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.     

Aromatic hydrocarbon metabolites in fish: automated extraction and high-performance liquid chromatographic separation into conjugate and non-conjugate fractions

An automated extractor-concentrator was used to extract metabolites of naphthalene, 2,6-dimethylnaphthalene, and benzo[a]pyrene from serum, bile and liver homogenate of rainbow trout (Salmo gairdneri). The extracts were analyzed by reversed-phase high-performance liquid chromatography (HPLC) with fluorescence detection. Recoveries of naphthalene and 2,6-dimethylnaphthalene metabolites from all matrices were generally greater than 90%; however, the recoveries of benzo[a]pyrene metabolites from serum ranged from 37-99%. In addition, conjugated metabolites of polycyclic aromatic hydrocarbons (PAHs) were separated from non-conjugated metabolites and parent PAHs by using two diol columns with normal-phase HPLC. The extraction and separation techniques were also applied to isolate metabolites in samples from fish fed 2,6-dimethylnaphthalene.     

Comparison of liquid chromatography-mass spectrometry interfaces for the analysis of polar metabolites of benz[a]pyrene

 The performance of two liquid chromatography-mass spectrometry (LC/MS) interfacing techniques, thermospray (TSP) and atmospheric pressure chemical ionization (APCI), for the analysis of benzo[a]pyrene (BaP) metabolites (hydroxy, epoxy and quinone derivatives) was compared. Interface and detection parameters such as source temperature, eluent composition or flow rate were optimized using negative ion mode. In TSP, the main ions are mostly [M]^-, [M−H₂O]^- or [M+CH₃COO]^-, whereas APCI gives mainly the [M]^- and [M−H]^- ions. Quantification was carried out by flow injection. Calibration graphs were linear in the range of 10 ng to 1000 ng in TSP and 0.1 ng to 10 ng in APCI. Detection limits were in the range of 1 ng to 20 ng in TSP and 0.002 ng to 0.2 ng in APCI. The presence of BaP-1,6-dione, BaP-3,6-dione, and BaP-6,12-dione was confirmed in environmental samples of air particulate matter. Received: 6 January 1997/Accepted: 18 April 1997     

Comparison of liquid chromatography-mass spectrometry interfaces for the analysis of polar metabolites of benz[a]pyrene

 The performance of two liquid chromatography-mass spectrometry (LC/MS) interfacing techniques, thermospray (TSP) and atmospheric pressure chemical ionization (APCI), for the analysis of benzo[a]pyrene (BaP) metabolites (hydroxy, epoxy and quinone derivatives) was compared. Interface and detection parameters such as source temperature, eluent composition or flow rate were optimized using negative ion mode. In TSP, the main ions are mostly [M]^-, [M−H₂O]^- or [M+CH₃COO]^-, whereas APCI gives mainly the [M]^- and [M−H]^- ions. Quantification was carried out by flow injection. Calibration graphs were linear in the range of 10 ng to 1000 ng in TSP and 0.1 ng to 10 ng in APCI. Detection limits were in the range of 1 ng to 20 ng in TSP and 0.002 ng to 0.2 ng in APCI. The presence of BaP-1,6-dione, BaP-3,6-dione, and BaP-6,12-dione was confirmed in environmental samples of air particulate matter. Received: 6 January 1997/Accepted: 18 April 1997     

Ozonation of pyrene and benzo[a]pyrene in silica and soil –14C-mass balances and chemical analysis of oxidation products as a first step to ecotoxicological evaluation

Ozonation of polycyclic aromatic hydrocarbons (PAHs) in soil is a process that can be used for in-situ soil remediation or in combination with bioremediation techniques. First steps to a comprehensive ecotoxicological evaluation of this method is done by ozonation of radioactively labeled (¹⁴C) pyrene and benzo[a]pyrene in a silty soil (LUFA 2.2) under mass-balancing conditions and GC-MS analysis of aromatic ozonation products. ¹⁴C-Mass-balances for pyrene and benzo[a]pyrene (b[a]p) in soil showed that, apart from ¹⁴CO₂ formation, considerable percentages of both PAHs are oxidized to water soluble substances (20–30%) or to non-extractable or bound residues (10% for pyrene, 30% for b[a]p). TLC and GC-MS analysis of ozonation products extractable from artificially contaminated silica and soil by organic solvents revealed a large number of aromatic substances. PAH-quinones and ten ring fission products with formyl- and carboxy-groups of both pyrene and b[a]p could be identified. Received: 11 March 1997 / Accepted: 25 April 1997     

