Enrichment of benzo[a]pyrene in vegetable oils and determination by HPLC-FL

We have developed a simple method for the determination of the carcinogen Benzo[a]pyrene (BP) in vegetable oils. The method consists of extraction of the vegetable oil in acetonitrile, concentration to dryness in rotary evaporator and redissolution of the residue in hexane. The purification of the hexane extract was on Sep-Pack Silica Plus cartridges, and the determination of the BP in the isolated extract was by HPLC-FL. Detection and quantification limits were 0.23 and 0.32 μg kg⁻¹ of olive oil, respectively. Recovery (>93%) and RSD (<4%) were satisfactory. When applied to 18 oil samples, BP levels varied from not detected to 1.99 μg kg⁻¹.     

Characterization of antibodies against benzo[a]pyrene with thermodynamic and kinetic constants

Antibodies of a polyclonal antiserum against benzo[a]pyrene were characterized by determining thermodynamic and kinetic constants of the antigen–antibody reaction. Label-free binding assays with optical detection based on reflectometric interference spectroscopy were performed to determine these constants. Different evaluation methods for kinetic measurements were compared. Also, cross-reactivity against two other polycyclic aromatic hydrocarbons, chrysene and pyrene, was checked. The affinity constant between the antibodies and benzo[a]pyrene in homogeneous phase was determined to be K=(5.3±0.3)×10⁷ M⁻¹ which was in the middle of the usual range of antibody affinities. The association rate constant for the reaction at the surface was determined to be (3.8±0.9)×10⁵ M⁻¹ s⁻¹, the dissociation rate constant as (9.7±0.5)×10⁻³ s⁻¹. Different evaluation methods applied to the kinetic measurements led to the same results. This antiserum would be suitable for the selective determination of benzo[a]pyrene in concentrated samples.     

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.     

The solid-matrix phosphorescence of (±)-anti-dibenzo[a,l]pyrene diol epoxide-DNA adducts and benzo[e]pyrene

New solid-matrix phosphorescence (SMP) methods for (±)-anti-DB[a,l]PDE-DNA adducts and B[e]P were developed. The methods can be used to detect and characterize (±)-anti-DB[a,l]PDE-DNA adducts and B[e]P by employing SMP spectra, intensities, and lifetimes acquired with the heavy-atom salt, TlNO₃, on Whatman 1PS paper. With the SMP data, a number of photophysical parameters were calculated such as biexponential SMP decay curves, pre-exponential factors, and fractional contribution to SMP decay curves. The SMP results were compared with earlier SMP data for (±)-anti-BPDE-DNA adducts and tetrol I-1. The SMP results show that small molecular-weight compounds like B[e]P can be readily detected and characterized by SMP. For example, the limit of detection for B[e]P was 0.60 pmol. Comparison of the SMP properties of the (±)-anti-DB[a,l]PDE-DNA adducts with earlier SMP data for the (±)-anti-BPDE-DNA adducts showed major differences in the SMP spectra, intensities, and lifetimes. The methods developed are important for the comparison of the SMP properties of different diol epoxides of PAH bonded to DNA.     

The selective cleanup of complex matrices and simultaneous separation of benzo[a]pyrene by solid-phase extraction with MgO microspheres as sorbents

A new method for the selective cleanup of complex matrices and simultaneous separation of benzo[a]pyrene (BaP) was developed in this study. This method was based on solid-phase extraction (SPE) using magnesium oxide microspheres as sorbents, and it eliminated interferences from various impurities, such as lipids, sulphur, pigments, halobenzenes, polychlorodibenzo-p-dioxins and polychlorodibenzofurans. Several parameters, including the volume of rinsing and eluting solvents, the type of loading solvents and SPE sorbents, were optimized systematically. The capability for impurity removal was verified by gel permeation chromatography, gas chromatography, and liquid chromatography. Compared to commercial sorbents (silica gel, florisil and alumina), MgO microspheres exhibited excellent performance in the selective isolation of BaP and removal of impurities. The proposed method was applied to detect BaP in complex samples (sediments, soils, fish, and porcine liver). The limit of quantification (LOQ) was 1.04 ngL(-1), and the resulting regression coefficient (r(2)) was greater than 0.999 over a broad concentration range (9.5-7600 ngL(-1)). In contrast to traditional methods, the proposed method can give rise to higher recovery (85.1-100.8%) and better selectivity with simpler operation and less consumption of organic solvents (20-40 mL).     

