Quantification of PAH4 in roasted cocoa beans using QuEChERS and dispersive liquid-liquid micro-extraction (DLLME) coupled with HPLC-FLD.

Roasting is an important process in cocoa production which may lead to formation of non-desirable compounds such as polycyclic aromatic hydrocarbons (PAHs). Therefore, PAH4 (sum of four different polycyclic aromatic hydrocarbons; benz[a]anthracene, chrysene, benzo[b]fluoranthene, and benzo[a]pyrene) in roasted cocoa beans was determined using a modified method (combination of QuEChERS and DLLME), and quantified by HPLC-FLD. The modified method was validated and met the performance criteria required by the EU Regulation (No. 836/2011). Results show a significant (p < 0.05) increase of PAH4 (0.19-7.73 ng/g) with an increase in temperatures (110-190 °C) and duration (10-50 min). The PAHs content in whole cocoa bean roasting was detected even at the lowest temperature (110 °C) compared to nib roasting detected at 150 °C which indicates that PAHs was transferred from dried shells to roasted cocoa beans during the roasting process. The data obtained may help to control and minimize PAH4 formation during cocoa processing.

Enhancement of mixture pollutant biodegradation efficiency using a bacterial consortium under static magnetic field.

One of the main challenges of bioremediation is to define efficient protocols with low environmental impact and high removal rates, such as static magnetic field (SMF). The aim of this study was to evaluate the effect of SMF exposure on the biodegradation rate of a mixture of pollutants using three bacterial strains which were isolated and identified from the Bizerte lagoon: Pseudomonas stutzeri LBR (KC157911), Cupriavidus metallidurans LBJ (KU659610) and Rhodococcus equi LBB (KU743870). To recognize the improvement role of SMF, the culture was submitted to a pre-treatment with SMF with an induction equal to 200 mT for 5 hours, after that the degradation experiment was followed with individual strains and with a consortium. Results showed an increase by 20% in the growth of the exposed bacterial population compared to controls, and 98% of biodegradation of DDT and 90% for BaP after 30 days of follow-up. This encouraging data opens new perspectives for a bioremediation bioprocess using SMF.

Polycyclic aromatic hydrocarbons in teas using QuEChERS and HPLC-FLD.

Polycyclic aromatic hydrocarbons (PAHs) are food-processing contaminants considered to be carcinogenic and genotoxic. Due to its drying process stage, teas may be contaminated with PAHs. The aim of the study was to validate an analytical method involving QuEChERS and HPLC-FLD for the determination of PAH4 in teas and evaluate the contamination levels in 10 different types of teas from Brazil. Recoveries varied from 54% to 99% and relative standard deviations from 1% to 21%. Limits of detection and quantification were from 0.03 to 0.3 µg/kg and 0.1 to 0.5 µg/kg, respectively. Mate tea presented the highest PAH levels, with PAH4 varying from 194 to 1795 µg/kg; followed by black (1.8-186 µg/kg), white (24-119 µg/kg), and green teas (3.1-92 µg/kg). Teas with lowest PAH4 were strawberry, lemongrass, peppermint, and boldo. Only trace levels of PAHs were detected in tea infusions, so apparently it would not affect PAH intake by Brazilian population.

Comparative proteomic analysis and characterization of benzo(a)pyrene removal by Microbacterium sp. strain M.CSW3 under denitrifying conditions.

High-molecular-weight polycyclic aromatic hydrocarbons are persistent organic pollutants with great environmental and human health risks and the associated bioremediation activities have always been hampered by the lack of powerful bacterial species under redox conditions. A Microbacterium sp. strain capable of using benzo(a)pyrene as sole carbon and energy sources under denitrifying conditions was isolated. The difference in protein expression during BaP removal and removal characterization were investigated. A total of 146 proteins were differentially expressed, 44 proteins were significantly up-regulated and 102 proteins were markedly down-regulated. GO and COG analysis showed that BaP removal inhibited the expression of proteins related to glucose metabolism at different levels and activated other metabolic pathway. The proteins associated with catalytic activity and metabolic process were altered significantly. Furthermore, the BaP removal might be occurred in certain organelle of M.CSW3. The strain removed BaP with a speed of 0.0657-1.0072 mg/L/day over the concentrations range 2.5-100 mg/L. High removal rates (>70%) were obtained over the range of pH 7-11 in 14 days. Carbohydrates and organic acids which could be utilized by the strain, as well as heavy metal ions, reduced BaP removal efficiency. However, phenanthrene or pyrene addition enhanced the removal capability of M.CSW3. The strain was proved to have practical potential for bioremediation of PAHs-contaminated soil and this study provided a powerful platform for further application by improving production of associated proteins.

