fluoranthene, respectively. The degree of correlation between ELISA and HPLC results for collected samples of air particulate matter was inversely dependent on the distance between the sampling location and the source. Received: 20 August 1997 / Revised: 25 November 1997 / Accepted: 27 November 1997     

Solid-Phase microextraction with Whatman 1PS paper and direct room-temperature solid-matrix luminescence analysis

Solid-phase microextraction has been combined with solid-matrix luminescence for the detection of a variety of compounds at sub-nanogram levels for the first time. Whatman 1PS paper was placed in water solutions of polar and nonpolar compounds for the selective removal of the nonpolar compounds such as benzo(a)pyrene. Distribution constants were obtained for 4-phenylphenol, benzo(f)quinoline, benzo(h)quinoline, phenanthrene, and benzo(a)pyrene. The distribution constants showed that phenanthrene and benzo(a)pyrene in water had a very strong affinity for the 1PS paper. Once the solutes were extracted for a fixed period of time, the 1PS paper was dried, and either the solid-matrix fluorescence or solid-matrix phosphorescence was detected from the adsorbed lumiphors by using the appropriate excitation wavelengths. It was a simple matter to detect at least three adsorbed compounds on the 1PS paper by solid-matrix luminescence. Benzo(a)pyrene was easily detected at a level of 0.02 ng ml⁻¹ in water.     

Bioaccumulation and biotransformation of 1-hydroxypyrene by the marine whelk Neptunea lyrata

In the marine environment, organisms can be exposed to oxidised forms of polycyclic aromatic hydrocarbons. Bioaccumulation and biotransformation of these derivatives has rarely been investigated and would lead to a better understanding of the overall fate of polycyclic aromatic hydrocarbons and that of other phenolic contaminants. The marine whelk Neptunea lyrata was exposed to 1-hydroxypyrene through its diet over 35 days. Extracts from the muscle and visceral mass of each animal were analysed by liquid chromatography with fluorescence detection. The quantified compounds included 1-hydroxpyrene and the phase II metabolites pyrene sulphate, pyrene glucuronide and one isomer of pyrenediol disulphate. The hydroxylated hydrocarbon was highly retained with 78% of the exposure amount recovered primarily from the visceral mass of the whelks, while the muscle accounted for 4% of the body burden. Whelks efficiently biotransformed 1-hydroxypyrene with a mean of 81% of the compound detected as phase II metabolites. The novel biotransformation product, pyrenediol disulphate, accounted for the largest proportion of the 1-hydroxypyrene derivatives detected at body burdens below 200?ng. At higher body burdens, bioaccumulation increased. Control animals showed trace levels of pyrene derivatives with 76% represented by metabolites. This study highlights the importance of investigating the multiple fates of reactive chemicals in order to interpret exposure.