Preliminary experiments for the analysis of tetrahydrotetrols of benz[a]anthracene and benzo[a]pyrene derived from their hemoglobin adducts using a coupled-column high-performance liquid chromatographic system

Improved technologies for the detection of polycyclic aromatic hydrocarbon adducts are required for human biomonitoring. Therefore, a coupled-column high-performance liquid chromatographic method, with system-integrated sample processing, has been developed and its applicability for determination of tetrahydrotetrols of polycyclic aromatic hydrocarbons in acid hydrolysates of human hemoglobin has been investigated. A novel column-switching technique applying ‘thermotransfer’ is used to separate tetrahydrotetrols of benzo[a]pyrene and benz[a]anthracene more efficiently. Derivatives of polycyclic aromatic hydrocarbons from blood hydrolysates are concentrated on a pre-column and then transferred to the analytical column by applying an electrical current to heat the solvent eluting the pre-column. This method allows for rapid and quantitative transfer of the analytes from the pre-column to the analytical column, after HPLC-integrated sample processing.     

Magnesium oxide microspheres as a novel solid‐phase extraction sorbent for the determination of benzo[a]pyrene in environmental water samples

Magnesium oxide microspheres were developed as a novel SPE sorbent for the determination of benzo[a]pyrene (BaP), one of the most potent carcinogenic agents, in environmental water samples. The parameters controlling the extraction efficiency, such as elution volume, flow rate, pH values, and breakthrough volume, were investigated in detail. Considering the facile preparation and satisfying recovery, a corresponding analytical method has been developed to determine the concentration of BaP in real tap water, river water, and seawater. The recoveries for the spiked BaP were excellent (94–101%).     

A selective method for the determination of benzo[a]pyrene in soil using porous graphitic carbon liquid chromatography columns

A back-flushing procedure using porous graphitic carbon (PGC) HPLC columns has been used successfully for the cleanup of soil samples for the determination of benzo[a]pyrene in ppb levels by an ODS-fluorescence HPLC column. The procedure was tested on nine random soil samples taken from an industrial area of the Kingdom of Bahrain. The mean percent recovery from the PGC column was 96% and the average coefficient of variation for the whole method was 5.2%.     

Chemical sensing of Benzo[a]pyrene using Corchorus depressus fluorescent flavonoids

Plant phytochemicals, such as flavonoids are in use for the development of optical biosensor. Benzo[a]pyrene (B[a]P), is a pervasive environmental and dietary carcinogen. A fluorescent assay is developed using plant isolated flavonoid for the detection of B[a]P. High content saponins are excluded from the flavonoid-containing methanolic extract of Corchorus depressus by implying reduction of silver ions by saponins resulting in formation of silver nanoparticles. Isolated plant flavonoids are used to develop a spectrofluorometric assay for the detection of B[a]P. Decrease in the flavonoid fluorescence intensity by B[a]P is found to be based on both static and dynamic quenching. Specificity of the assay for B[a]P was tested for other carcinogens belonging to different classes of compounds. Flavonoids-mediated sensing can be implied for the development of new generation of nanoparticle-based biosensors that can be more sensitive and less susceptible to external factors, such as temperature and humidity.     

Analysis of benzo[a]pyrene diol epoxide-DNA adducts by capillary zone electrophoresis- electrospray ionization-mass spectrometry in conjunction with sample stacking

Benzo[a]pyrene diol epoxide (BPDE) was reacted in vitro with (2'-deoxy)nucleotides and with calf thymus DNA. The modified DNA was enzymatically hydrolyzed to the 5'-monophosphate nucleotides using deoxyribonuclease I (DNA-ase I), nuclease P1 and snake venom phosphodiesterase (SVP). Most of the unmodified nucleotides were removed using solid phase extraction (SPE) in a polystyrene divinylbenzene copolymer. Three adducts could be detected and identified using capillary zone electrophoresis(negative)-ion electrospray ionization-mass spectrometry (CZE-(-)-ESI-MS) in conjunction with sample stacking. This way, not only a BPDE-dGMP adduct [(M-H)(-) at m/z 648], a well-known nucleotide adduct, could be retrieved, but also a BPDE-dAMP [(M-H)(-) at m/z 632] and a BPDE-dCMP adduct [(M-H)(-) at m/z 608] could be detected for the first time. The presence of the prominent ion at m/z 195 (the deoxyribose-phosphate ion) in the three low-energy collision-activated decomposition (CAD) spectra indicated that the adducts were formed through base-alkylation. CZE-positive ion ESI-MS/MS experiments were performed to obtain further information on base-alkylation. The absence of the loss of NH(3) from the nucleobase in each CAD spectrum points to an alkylated exocyclic NH(2) position of the nucleobase. So, the three adducts could be identified as BPDE-N(2)-dGMP, BPDE-N(6)-dAMP and BPDE-N(4)-dCMP using CZE-ESI-MS and CZE-ESI-MS/MS.     