Optimization of hydrogen peroxide-to-hemoglobin ratio for biocatalytic mineralization of polycyclic aromatic hydrocarbons (PAHs)-contaminated soils.

This study investigates the efficiency of hemoglobin (Hb)-catalyzed biocatalytic reactions for removal of polycyclic aromatic hydrocarbons (PAHs) in a historically PAHs-contaminated soil and of benzo(a)pyrene (BaP) in an artificially BaP-contaminated soil. PAHs removal tests at various H2O2-to-Hb mass ratios (0-3.7) showed that the PAHs removal was greater at H2O2-to-Hb mass ratio of ≥3. This was attributed to the greater removal of high molecular weight PAHs at higher H2O2-to-Hb mass ratios. The BaP removal increased from 36% to 85% with increasing H2O2-to-Hb mass ratio from 1 to 3, and further increase in H2O2-to-Hb mass ratio decreased the BaP removal. Thus, the optimal H2O2-to-Hb mass ratio for BaP removal was determined to be 3 in the artificially BaP-contaminated soil. The BaP removal in the presence of only Hb can be attributed to the capture of BaP by Hb. The increased BaP removal in the presence of H2O2 is likely due to BaP mineralization as the BaP removal and the CO2 generated showed a strong positive correlation (R2 = 0.999). Overall, this study shows that the Hb-catalyzed biocatalytic reactions can effectively remove PAHs in soil.

Optimisation of extraction methods and quantification of benzo[a]pyrene and benz[a]anthracene in yerba maté tea by isotope dilution mass spectrometry.

A gas chromatography-isotope dilution mass spectrometry (GC-IDMS) technique was developed for the quantification of two heavy polyaromatic hydrocarbons (PAHs), benz[a]anthracene and benzo[a]pyrene, in yerba maté tea (maté). The optimisation of two extraction methods, namely liquid-liquid extraction and accelerated solvent extraction, was carried out. Both optimised methods were validated using a certified reference material of fine dust and the results were within the expanded uncertainties at 95% confidence level. Recoveries of 99.2-106.7% with RSD of measurements of 1.1-2.3% were achieved for benz[a]anthracene. Recoveries of 95.7-101.9% with RSD of measurements of 0.4-1.4% were achieved for benzo[a]pyrene. The validated methods were applied for the extraction of benz[a]anthracene and benzo[a]pyrene in maté powder from NIST. A metrological approach was undertaken to ensure the traceability of measurement results. The uncertainties associated with the results were rigorously evaluated and also reported herein. Graphical abstract Quantification of benz[a]anthracene and benzo[a]pyrene using IDMS.

Development of a QuEChERS-Based Method for Determination of Carcinogenic 2-Nitrofluorene and 1-Nitropyrene in Rice Grains and Vegetables: A Comparative Study with Benzo[a]pyrene.

Nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) are ubiquitous environmental pollutants attracting increasing attention because of their potent mutagenicity to humans. Previous studies of nitro-PAHs focused on investigating their formation mechanisms and detecting them in atmospheric environment; however, few studies have reported their occurrence in food samples, and regulations on nitro-PAHs are still lacking. We report in this study the development and application of a quick, easy, cheap, effective, rugged, and safe (QuEChERS) method for determination of nitro-PAHs in rice and vegetable samples. Analysis of the collected samples by the validated method revealed 1-nitropyrene and 2-nitrofluorene were widespread food contaminants. A comparative study with benzo[a]pyrene, the commonly used marker for PAH exposure, showed that carcinogenic nitro-PAHs existed in rice and vegetables at similar concentrations. Dietary exposure risk, which was estimated based on the surveillance data, suggested 3.28-5.03 ng/kg/day of nitro-PAHs exposure for Hong Kong citizens from rice grains and vegetables.

