Determination of benzo[a]pyrene tetrols by column-switching capillary liquid chromatography with fluorescence and micro-electrospray ionization mass spectrometric detection

The present work displays capillary liquid chromatographic column switching methodology tailored for determination of benzo[a]pyrene tetrol isomers in biological matrices using on-line fluorescence and micro-electrospray ionization mass spectrometric detection. A well-established off-line crude solid phase extraction procedure was used in order to make the method compatible with several biological matrices. The solid phase extraction eluates were evaporated to dryness, redissolved in 1.0 ml methanol:water (10:90, v/v), loaded onto a 0.32 mm I.D. x 40 mm 5 microm Kromasil C(18) pre-column for analyte enrichment and back-flushed elution onto a 0.30 mm I.D. x 150 mm 3.5 microm Kromasil C(18) analytical column. The samples were loaded with a flow rate of 50 microl min(-1) and the tetrols were separated at a flow rate of 4 microl min(-1) with an acetonitrile:10 mM ammonium acetate gradient from 10 to 90%. A sample loading flow rate up to 50 microl min(-1) was allowed. The fluorescence excitation and emission were set to 342 and 385 nm, respectively, while mass spectrometric detection of the benzo[a]pyrene tetrols was obtained by monitoring their [M - H](-) molecular ions at m/z 319. The method was validated over the concentration range 0.1-50 ng ml(-1) benzo[a]pyrene tetrols in a cell culture medium with 100 microl injection volume, fluorescence detection and the first eluting tetrol isomer as model compound, resulting in a correlation coefficient of 0.993. The within-assay (n= 6) and between-assay (n= 6) precisions were determined to 2.6-8.6% and 3.8-9.6%, respectively, and the recoveries were determined to 97.9-102.4% within the investigated concentration range. The mass limit of detection (by fluorescence) was 3 pg for all the tetrol isomers, corresponding to a concentration limit of detection of 30 pg ml(-1) cell culture medium. The corresponding mass spectrometric mass limits of detection were 4-10 pg, corresponding to concentration limits of detection of 40-100 pg ml(-1) cell culture medium.     

Combination of micellar extraction of polycyclic aromatic hydrocarbons from aqueous media with detection by synchronous fluorescence

Summary The application of different non-ionic surfactants for the micellar extraction and enrichment of PAHs from aqueous media was tested. Recoveries were up to 100%. A spectroscopic method for the simultaneous detection of PAH-mixtures by synchronous fluorescence in the micellar phase was developed, with detection limits of 6.8 and 2.6 ng/l for benzo(k)fluoranthene and benzo(a)pyrene, respectively. The method was applied to the extraction and detection of benzo(k)fluoranthene/benzo(a)pyrene mixtures from aqueous solutions and soil suspensions. Genapol X-80 was found to suppress PAH-adsorption on bentonite at surfactant concentrations above 0.1%.Dedicated to Prof. Dr. V. Krivan on the occasion of his 60th birthday.     

Optimization of the extraction of polycyclic aromatic hydrocarbons from wood samples by the use of microwave energy

A simple and rapid microwave-assisted extraction (MAE) procedure was developed and optimized for benzo[a]anthracene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene and benzo[a]pyrene in wood samples. The spiked wood used was prepared 3 months before analysis to simulate weathering processes and to allow the formation of analyte-matrix interaction. The samples, immersed in acetonitrile were irradiated with microwaves in a closed-vessel system. Optimization of the method was achieved by using a factorial design approach on parameters such as extraction time, temperature and sample amount. The analysis of extracts has been carried out by reversed-phase high-performance liquid chromatography with fluorescence detection for quantification and UV-diode-array detection for confirmation. The MAE procedure yielded extracts that could be analyzed directly without any preliminary clean-up or solvent exchange steps.     