高效液相色谱荧光检测法快速测定天然生育酚中痕量苯并[a]芘

建立了一种快速、新颖的高效液相色谱荧光检测法测定天然生育酚中痕量苯并[a]芘.天然生育酚样品经中性氧化铝柱色谱富集和净化后在 XDB-C,18反相柱上,采用水-乙腈(体积比20比80)为流动相进行分离,采用荧光检测器进行检测,其激发波长为 260 nm,发射波长为 408 nm.苯并[a]芘的定量下限为 0.50 ng·g-1.加标回收率大于 77.9%.分析了 3 个不同试样,测定结果的相对标准偏差在 3.0%～10.6%之间.     

Determination of benzo[a]pyrene in cigarette mainstream smoke by using mid-infrared spectroscopy associated with a novel chemometric algorithm

Determination of benzo[a]pyrene (BaP) in cigarette smoke can be very important for the tobacco quality control and the assessment of its harm to human health. In this study, mid-infrared spectroscopy (MIR) coupled to chemometric algorithm (DPSO-WPT-PLS), which was based on the wavelet packet transform (WPT), discrete particle swarm optimization algorithm (DPSO) and partial least squares regression (PLS), was used to quantify harmful ingredient benzo[a]pyrene in the cigarette mainstream smoke with promising result. Furthermore, the proposed method provided better performance compared to several other chemometric models, i.e., PLS, radial basis function-based PLS (RBF-PLS), PLS with stepwise regression variable selection (Stepwise-PLS) as well as WPT-PLS with informative wavelet coefficients selected by correlation coefficient test (rtest-WPT-PLS). It can be expected that the proposed strategy could become a new effective, rapid quantitative analysis technique in analyzing the harmful ingredient BaP in cigarette mainstream smoke.     

在线液相-气相二维色谱测定卷烟主流烟气中的苯并[a]芘

建立了在线液相-气相二维色谱测定卷烟主流烟气中的苯并[a]芘方法.采用剑桥滤片捕集烟气粒相物,环己烷萃取,以D12-苯并[a]芘为内标,然后用在线液相-气相二维色谱测定:样品直接进样进入液相色谱,经微型硅胶柱分离,含苯并[a]芘的部位切割进人气相色谱,排干溶剂后启动气相色谱升温,经毛细管柱进行分离,用质谱检测.本方法将烟气苯并[a]芘测定中的硅胶柱层析和气相色谱-质谱分析在线连接起来,可不经样品前处理净化直接进样分析;而且每次进样可达40 μL,与常规气相色谱-质谱分析最大进样2.0 μ.L相比,分析灵敏度提高了20倍.方法线性范围达0.08～ 50 ng/L,相关系数为r2=0.999,回收率为94.2％～105.5％;检出限和定量限分别为0.09和0.30 ng/支,应用本方法对14个不同类型市售卷烟和2R4F标准烟进行了测定,结果与GB/T21130-2007测定结果相符合.     

Ring-contraction from benzo[b][1,4]thiazines to benzo[d]thiazolines induced by oxygen

An unexpected ring-contraction from benzo[b]pyrazino[1,2-d][1,4]thiazine-1,4-diones (6) to benzo[4,5]thiazolo[3,2-a]pyrazine-1,4-diones (7) has been developed. The preliminary mechanistic studies showed the transformation contained two independent steps: the first step is the formation of a Michael adduct upon the addition of the protic solvent, in the presence of base, to the C-2-C-3 double bond of compound 6, and the second step is a ring-contraction induced by oxygen via the migration of sulfur atom from C-2 to C-3 position. And its scope is also studied.     

Electrochemical DNA biosensor for the determination of benzo[a]pyrene–DNA adducts

The metabolites of the environmental pollutant, benzo[a]pyrene (BaP) are carcinogenic and mutagenic agents. Thus, the determination of additional products (adducts) of the interaction between DNA and BaP, attracts great interest in cancer research.

