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     

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.     

Mass spectrometric structure determination of metabolites of 3-[2-(N,N-dimethylaminomethyl)phenylthio]phenol

Metabolites of 3-[2-(N,N-dimethylaminoethyl)phenylthio]phenol (I) were isolated from the urine of rats, mice, rabbit and dog and from the faeces of rats by extraction and thin-layer chromatography. From the mass spectrum of I and using characteristics mass shifts in the spectra of metabolites, the structures of four metabolites were determined, whereas for two other isomeric metabolites the structures were resolved by means of IR spectra. Conclusions regarding the structures derived from the spectra were confirmed by comparing them with synthetic standards. Generally, metabolic changes of I can be characterized by demethylation, hydroxylation combined with methylation, S-oxidation and by a combination of these metabolic reactions.     

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.     

Detection of benzo[a]pyrene sulfate and glucuronide conjugates in cell culture medium by directly coupled microbore high-performance liquid chromatography-fast atom bombardment mass spectrometry.

An improved method is described for detecting glucuronide and sulfate conjugates of benzo[a]pyrene in medium from cell cultures treated with benzo[a]pyrene. This method is based on a microbore high-performance liquid chromatograph directly coupled to a high-resolution continuous-flow fast atom bombardment mass spectrometer. Sulfate and glucuronide conjugates, as well as some structural isomers of glucuronide conjugates, were fully separated by the reversed-phase microbore high-performance liquid chromatography conditions used in this study. Since the method does not rely on the use of radiolabeled materials, it may be used to detect conjugates of a wide variety of hydrocarbons. The high sensitivity and selectivity of the method were demonstrated by detecting conjugates in the media of cell cultures derived from mice, hamsters and humans.     

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.     

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。     

改进的QuEChERS-液相色谱法检测蔬菜中的苯并(α)芘

采用改进的QuEChERS,对蔬菜样品进行提取、净化,用高效液相色谱仪/荧光检测器检测,建立了蔬菜中苯并(α)芘的测定方法.样品用乙腈提取,PSA+中性Al2O3小柱净化,以10mL正己烷-乙腈(95:5,体积比)洗脱小柱,外标法定量.苯并(α)芘的含量在1.0～20.0μg/kg范围内与峰面积呈良好线性,相关系数(r...     

Validation of a method for extraction of polycyclic aromatic hydrocarbons from sewage sludge and analysis by GC-MS

In the present study, the solid–liquid extraction with low temperature purification was validated for the determination of 16 polycyclic aromatic hydrocarbons from sewage sludge by gas chromatography-mass spectrometry. Recoveries ranged 70–114% for naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene and chrysene, while the compounds benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene, dibenzo[a,h]anthracene and benzo[g,h,i]perylene showed recoveries of between 40 and 70%. The relative standard deviation was less than 13% for all of the compounds. Negative matrix effect was observed on the 10 compounds with less retention time in the chromatographic analysis and positive matrix effect noticed on the others. The limits of quantification were from 2 to 20 μg kg^?1, about 30 times less than the maximum residue limit allowed in sludge by the European Union. The validated method produced quantification of 11 PAHs in one sludge sample at concentrations ranging 20–2000 μg kg^?1.     

超高效液相色谱-大气压化学电离-三重四极杆质谱联用法快速测定食品中苯并[a]芘

建立了快速检测食品中苯并[a]芘的超高效液相色谱-三重四极杆质谱联用(UPLC-MS/MS)分析方法。样品用正己烷提取后,经分子印迹固相萃取柱净化,以甲醇和水作为流动相进行梯度洗脱,在XBridge BEH C18柱上实现分离,大气压化学电离(APCI)-三重四极杆质谱正离子MRM方式检测,以苯并[a]芘-d12作为内标的稳定同位素稀释法定量。方法的线性范围为0.07~50μg/kg,定量限为0.07μg/kg。平均加标回收率为86%~104%,相对标准偏差为2.3%~14%。该方法灵敏、准确,适用于食品中苯并[a]芘的测定,已应用于实际样品的检查。     

ENHANCED GROWTH AND EXPERIMENTAL METASTASIS OF CHEMICALLY INDUCED TUMOR IN ULTRAVIOLET IRRADIATED SYNGENEIC MICE

Abstract— Recent studies have shown that ultraviolet (UV) irradiation induces a systemic effect which enhances subsequent tumor induction by benzo[a]pyrene in a manner which is dependent on the dose of benzo[a]pyrene. The present study was designed to test whether UV-B irradiation renders mice susceptible to subcutaneous or intravenous injection of a regressor tumor induced by benzo[a]pyrene. The sources of UV-B irradiation were banks of 6 Westinghouse FS-40 sunlamps, situated 20 cm above the mouse cages. Female BALB/cAnNHsd received five 30-min dorsal UV-B radiation treatments per week for 12 weeks, resulting in a total dose of approx. 6.4 × 10⁵ J m^-2. Two to seven days after termination of UV treatments, syngeneic regressor tumor cells (BP2) induced by benzo[a]pyrene were injected subcutaneously or intravenously into irradiated mice and unirradiated controls. By 38 days post subcutaneous implantation, 24/30 and 3/30 BP2 implants were detectable in the irradiated and unirradiated mice, respectively. Ultraviolet irradiated mice were also unable to reject lung colonies resulting from intravenous administration of BP2 cells, although they were rejected by unirradiated mice. The mean number of lung colonies per mouse was 16- to 35-fold greater in UV irradiated mice than in unirradiated controls, at 14 to 17 days post injection. Thus, UV irradiation rendered mice, with no known exposure to benzo[a]pyrene, susceptible to a subcutaneous or intravenous injection of a regressor tumor induced by benzo[a]pyrene.     