Screening potential of solid-phase extraction and solid surface room temperature fluorimetry for polycyclic aromatic hydrocarbons in water samples

The analytical potential of solid-phase extraction and room temperature fluorimetry for screening polycylic aromatic hydrocarbons in water samples is evaluated. Solid-phase extraction was performed via a syringe procedure previously reported (Talanta 52 (2000) 727). The simplicity of fluorescence measurements on the solid substrate is equivalent to solution measurements. Since oxygen quenching of fluorescence is not significant, placing the extraction membrane in the substrate holder of the spectrometer rapidly performs fluorescence measurements. Limits of detection at the pg ml(-1) level were estimated for several pollutants. With a commercial spectrofluorimeter, benzo(a)pyrene was quantitatively determined at the 5 pg ml(-1) concentration level. This concentration compared favorably to limits of detection estimated by laser-induced fluorimetry. A unique advantage of this approach was the possibility of adjusting the volume of extracted water to reach concentration levels below instrumental detection levels. Since SPE procedure was rapid and simple the trade-off of including an additional experimental step to lower limits of detection was advantageous. Although the selectivity of this approach was not fully investigated, our studies showed that selective excitation was sufficient to identify benzo(a)pyrene in a seven-component mixture and spiked Red River water of unknown composition.     

Admicelle-enhanced synchronous fluorescence spectrometry for the selective determination of polycyclic aromatic hydrocarbons in water

A simple and rapid method for the highly sensitive determination of polycyclic aromatic hydrocarbons (PAHs) in water was developed. Benzo[a]pyrene, benzo[k]fluoranthene, perylene, and pyrene in water were concentrated into sodium dodecyl sulfate (SDS)-alumina admicelles. The collection was performed by adding SDS and alumina particles into the sample solution at pH 2. After gentle mixing, the resulting suspension was passed through a membrane filter to collect the SDS admicelles containing highly concentrated PAHs. The filter was placed on a slide glass and then covered admicellar layer with a fused silica glass plate before setting in a fluorescence spectrometer. Benzo[a]pyrene, benzo[k]fluoranthene, perylene, and pyrene were selectively determined by the synchronous fluorescence scan (SFS) analysis with keeping wavelength intervals between excitation and emission to 98, 35, 29, and 45 nm, respectively. Because of the minimum spectral overlapping, 1-40 ng l⁻¹ of benzo[a]pyrene, benzo[k]fluoranthene, and perylene as well as 10-150 ng l⁻¹ of pyrene were selectively determined with eliminating the interferences of other 12 PAHs. The detection limits were 0.3 ng l⁻¹ for benzo[a]pyrene, benzo[k]fluoranthene, and perylene, and 1 ng l⁻¹ for pyrene. They were 2-3 orders of magnitude lower than the detection limits in normal aqueous micellar solutions. The application to water analysis was studied.     

Sensitive and selective determination of metabolically formed trans-dihydrodiols and phenols of benzo[a]pyrene in water and urine samples by HPLC with amperometric detection

A method has been developed to separate hydroxylated metabolites of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene, i. e. trans-4,5-, 7,8-, 9,10-dihydrodiol and 1-, 3-, 7-, and 9-phenol, by HPLC with amperometric detection employing an isocratic methanol/water eluent (70:30, v/v) containing 0.5 g/L sulfuric acid and 1 g/L lithium perchlorate. Compared with the usually applied fluorescence (λ_ex = 265 nm, λ_em = 460 nm) and ultraviolet (λ = 265 nm) detection, the amperometric technique is about 2–12 times more sensitive for the determination of all metabolites investigated. The method was applied to the determination of the seven metabolites of benzo[a]pyrene in different water samples and in urine after solid-phase extraction (SPE). The results obtained by HPLC with amperometric detection after SPE enrichment from an aqueous extract of a soil sample and from the urine of a rat intragastrically treated with benzo[a]pyrene agreed well with the values determined with fluorescence and/or UV detection. Received: 20 December 1996 / Revised: 10 March 1997 / Accepted: 30 April 1997     