In this study, the determination of interaction between BaP and calf thymus double-stranded DNA (dsDNA) was performed by using differential pulse voltammetry (DPV) and constant current chronopotentiometric stripping analysis (PSA) in connection with carbon paste electrode (CPE) or glassy carbon electrode (GCE). As a result of interaction of BaP with dsDNA, the signal obtained from the oxidation of guanine decreased and a new adduct signal at a more positive potential appeared. This new peak is attributed to the formation of an adduct from the interaction of guanine with BaP. The chemically prepared anti-7,8,9,10-tetrahydrobenzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) adduct by using iodine oxidation was analyzed and the electrochemical signal of the adduct was observed. When the dsDNA modified GCE was immersed into various concentrations of BaP solution, the oxidation peak of guanine decreased and the adduct peak increased with the increasing BaP concentration. The partition coefficient was also obtained from the peak of BaP with dsDNA. The results revealed that the formation of adducts could be determined by using electrochemical DNA biosensors, which are fast, simple and cost-effective devices. Furthermore, this study promises that the analysis of other important adducts would benefit from the introduction of electrochemical methods.     

Degradation of anthracene, pyrene and benzo[a]-anthracene in aqueous solution by chlorine dioxide

Polycyclic aromatic hydrocarbons (PAHs) constitute an important group of micropollutants, which are known to be mutagenic, carcinogenic and/or co-carcinogenic and relatively persistent in the environment. The effects of chlorine dioxide (ClO2) on the degradation of anthracene (ANTH), pyrene (PYR) and benzo[a]anthracene (BaA) in aqueous solution were investigated using high performance liquid chromatography (HPLC). In preliminary experiments, it was observed that ClO2 could remove these three PAHs effectively within a short time. Several factors including reaction time, the concentration of ClO2 and pH of the reaction mixture influencing the degradation ratio of PAHs have been studied by batch experiments. The results showed that the degradation ratio of PAHs was affected by reaction time and the concentration of ClO2 instead of pH. The degradation ratio of ANTH, PYR and BaA could reach their maximum as approximately 99.0%, 67.5% and 89.5%, respectively, under the condition as follows: reaction time 30, 60 and 120 min, the concentration of ClO2 0.1, 0.4 and 0.5 mmol·L-1, and pH 7.2. ANTH was selected as the representative to study the reaction mechanism with ClO2. The oxidation products formed in the reaction of ANTH with ClO2 were tentatively identified by gas chromatography-mass spectrometry (GC-MS), and the results showed that the main product was 9, 10-anthraquinone, which could be biodegraded more easily and quickly than ANTH. Through analyzing the reaction properties of ANTH and ClO2, the possible pathway for the ANTH-ClO2 reaction was proposed based on the theory of single electron transfer (SET).     

Uncertainty evaluation for the quantification of low masses of benzo[a]pyrene: Comparison between the Law of Propagation of Uncertainty and the Monte Carlo method

A proper evaluation of the uncertainty associated to the quantification of micropollutants in the environment, like Polycyclic Aromatic Hydrocarbons (PAHs), is crucial for the reliability of the measurement results. The present work describes a comparison between the uncertainty evaluation carried out according to the GUM uncertainty framework and the Monte Carlo (MC) method. This comparison was carried out starting from real data sets obtained from the quantification of benzo[a]pyrene (BaP), spiked on filters commonly used for airborne particulate matter sampling. BaP was chosen as target analyte as it is listed in the current European legislation as marker of the carcinogenic risk for the whole class of PAHs.     

Determination of benzo[a]pyrene in edible oils using phase‐transfer‐catalyst‐assisted saponification and supramolecular solvent microextraction coupled to HPLC with fluorescence detection

For the analysis of edible oils, saponification is well known as a useful method for eliminating oil matrices. The conventional approach is conducted with alcoholic alkali; it consumes a large volume of organic solvents and impedes the retrieval of analytes by microextraction. In this study, a low‐organic‐solvent‐consuming method has been developed for the analysis of benzo[a]pyrene in edible oils by high‐performance liquid chromatography with fluorescence detection. Sample treatment involves aqueous alkaline saponification, assisted by a phase‐transfer catalyst, and selective in situ extraction of the analyte with a supramolecular solvent. Comparison of the chromatograms of the oil extracts obtained by different microextraction methods showed that the supramolecular solvent has a better clean‐up effect for the unsaponifiable matter from oil matrices. The method offered excellent linearity over a range of 0.03– 5.0 ng mL⁻¹ (r > 0.999). Recovery rates varied from 94 to 102% (RSDs <5.0%). The detection limit and quantification limit were 0.06 and 0.19 μg kg⁻¹, respectively. The proposed method was applied for the analysis of 52 edible oils collected online in China; the analyte contents of 23 tested oil samples exceeded the maximum limit of 2 μg kg⁻¹ for benzo[a]pyrene set by the Commission Regulation of the European Union.