Mimicking enzymatic effects of cytochrome P450 by an efficient biosensor for in vitro detection of DNA damage.

A novel biosensor for detecting DNA damage induced by benzo(a)pyrene (BP) and its metabolite was presented in this work. The nafion-solubilized single wall carbon nanotubes-ionic liquid (SWCNTs-NA-IL) composite film was prepared and then horseradish peroxidase (HRP) and double-stranded DNA were alternately assembled on the composite film by the layer-by-layer method. The biosensor was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), differential pulse voltammetry (DPV), scanning electron microscopy (SEM) and computational methods. UV-vis spectrophotometry was also used to investigate DNA damage induced by BP and its metabolites in solution. The DNA biosensor was treated separately in BP, hydrogen peroxide (H2O2) and in their mixture, respectively. The EIS analysis showed a decrease in the charge transfer resistance at the DNA/HRP/SWCNTs-NA-IL/GCE incubated in a mixture of HRP and H2O2, because HRP in the presence of H2O2 could mimic enzymatic effects of cytochrome P450 (CYP450) to metabolize BP which could cause significant DNA damage and the exposed DNA bases reduced the electrostatic repulsion of the negatively charged redox probe and leads to Faradaic impedance changes. Finally, a novel biosensor for BP determination was developed and this method provided an indirect, and quantitative estimation of DNA damage in vitro.

Effect-directed analysis of Elizabeth River porewater: developmental toxicity in zebrafish (Danio rerio).

In the present study, effect-directed analysis was used to identify teratogenic compounds in porewater collected from a Superfund site along the Elizabeth River estuary (VA, USA). Zebrafish (Danio rerio) exposed to the porewater displayed acute developmental toxicity and cardiac teratogenesis, presumably because of elevated sediment levels of polycyclic aromatic hydrocarbons (PAHs) from historical creosote use. Pretreatment of porewater with several physical and chemical particle removal methods revealed that colloid-bound chemicals constituted the bulk of the observed toxicity. Size-exclusive chromatography and normal-phase high-performance liquid chromatography were used to fractionate Elizabeth River porewater. Acute toxicity of porewater extracts and extract fractions was assessed as the pericardial area in embryonic zebrafish. The most toxic fraction contained several known aryl hydrocarbon receptor (AhR) agonists (e.g., 1,2-benzofluorene and 1,2-benzanthracene) and cytochrome P450 A1 (CPY1A) inhibitors (e.g., dibenzothiophene and fluoranthene). The second most toxic fraction contained known AhR agonists (e.g., benzo[a]pyrene and indeno[1,2,3-cd]pyrene). Addition of a CYP1A inhibitor, fluoranthene, increased toxicity in all active porewater fractions, suggesting synergism between several contaminants present in porewaters. The results indicate that the observed acute toxicity associated with Elizabeth River porewater results from high concentrations of AhR agonistic PAHs and mixture effects related to interactions between compounds co-occurring at the Elizabeth River site. However, even after extensive fractionation and chemical characterization, it remains plausible that some active compounds in Elizabeth River porewater remain unidentified.

Benzo(a)pyrene penetration on a smoked meat product during smoking time.

The Codex Alimentarius gives recommendations to prevent carcinogenic polycyclic aromatic hydrocarbons (PAH) (represented by benzo(a)pyrene (BaP)) contamination during processing of meat products, including the control of smoking time. The influence of direct smoking time (0, 1, 3, 5 and 7 days) on the relationship between the BaP and moisture content of a typical Spanish smoked meat product called chorizo and the mechanism of BaP penetration and water release from four different depths in the product was studied. Chorizo was studied from the Principality of Asturias, a location never before tested. An analytical method was developed for this purpose consisting of PAH extraction assisted by sonication followed by solid-phase extraction (SPE) sample clean-up and analytical determination using GC-MS. Results show that an increase in smoking time produced contradictory and independent effects on the moisture and BaP content (µg kg⁻¹) of chorizos. The moisture content decreased from 49.9% to 31.3%. On the other hand, the BaP content increased from less than 0.24 µg kg⁻¹ to 0.75 µg kg⁻¹, finally stabilising after 5 days of smoking. After this time, the natural pores of the casing could be blocked by the large size tar particles from smoke, preventing the continued penetration of PAHs. The BaP content decreased and the moisture content increased progressively from the casing to the centre of the meat product. BaP mainly accumulated in the smoked casing, being four times in excess of the legal limit. This paper analyses the mechanism for preventing PAHs contamination during the process of smoking meat products.