固相萃取-高效液相色谱-荧光法测定方便面和烤肠中的苯并[a]芘

建立了固相萃取-高效液相色谱-荧光法检测方便面和烤肠中苯并[a]芘的方法。采用正己烷作为提取溶剂,经苯并[a]芘专用固相萃取柱HiCapt Benzo富集净化,高效液相色谱-荧光法对样品中苯并[a]芘进行分离分析。苯并[a]芘的质量浓度在0.5~20.0μg/kg范围内与色谱峰面积呈良好的线性关系,相关系数R2为0.9997。方便面和烤肠中苯并[a]芘的加标回收率分别为92.2%~98.3%和95.9%~97.9%,日内和日间相对标准偏差分别为3.34%~5.01%和2.11%~4.07%。与传统方法相比,该方法快速简单、有机溶剂消耗少,在油炸烟熏食品的苯并[a]芘分析中具有较大应用前景。     

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.     

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.     

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.     

Studies on the chromatographic fractionation of metabolites of benzo[a]pyrene in faeces and urine from germfree and conventional rats

Rats, germfree and conventional, were dosed with 14C-labelled benzo[a]pyrene. Faeces and urine were collected. Metabolites in faeces were effectively extracted with a new method using a combination of solvents and solid sorbents. Metabolites in urine were extracted with octadecylsilane-bonded silica. The metabolites were fractionated into groups by chromatography on a cation exchanger (SP-LH-20 or SP-Sephadex C-25) and an anion exchanger (TEAP-LH-20). Some of the groups were further purified by column chromatography and analysed by HPLC and TLC. The analyses show a complex pattern of metabolism. A large part of the metabolites (9-24% depending on animal type and route of excretion) had amphoteric properties, e.g. like glutathione and cysteine conjugates. The abundance of conjugates sensitive to beta-glucuronidase and sulphatase was low. The relative amount of acidic conjugates in faeces was much higher in the germfree than in the conventional rats indicating the influence of the intestinal flora on the metabolism. The results support the view that the mercapturic acid pathway is a quantitatively important metabolic route for benzo[a]pyrene in rats. The methods of extraction and group fractionation were designed to be generally applicable to the analysis of lipophilic xenobiotics and their metabolites.     

Analysis of N7-(benzo[a]pyrene-6-yl)guanine in urine using two-step solid-phase extraction and isotope dilution with liquid chromatography/tandem mass spectrometry

Analysis of urinary N7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua), a DNA adduct induced by benzo[a]pyrene, may serve as a risk-associated biomarker for exposure to polyaromatic hydrocarbons (PAHs). In this study a highly sensitive and specific analytical method, incorporating on-line sample preparation coupled with isotope-dilution liquid chromatography and tandem mass spectrometry (LC/MS/MS), was developed to quantitate this adduct in human urine. In order to achieve accurate quantitation, 15N5-labeled BP-6-N7Gua was synthesized to serve as the internal standard, and a two-step solid-phase extraction (SPE) procedure using C8 and SCX cartridges was used for sample cleanup. BP-6-7-N7Gua was analyzed using positive ion LC/MS/MS operated in multiple reaction monitoring (MRM) mode. The [M+H]+ ions at m/z 402 and 407 and the common fragment ion of [M+H]+ at m/z 252 were monitored for quantification. The recovery of this analyte after two-step SPE was 90%, and the limit of detection was 2.5 fmol/mL in 10 mL of urine. This highly specific and sensitive method for BP-6-N7Gua in urine may be applied to assess exposure to PAHs in coke-oven workers for future molecular epidemiology studies on health effects of PAHs.     

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.     

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。     

固相萃取和气相-质谱法测定主流烟气中苯并[a]芘的研究

 提出了一种用于测定主流烟气中痕量苯并 [a]芘的方法。以甲醇 正庚烷萃取体系初步处理主流烟气中的总粒相物 (TPM) ,接着以KOH甲醇溶液数毫升洗涤正庚烷萃取液 ,再将正庚烷萃取液适当浓缩后经过酸化的硅胶固相萃取小柱 ,流出液用N2 吹干后以乙酸乙酯 2 0 0 μL定容 ,对苯并 [a]芘进行选择离子监测方式下的气相 质谱法定量测定。方法的分离效果及重现性好 ,可用于复杂体系中痕量组分苯并 [a]芘的定量测定。     

Rapid SPE-HPLC determination of the 16 European priority polycyclic aromatic hydrocarbons in olive oils

Polycyclic aromatic hydrocarbons (PAHs) are a large class of organic compounds. It has been established that the main source of exposure to these compounds for human beings is through food, particularly fats and oils, due to the lipophilic nature of these polycyclic compounds. The aim of this work was to optimise and validate a method involving SPE and HPLC for rapid determination of the 16 European Union (EU) priority PAHs (required by the Recommendation 2005/108/EC) in vegetable oils. Two spectrofluorometric detectors and a UV-Visible detector in series were used to identify and quantify the target compounds. Linearity, recoveries, LOD, and LOQ were found to be in agreement with the performance criteria for benzo[a]pyrene (BaP) analysis as required by the Commission Directive 2005/10/EC, and satisfactory for all the compounds of interest, except for cyclopenta[c,d]pyrene, which presented a very low signal in the UV. Optimised chromatographic conditions for the separation of 25 PAHs, comprising both EPA and EU priority PAHs plus benzo[e]pyrene and benzo[b]chrysene, have been also proposed.     