采用新型固相萃取柱快速测定食用植物油中苯并［a］芘

研究了Bond Elut ENV新型固相萃取柱在食用植物油中苯并[a]芘快速检测中的应用,建立了快速测定食用植物油样品中苯并[a]芘残留量的固相萃取/液相色谱/荧光检测法。样品用正己烷溶解,固相萃取净化,SUPELCOSILTMLC-PAH(25 cm×4.6 mm,5μm)色谱柱分离,以乙腈-水(95∶5)为流动相,荧光检测(λex=297 nm,λem=408 nm),外标法定量。苯并[a]芘的检出限为0.3μg/kg,在1.0～50.0μg/L范围内线性关系良好,相关系数为0.999 6,方法的回收率为79%～102%,相对标准偏差不高于9.4%。该方法准确、实用、简便、快速,在食用植物油的苯并[a]芘残留量检测方面有广泛的应用前景。     

Exploration of coordination polymer as sorbent for flow injection solid-phase extraction on-line coupled with high-performance liquid chromatography for determination of polycyclic aromatic hydrocarbons in environmental materials

The copper(II) isonicotinate (Cu(4-C5H4N-COO)2(H2O)4) coordination polymer was prepared, characterized and explored as sorbent for flow injection solid-phase extraction on-line coupled with high-performance liquid chromatography (HPLC) for determination of trace polycyclic aromatic hydrocarbons (PAHs) in environmental matrices. Naphthalene, phenanthrene, anthracene, fluoranthene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene and benzo(ghi)perylene with various shape, size and hydrophobicity were used as model analytes. The porosity of the coordination polymer allows these guest PAHs molecules to diffuse into the buck structure, and the shape and size of the pores lead to shape- and size-selectivity over the guests. The precolumn packed with the coordination polymer was shown to be promising for solid-phase extraction of PAHs in environmental samples with subsequent HPLC separation and UV detection. With extraction of 50 ml of sample solution, the enhancement factors for the PAHs studied ranged from 200 to 2337, depending on the shape, size and hydrophobic property of the PAHs. The detection limits (S/N = 3) of 2-14 ng l(-1) and the sample throughput of 3 samples h(-1) were obtained. The developed method was applied to the determination of trace PAHs in a certified reference material (coal fly ash) and local water samples.     

New approach for screening polycyclic aromatic hydrocarbons in water samples

For the first time, solid-phase extraction (SPE) has been combined to room-temperature phosphorimetry (RTP) to determine the 16 polycylic aromatic hydrocarbons related as major pollutants by the US Environmental Protection Agency (EPA). These include naphthalene, anthracene, acenaphthylene, acenaphthene, fluorene, fluoranthene, benzo(a)anthracene, benzo(k)fluoranthene, benzo(b)fluoranthene, benzo(a)pyrene, indeno(1,2,3-cd)pyrene, pyrene, chrysene, phenanthrene, benzo(g,h,i)perylene and dibenzo(a,h)anthracene. The pre-concentration factor obtained by SPE, combined with the sensitivity of RTP, resulted in calibration curves with linear dynamic ranges at the parts-per-billion level (ng ml(-1)). The limits of detection were estimated at the parts-per-trillion level (pg ml(-1)). Several pollutants usually encountered in water samples were tested for interference. These included polychlorinated biphenyls, pesticides, and volatile organic compounds. As a result of the appropriate combination of excitation wavelength (330 nm) and phosphorescence enhancers (0.1 M TlNO(3) and 0.05 M sodium dodecyl sulfate, SDS), no interference was observed. The results demonstrate the potential of SPE-RTP for screening polycyclic aromatic hydrocarbons (PAHs) in environmental waters.     

Coupling micelle-mediated extraction using mixtures of surfactants and fluorescence measurements with a fiber-optic for the screening of PAHs in seawater

The screening of PAHs from seawater samples using cloud-point extraction (CPE) as a step prior to their determination by fluorescence measurements with a fiber-optic is proposed. The CPE is carried out with the nonionic surfactants mixture POLE and Brij 30. The fluorescence measurement parameters were optimized, allowing selection of benzo(a)pyrene (B(a)Py) and benzo(k)fluoranthene (B(k)Ft) as the target analytes for the screening. The reproducibility of the whole screening system, expressed as relative standard deviation, was 9.0% for B(a)Py and 12.1% for B(k)Ft (both for n = 7). The reliability of the method was established at five concentrations for B(a)Py (between 0.5 and 3.3 times the detection limit: 0.31 ng ml(-1)) and at three concentrations for B(k)Ft (between 0.6 and 2.5 times the detection limit: 0.56 ng ml(-1)). The resolution of binary mixtures of these PAHs at different levels of concentration, and a study of the interferences with the rest of the PAHs were also carried out.     