Benzo[a]pyrene-specific online high-performance liquid chromatography fractionation of air particulate extracts - a tool for evaluating biological interactions.

Benzo[a]pyrene (B[a]P) is a known human carcinogen and is commonly used as a surrogate for assessing the carcinogenic risk posed by complex mixtures of polycyclic aromatic hydrocarbons (PAHs) present in air particulate matter (PM). However, studies have shown that using B[a]P as a surrogate may underestimate the carcinogenic potential of PAH mixtures, as the risk assessment approach does not consider interaction effects. Thus, toxicological studies using B[a]P to assess its carcinogenic potential in environmentally derived complex mixtures, as opposed to single compound experiments, could improve risk assessment. The intention of the present study was to develop an online HPLC fractionation system for the selective removal of B[a]P from air PM extracts. Two serial pyrenylethyl (PYE) columns enabled selective separation of B[a]P from its isomers and other PAHs as well as a short fractionation cycle of 30min. One run consisted of three collection steps: the first fraction contained PAHs eluting earlier than B[a]P, the second contained B[a]P and the last contained later-eluting PAHs. The selectivity and recovery of the system was investigated using extracts of Stockholm air PM samples. The overall recovery for all PAHs was approximately 80%, and the system proved to be selective, as it removed 94% of B[a]P and less than 3% of benzo[b]fluoranthene from the complex PAH mixture. Exposing human cells to blanks generated by the fractionation system did not induce cytotoxicity or DNA damage signalling. In conclusion, the online HPLC system was selective for B[a]P fractionation whilst minimising run-to-run variation and allowing repeated fractionations for larger samples due to its relatively short run time.

[Isolation of an effective benzo [a] pyrene degrading strain and its degradation characteristics].

A strain which could utilize BaP as a sole carbon and energy source and efficiently degrade benzo[a] pyrene (BaP) was isolated from the contaminated sediments of Guiyu, Guangdong province, China. The strain was identified as Brevibacillus brevis based on physiological and biochemical experiments together with 16S rRNA sequence analysis. The experimental results showed that the biodegradation rate of BaP by B. brevis in 7 days was 51.35%. The study also demonstrated that pH, temperature, bacterial dosage, initial concentration of BaP and processing time were important factors for BaP degradation. B. brevis could tolerate wide pH and temperature ranges, from 2 to 12 degrees C and 25 to 40 degrees C, respectively. The optimum condition for BaP degradation was pH 7 and 25 degrees C. With the increase of B. brevis inoculation amount, the degradation efficacy displayed an initial increasing trend and then came to a plateau. And the increase of BaP concentration led to the enhancement of BaP degradation. Addition of salicylic, succinate and phthalate showed no obvious positive effect on BaP biodegradation. After degradation of BaP, the surface of B. brevis was wrinkled, and became depressed and deformed over time.

[Isolation of highly-effective benzo[a]pyrene degrading strain and its degradation capacity].