高效液相色谱法测定生活饮用水中的苯并[a]芘

建立高效液相色谱二极管阵列检测器检测生活饮用水苯并[a]芘的测定方法。采用C18反相色谱柱(150mm×4.6 mm,5μm),在流动相为甲醇–水(体积比为90∶10)、流量1.0 mL/min、检测波长295 nm、柱温35℃、进样体积20μL的条件下测定生活饮用水中苯并[a]芘。该方法检出限为6 ng/L,线性范围0～100 ng/mL,加标回收率为88.1%～93.4%,测定结果的相对标准偏差为1.06%(n=9)。该法样品预处理简单,分离度高,分析时间短,适用于生活饮用水中苯并[a]芘的准确定性定量测定。     

Two Stage Solid Phase Extraction with Subsequent HPLC Isolation for Structure Elucidation of Major Metabolites of (+)-(R)-2-{3-(benzo[1,4]dioxan-2-yl-methylamino)-1-propyl}-3(2H)-pyridazinone (GYKI-16084)

GYKI-16084 – (+)-(R)-2-{3-(benzo[1,4]dioxan-2-yl-methylamino)-1-propyl}-3(2H)-pyridazinone hydrochloride – is a new drug candidate for the treatment of benign prostatic hyperplasia. In our study the major metabolites formed in the rat and dog were isolated from dog and rat urine, then their structures were elucidated by means of MS and NMR. A two stage solid phase extraction (SPE) procedure and a semi-preparative HPLC method were developed utilizing various mechanisms of separation. The major metabolites proved to be isomeric glucuronides of the benzodioxane moiety hydroxylated at positions 6 or 7 and {2-(2-carboxyethyl)-3(2H)-pyridazinone}.     

全自动固相萃取-气相色谱-串联质谱法测定卷烟主流烟气中的3种多环芳烃

以全自动固相萃取技术净化主流烟气萃取液,建立了卷烟主流烟气中苯并［a］芘、苯并［a］蒽和屈艹 3种多环芳烃的气相色谱-串联质谱(GC-MS/MS)测定方法。以吸烟机抽吸卷烟,并以剑桥滤片捕集卷烟主流烟气,然后以含氘代苯并［a］芘内标的环己烷溶液萃取滤片,萃取液经全自动固相萃取仪净化后以GC-MS/MS分离检测。结果表明,苯并［a］芘、苯并［a］蒽和屈艹 的检出限分别为0.05、0.16和0.23 ng/cig,回收率为91.5%～102.1%,相对标准偏差(RSD)均小于5%。该方法的自动化程度高、操作简便、检出限低、重复性好,适用于卷烟主流烟气中苯并［a］芘、苯并［a］蒽 3种多环芳烃释放量的检测。     

Humic acid-bonded silica as a novel sorbent for solid-phase extraction of benzo[a]pyrene in edible oils

A novel solid-phase extraction (SPE) sorbent, humic acid-bonded silica (HAS), was prepared. Humic acids (HAs) were grafted onto silica matrices via an amide linkage between humyl chloride and the amido terminus of 3-aminopropyltrimethoxysilane (APTS)-silica gel. The resulting material was characterized by Fourier transform infrared spectrometer, elemental analysis, and nitrogen adsorption analysis. This sorbent exhibits an excellent adsorption capacity for some electron-abundant analytes owing to its peculiar structure. In this paper, we choose benzo[a]pyrene (BaP) in oil as a probe to validate the adsorption capacity of the material. Thus a fast, cheap and simple SPE method with humic acid-bonded silica cartridge for edible oil clean-up, followed by high-performance liquid chromatography (HPLC) with fluorescence detection was established. The effects of experimental variables, such as washing and elution solvents, and the amount of sorbents have been studied. The recoveries of BaP in edible oils spiked at 0.2-100 μg kg⁻¹ were in the range of 78.8-102.7% with relative standard deviations ranging between 1.3 and 9.3%; the limit of detection was -0.06 μg kg⁻¹.