Time-resolved laser-induced fluorimetry for screening polycylic aromatic hydrocarbons on solid-phase extraction membranes

The potential of solid-phase extraction (SPE) time-resolved laser-induced fluorimetry (TRLIF) is evaluated for screening polycyclic aromatic hydrocarbons (PAHs) in aqueous samples. Octadecyl membranes are used with the dual purpose of extracting the pollutants from the water sample and serving as the solid substrate for fluorescence detection. Excitation of fluorescence is performed with a Nd:YAG pumped tunable dye laser pumped with a pulsed source for time-resolving spectral interference. Wavelength time matrices (WTMs) and real time-resolved fluorescence spectra are recorded with a pulsed delay generator, a spectrograph and an intensified charge-coupled device (ICCD). In comparison to SPE solid-matrix luminescence (SML) with conventional instrumentation, this approach provides better limits of detection (LOD) and selectivity. The improvement in LOD is of one order of magnitude, reaching the parts-per-trillion level with 10 ml of water sample. The improvement in selectivity allows the direct determination of target compounds in complex samples. The direct determination of benzo[a]pyrene from a spiked river water sample of unknown composition is presented.     

Flow-through sensor based on derivative synchronous fluorescence spectrometry for the simultaneous determination of pyrene,benzo(e)pyrene and benzo(ghi)perylene in water

A flow-through/first derivative synchronous spectrofluorimetric sensor for the determination of PAH has been described. This sensor has been used for the simultaneous determination of PAH mixtures (pyrene, benzo(e)pyrene and benzo(ghi)perylene). Linear calibration ranges between 10 and 500 ng/ml with acceptable precision (repeatability, expressed as relative standard deviation, smaller than 4.6%, and sampling frequency of 12 h(-1)) have been obtained. The method has been applied to the determination of the target analytes in spiked water samples with excellent results (recoveries between 94 and 108%).     

Fast sequential injection determination of benzo[A]pyrene using variable angle fluorescence with on-line solid-phase extraction

A methodology for the analysis of drinking water for one of the most potent carcinogenic agents known; benzo[a]pyrene (BaP), in the presence of other interfering PAHs is presented. The methodology described is based on the sequential injection analysis of the sample on to a microcolumn (containing 5 mg of C18) where extraction and preconcentration of BaP takes place, followed by elution of BaP with 1 ml of 1,4-dioxane and subsequent detection by using variable angle fluorescence. The advantages of the method include the small amount of stationary phase employed together with the possibility of re-using the phase in order to carry out a large number of injections without the need for column re-packing. Also noteworthy is the small volume of 1,4-dioxane used to elute the BaP retained on the column and the small sample volumes required (9-10 ml) for achieving detection limits at the ng l-1 level. Thus, a methodology for BaP determination is obtained which complies with the requirements of the 98/83/EC Directive which fixes a maximum admissible concentration for this pollutant in waters for public consumption of 10 ng l-1. The variable angle spectra obtained are further processed by means of the multiple linear regression technique. The detection limit for BaP is 2.5 ng l-1, and the linear range is between 7.5 and 280 ng l-1.     

Sensitive and selective determination of metabolically formed trans-dihydrodiols and phenols of benzo[a]pyrene in water and urine samples by HPLC with amperometric detection

A method has been developed to separate hydroxylated metabolites of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene, i. e. trans-4,5-, 7,8-, 9,10-dihydrodiol and 1-, 3-, 7-, and 9-phenol, by HPLC with amperometric detection employing an isocratic methanol/water eluent (70:30, v/v) containing 0.5 g/L sulfuric acid and 1 g/L lithium perchlorate. Compared with the usually applied fluorescence (λ_ex = 265 nm, λ_em = 460 nm) and ultraviolet (λ = 265 nm) detection, the amperometric technique is about 2–12 times more sensitive for the determination of all metabolites investigated. The method was applied to the determination of the seven metabolites of benzo[a]pyrene in different water samples and in urine after solid-phase extraction (SPE). The results obtained by HPLC with amperometric detection after SPE enrichment from an aqueous extract of a soil sample and from the urine of a rat intragastrically treated with benzo[a]pyrene agreed well with the values determined with fluorescence and/or UV detection.     