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.     

Synthesis of Oxidized Derivatives of 10-Deuterobenzo[A]Pyrene

A series of oxidized derivatives of 10-deuterobenzol[a]pyrene (10-D-BaP) were synthesized. Oxidation of 10-D-BaP with sodium dichromate dihydrate yielded the 1,6-, 3,6-, and 6,12-quinones of 10-D-Bap. Upon electrophilic aromatic substitution of 10-D-BaP with sodium nitrate, N-bromosuccinimide, and N-chlorosuccinimide we were afforded 6-nitro-10-D-BaP, 6-bromo-10-D-BaP, and 6-chloro-10-D-BaP, respectively, in high yields. The Vilsmier-Haack reaction of 10-D-BaP provided 6-formyl-10-D-BaP which upon reduction with sodium borohydride, yielded 6-hydroxymethyl-10-D-BaP. Reduction with hydrazine in n-amyl alcohol gave 6-methyl-10-D-BaP. Reduction of 6-nitro-10-D-BaP with hydrazine and Pd/C yielded 6-amino-10-D-BaP. The synthesized compounds, which are known metabolites of benzo[a]pyrene and are useful substrates for metabolism, are important compounds for mechanistic study of the chemical carcinogenesis by benzo[a]pyrene.     

High-performance liquid chromatographic method for the determination of polycyclic hydrocarbon metabolite distributions

Analytical separations and quantification of eight metabolites of benzo[a]pyrene, nine metabolites of 7-methylbenz[c]acridine and nine metabolites of dibenz[a,j]acridine have been carried out on two reversed-phase high-performance liquid chromatographic systems using 3- and 10-microns C8 packings. Quantitative results for the distributions of primary metabolites for the two systems closely agreed and showed that up to 70% savings in chromatographic time could be achieved with the 3 micron particle size columns.     

Error propagation as a factor in selection of measurement intervals for the determination of polycyclic aromatic hydrocarbons by second-derivative spectrofluorimetry

The most suitable wavelength intervals were selected for the determination of 4 polycyclic aromatic hydrocarbons (PAHs; benzo[g,h,i]perylene, dibenzo[a,h]anthracene, pyrene, and triphenylene) in very complex mixtures of 11 PAHs: anthracene, benz[a]anthracene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[g,h,i]perylene, benzo[k]fluoranthene, chrysene, dibenz[a,h]anthracene, phenanthrene, pyrene, and triphenylene. The multiple linear regression algorithm was applied to measurements made in several wavelength intervals previously selected on the basis of sensitivity and minimum number of interfering compounds. Of the different models obtained, those displaying minimum error propagation in the analytical result were selected. By applying the models proposed in this study, we precisely and accurately determined benzo[g,h,i]perylene, dibenz[a,h]anthracene, pyrene, and triphenylene in complex mixtures--a feat that could not be achieved by the use of constant-wavelength spectrofluorimetry in combination with second-derivative techniques.     

Synthesis of the o-quinones and other oxidized metabolites of polycyclic aromatic hydrocarbons implicated in carcinogenesis

Efficient new syntheses of the o-quinone derivatives of benzo[a]pyrene (BPQ), 7,12-dimethylbenz[a]anthracene (DMBAQ), and benz[a]anthracene (BAQ), implicated as active carcinogenic metabolites of the parent polycyclic aromatic hydrocarbons (PAHs), are reported. These PAH quinones also serve as starting compounds for the synthesis of the other active metabolites of these PAHs thought to be involved in their mechanism(s) of carcinogenesis. The latter include the corresponding o-catechols, trans-dihydrodiols, and the corresponding anti- and syn-diol epoxides.     

The development of solid-surface fluorescence characterization of polycyclic aromatic hydrocarbons for potential screening tests in environmental samples

This paper presents the characterization of polycyclic aromatic hydrocarbons (PAHs) in solid-surface fluorescence as the first step for obtaining new optical sensors for PAHs screening. The fluorescence properties of the EPA-PAHs (naphthalene, acenaphthene, acenaphthylene, 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) on five types of solid-surfaces were evaluated. The experimental variables (pH and percentage of organic solvent in samples) were studied, obtaining different possibilities for making individual sensors for some of these PAHs and the best conditions for developing sensors for PAH screening were also studied.     

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.     

Determination of propiverine and its metabolites in rat samples by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometric (LC-MS-MS) method was developed and validated for the determination of the anticholinergic and antimuscarinc drug propiverine and eight of its metabolites in serum, urine, faeces and different tissue samples of rats. Samples containing propiverine and its metabolites in serum and urine and in the supernatants of faeces and tissue homogenates were extracted and cleaned up using an automated solid phase extraction (SPE) method. An external calibration was used. The analytes were measured employing the multiple reaction monitoring mode (MRM). A sufficient response over the range of 10-1000 ng/ml was demonstrated. The lower limit of quantification of the nine substances was 10 ng/ml. The presented method is suitable for pharmacokinetic or toxicokinetic studies. To look for additional unknown metabolites, the LC-MS-MS system operated in the precursor ion mode using typical product ions of propiverine and of its metabolites. With the help of the chromatographic behaviour and typical fragment ions of the unknown metabolites, it was possible to elucidate their structure. Five until now unknown metabolites were found in the urine and faeces samples. However, without reference substances, a quantification of these analytes was not possible.