One new mycete, which could degrade high concentration (up to 100 mg/L) of benzo[a]pyrene (BaP) in liquid, was isolated from contaminated soil of Beijing Coking Plant by gradually increasing the concentration of BaP in mineral salt medium (MSM) in order to get new microorganism species for remediation of BaP contamination. The strain was identified as Lasiodiplodia theobromae, and its biodegradation ability in liquid was further investigated. The results showed that L. theobromae could utilize BaP as sole carbon and energy sources. The experiment was conducted for 10 days, and the biodegradation rate of BaP was 52.5% +/- 1.5%. Compared to Czapek's mineral medium, MSM was more suitable for L. theobromae, and biodegradation rate was 2.8 percent greater than that by using Czapek's mineral media after 10 days' cultivation. Potato-dextrose nutrient medium could accelrate the biodegradation in early stage, and biodegradation rate of BaP increased by 19.2 percent in the second day. However, the accelration was not significant in the latter period, biodegradation rate was only increased by 5.4 percent after 10 days' cultivation. L. theobromae could tolerate a wide pH range, with the optimum pH of 5. Addition of salicylic and sodium succinate enhanced the biodegradation rates by 6.2 percent and 4.2 percent, respectively, after 10 days' cultivation. Besides BaP, L. theobromae could also degrade high concentration (200 mg/L) of phenanthrene and pyrene, and the biodegradation rates were 70.0% +/- 1.0%, 59.2% +/- 3.2%, and 52.5% +/- 1.5% when they were single substrate and were 21.6% +/- 2.1%, 14.5% +/- 5.5%, and 11.9% +/- 2.2% when they existed in mixture, respectively. The biodegradation rate followed an order of phenanthrene > pyrene > BaP. The co-existence of the three substrates led a reduction in biodegradation. This study provides a new microorganism species for remediation of polycyclic aromatic hydrocarbons (PAHs) contamination in the environment.

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).

Comparative evaluation of different cell lysis and extraction methods for studying benzo(a)pyrene metabolism in HT-29 colon cancer cell cultures.

Lysis and extraction of cells are essential sample processing steps for investigations pertaining to metabolism of xenobiotics in cell culture studies. Of particular importance to these procedures are maintaining high lysis efficiency and analyte integrity as they influence the qualitative and quantitative distribution of drug and toxicant metabolites in the intra- and extracellular milieus. In this study we have compared the efficiency of different procedures viz. homogenization, sonication, bead beating, and molecular grinding resin treatment for disruption of HT-29 colon cells exposed to benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH) compound and a suspected colon carcinogen. Also, we have evaluated the efficiency of various procedures for extracting BaP parent compound/metabolites from colon cells and culture media prior to High Performance Liquid Chromatography (HPLC) analyses. The extraction procedures include solid phase extraction, solid-supported liquid- liquid extraction, liquid-liquid extraction, and homogeneous liquid- liquid extraction. Our findings showed that bead-beating in combination with detergent treatment of cell pellet coupled with liquid-liquid extraction yielded greater concentrations of BaP metabolites compared to the other methods employed. Our method optimization strategy revealed that disruption of HT-29 colon cells by a combination of mechanical and chemical lysis followed by liquid-liquid extraction is efficient and robust enough for analyzing BaP metabolites from cell culture studies.

Fractionation of polycyclic aromatic hydrocarbon residues in soils.

Understanding the forms and availabilities of polycylic aromatic hydrocarbons (PAHs) would have considerable benefits for their risk assessment, and is of crucial importance for food security and remediation strategies in contaminated sites. In this work, the forms of six PAHs (fluorene, phenanthrene, fluoranthene, pyrene, benzo[a]anthracene, and benzo[a]pyrene) in soils were separated into three fractions including a desorbing fraction, a non-desorbing fraction, and a bound residual fraction using a sequential extraction mass balance approach. The desorbing and non-desorbing fractions were extracted with hydroxypropyl-beta-cyclodextrin (HPCD) and dichloromethane:acetone (1:1, vol/vol), respectively. The desorbing and non-desorbing fractions always dominated the total PAH content in soils. The proportion of bound PAH residue in nonsterilized soils was small (<16%), and even smaller (4.5%) in sterilized soils. The concentrations of the desorbing fraction of PAHs as well as the percentage of this fraction to the total PAH content in soils clearly decreased in 0-16 weeks, which may be due to microbial biodegradation and its transfer to other fractions in soils. The concentrations of the non-desorbing PAH fractions increased in sterilized soils, while remaining nearly constant or decreasing to some extent in nonsterilized soils after 16 weeks. The proportion of non-desorbing PAH fractions significantly increased in 16 week-incubation, and this proportion was positively correlated with the molecular weights of the PAHs tested, indicating that larger PAHs are more likely to be present in non-desorbing fractions. The bound PAH residue tended to increase at first and decrease thereafter over the 0-16-week period, and microbes played an important role in the formation of bound residue.