Flow-through sensor based on derivative synchronous fluorescence spectrometry for the simultaneous determination of pyrene, benzo(e)pyrene and benzo(ghi)perylene in water

A flow-through/first derivative synchronous spectrofluorimetric sensor for the determination of PAH has been described. This sensor has been used for the simultaneous determination of PAH mixtures (pyrene, benzo(e)pyrene and benzo(ghi)perylene). Linear calibration ranges between 10 and 500 ng/ml with acceptable precision (repeatability, expressed as relative standard deviation, smaller than 4.6%, and sampling frequency of 12 h^–1) have been obtained. The method has been applied to the determination of the target analytes in spiked water samples with excellent results (recoveries between 94 and 108%).     

Optimization of the matrix solid-phase dispersion sample preparation procedure for analysis of polycyclic aromatic hydrocarbons in soils: comparison with microwave-assisted extraction

A fast and simple preparation procedure based on the matrix solid-phase dispersion (MSPD) technique is proposed for the first time for the isolation of 16 polycyclic aromatic hydrocarbons (PAHs) from soil samples. Naphthalene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, chrysene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, benzo[g,h,i]perylene, and indeno[1,2,3-c,d]pyrene were considered in the study. Extraction and clean-up of samples were carried out in a single step. The main parameters that affect extraction yield, such as dispersant, type and amount of additives, clean-up co-sorbent and extractive solvent were evaluated and optimized. The addition of an alkali solution in MSPD was required to provide quantitative recoveries. Analytical determinations were carried out by high performance liquid chromatography (HPLC) with fluorescence detection. Quantification limits (between 0.01 and 0.6 ng g(-1) dry mass) were well below the regulatory limits for all the compounds considered. The extraction yields for the different compounds obtained by MSPD were compared with the yields obtained by microwave-assisted extraction (MAE). To test the accuracy of the MSPD technique, the optimized methodology was applied to the analysis of standard reference material BCR-524 (contaminated industrial soil), with excellent results.     

Supercritical fluid extraction of polycyclic aromatic hydrocarbons from liver samples and determination by HPLC-FL

An extraction/clean-up procedure by SFE was developed for isolating PAHs from liver samples for subsequent HPLC-FL determination of ten PAHs in the enriched extract. Recoveries (90-115%) and RSD % (< or =7.7) were satisfactory. When applied to 11 samples of bird of prey (Tyto alba) protected species and classified of special interest, from the Galicia (Northwest to Spain), benzo[ghi]perylene and indeno[1,2,3-cd]pyrene were undetectable; chrysene and benzo[a]pyrene are only detected in one sample; benzo[a]anthracene and benzo[k]fluoranthene are only quantified in one sample and benzo[b]fluoranthene in two samples. The other PAHs, anthracene, fluoranthene and pyrene are present in almost all the samples.     

Isotope dilution determination of polycyclic aromatic hydrocarbons in olive pomace oil by gas chromatography-mass spectrometry

A gas chromatographic (GC) method with mass spectrometry detection (MS) for the determination of eight polycyclic aromatic hydrocarbons (PAHs) in olive pomace oil has been developed. The oil was diluted with n-pentane and extracted by liquid-liquid partition with dimethyl sulphoxide (DMSO). After water addition and back-extraction with cyclohexane, a thin-layer chromatography on silica gel was performed as a further purification step. The PAHs spot was scraped off from the plate and the final extract was concentrated and analysed by GC-MS in full scan mode. The eight PAHs under investigation were determined in the presence of the corresponding labelled compounds added as internal standards to the sample at the beginning of the analytical process. The identified PAHs were then quantified by the isotope dilution methodology assuring the compensation of the concentration of each analyte for any variation in the sample preparation. The method precision was satisfactory with relative standard deviation (R.S.D.) values in the range 3.6-12.7% for all PAHs. The average recovery rates ranged from 69.0 to 97.5%. Accuracy was also calculated for benzo[k]fluoranthene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene and benzo[ghi]perylene by analysing a certified reference material (CRM 458, coconut oil) with adequate results. All response curves exhibited a linear fit from 0.1 to 10 microg ml(-1) and the determination coefficients R2 were better than 0.9942. The limits of detection (0.1-0.4 microg kg(-1)) were acceptable when compared with the maximum permitted limit of 2 microg kg(-1) for each of the eight considered PAHs and 5 microg kg(-1) for the sum of the eight PAHs established by the Italian legislation. Measurement uncertainty was finally calculated identifying and quantifying the uncertainty components of the analytical process. The relative expanded uncertainties (Uc), expressed as percent values were in the range 8.5-11.4% thus appropriate for residues quantification in the range of concentrations considered in the present study.     

Determination of Benzo(a)Pyrene and other Polynuclear Aromatic Hydrocarbons in Airborne Particulate Material by Ultrasonic Extraction and Reverse Phase High Pressure Liquid Chromatography

Abstract

Ultrasonic extraction of airborne particulate material on Hi-Vol filters is described. Almost all of the polar compounds are removed during the extraction by adsorption on the surface of the shredded glass fibers and controlled pore glass powder (CPG). The non-polar polynuclear aromatic hydrocarbons (PAH) in the extract are separated at room temperature by high pressure liquid chromatography (HPLC) on reverse phase Vydac using acetonitrile:water (70:30 v/v) as the chromatographic solvent. There is baseline separation of benzo(a)pyrene (BaP), benzo(k)fluoranthene (BkFt) and of benzo(e)pyrene (BeP), benzo(b)fluoranthene and perylene, the latter three present in one band. Extracts of airborne particulates show the same peaks. BaP elutes in approximately 14 minutes. Precision and accuracy measurements indicate full recovery of PAH and good extraction reproducibility. The detection limit of BaP at F 290/389 is less than 10 pg. Total analysis time is approximately 1 1/2 hr, most of which is waiting time.     

Cigarette filter as sorbent for on-line coupling of solid-phase extraction to high-performance liquid chromatography for determination of polycyclic aromatic hydrocarbons in water

An on-line solid-phase extraction (SPE) protocol using the cigarette filter as sorbent coupled with high-performance liquid chromatography (HPLC) was developed for simultaneous determination of trace naphthalene (NAPH), phenanthrene (PHEN), anthracene (ANT), fluoranthene (FLU), benzo(b)fluoranthene (BbF), benzo(k)fluoranthene (BkF), benzo(a)pyrene (BaP), and benzo(ghi)perylene (BghiP) in water samples. To on-line interface solid-phase extraction to HPLC, a preconcentration column packed with the cigarette filter was used to replace a conventional sample loop on the injector valve of the HPLC for on-line solid-phase extraction. The sample solution was loaded and the analytes were then preconcentrated onto the preconcentration column. The collected analytes were subsequently eluted with a mobile phase of methanol-water (95:5). HPLC with a photodiode array detector was used for their separation and detection. The detection limits (S/N = 3) for preconcentrating 42 mL of sample solution ranged from 0.9 to 58.6 ng L(-1) at a sample throughput of 2 samples h(-1). The enhancement factors were in the range of 409-1710. The developed method was applied to the determination of trace NAPH, PHEN, ANT, FLU, BbF, BkF, BaP and BghiP in local river water samples. The recoveries of PAHs spiked in real water samples ranged from 87 to 115%. The precisions for nine replicate measurements of a standard mixture (NAPH: 4.0 microg L(-1), PHEN: 0.40 microg L(-1), ANT: 0.40 microg L(-1), FLU: 2.0 microg L(-1), BbF: 1.6 microg L(-1), BkF: 2.0 microg L(-1), BaP: 2.0 microg L(-1), BghiP: 1.7 microg L(-1)) were in the range of 1.2-5.1%.