Mutagenicity of surface soil from residential areas in Kyoto city, Japan, and identification of major mutagens.

To clarify the mutagenic potential of surface soil in residential areas in Kyoto city, surface soil samples were collected twice or three times from 12 sites, and their organic extracts were examined by the Ames/Salmonella assay. Almost all (>92%) samples showed mutagenicity in TA98 without and with S9 mix, and 8/25 (32%) samples showed high (1000-10,000 revertants/g of soil) or extreme (>10,000 revertants/g of soil) activity. Moreover, to identify the major mutagens in surface soil in Kyoto, a soil sample was collected at a site where soil contamination with mutagens was severe and continual. The soil extract, which showed potent mutagenicity in TA98 without S9 mix, was fractionated by diverse column chromatography methods. Five major mutagenic constituents were isolated and identified to be 1,6-dinitropyrene (DNP), 1,8-DNP, 1,3,6-trinitropyrene (TNP), 3,9-dinitrofluoranthene (DNF), and 3,6-dinitrobenzo[e]pyrene (DNBeP) by co-chromatography using high performance liquid chromatography and spectral analysis. Contribution ratios of 1,6-DNP, 1,8-DNP, 1,3,6-TNP, 3,9-DNF, and 3,6-DNBeP to total mutagenicity of the soil extract in TA98 without S9 mix were 3, 10, 10, 10, and 6%, respectively. These nitroarenes were detected in surface soil samples collected from four different residential sites in other prefectures, and their contribution ratios to soil mutagenicity were from 0.7 to 22%. These results suggest that surface soil in residential areas in Kyoto was widely contaminated with mutagens and there were some sites where surface soils were heavily polluted. 1,6-DNP, 1,8-DNP, 1,3,6-TNP, 3,9-DNF, and 3,6-DNBeP may be major mutagenic constituents that contaminate surface soil in Kyoto and other residential areas.

[Desorption of polycyclic aromatic hydrocarbons in soils assisted by SPMD].

In order to develop a new method to study the desorption and bioavailability of hydrophobic organic chemicals (HOCs) in soils, a method using semi-permeable membrane device (SPMD) to study desorption of HOCs in soils has been set up, and assisted desorption of polycyclic aromatic hydrocarbons (PAHs), phenanthrene(PHE), pyrene(PYE), and benzo[a] pyrene (B[a]PYE) in three different kinds of soils was studied using SPMD. The results show that SPMD is a good measurement to study the desorption and bioavailability of HOCs in soils. SPMD assisted desorption of PAHs is highly dependent on the properties of the soils and the chemicals. PHE and PYE desorption percentages increase with the reduction of the content of soil organic matter (SOM), so that the desorption of the two chemicals increases from 56.45% and 48.28% to almost 100% when SOM content was reduced from 18.68% to 0.3%. However, clay has a significant holding effect on B[a]PYE, and PYE desorption is only 66.97% in Soil 3 with SOM of 0.3% and clay content of 39.05%. There is a great variety in the desorption among the different PAHs. With the reduction of SOM content and the elevation of contamination concentration, the difference between PHE and PYE decreases gradually, while B[a]PYE exhibits a significant difference from them. This could be attributed to the high lipophilicity and large molecular size of B[a]PYE, which make the molecule of B[a]PYE to be more easier to be held in the nanopores of clay and the dense region of SOM.

[Determination of benzopyrene in red clover extract by high performance liquid chromatography with spectrofluorescence detector].

OBJECTIVE: To establish a high performance liquid chromatography method with spectrofluorescence detector for the quantitative analysis of benzopyrene in red clover extract. METHOD: Analysis was performed on Hypersil C18 column (4. 6 mm x 200 mm, 5 microm) with acetonitrile and water (70: 30) as mobile phase; The excitation wavelength and emission wavelengh were 368 nm and 405 nm, respectively; Flow rate was 1.0 mL x min(-1). RESULT: The linear range was 0.93-37.2 ng x mL(-1), and r = 0.9992 (n = 5). The average recovery of benzopyrene was 85.2% (n = 9). CONCLUSION: The method was convenient and accurate with good recovery.