Dichlorobenzidine Research Articles

Simultaneous detection of hazardous skin whitening agents in Indian cosmetic products using a green chromatographic technique

The present work mainly highlights the simultaneous detection of four skin whitening agents i.e. hydroquinone (HQ), resorcinol (RS), catechol (CC) and 3,3′-dichlorobenzidine (DCB) in facial creams and body lotion. Among these, the first three are positional isomers of dihydroxybenzene so simultaneous separation is difficult with the conventional reverse-phase high-performance liquid chromatographic technique (RP-HPLC). The selected skin whitening agents were detected in facial cream and body lotion using micellar liquid chromatography coupled to a photodiode array detector (MLC-PDA). In the present study, optimization of the method was accomplished using response surface methodology (RSM) with central composite design (CCD). The second-order polynomial model for predicting the optimal chromatographic run time was evaluated by the analysis of variance (ANOVA) and 3D response surface plots for the interactions between three variables were constructed. Three experimental parameters which were chosen as independent variables were; surfactant concentration (SDS), the volume percentage of organic modifier (OM) and pH of the mobile phase. The second-order polynomial model for predicting the optimal chromatographic run time was evaluated by the analysis of variance (ANOVA) and 3D response surface plots for the interactions between three variables were constructed. Three experimental parameters which were chosen as independent variables were; surfactant concentration (SDS), percentage of organic modifier (OM) and pH of the mobile phase. The second-order polynomial model for predicting the optimal chromatographic run time was evaluated by the analysis of variance (ANOVA) and 3D response surface plots for the interactions between three variables were constructed. Three experimental parameters which were chosen as independent variables were; surfactant concentration (SDS), percentage of organic modifier (OM) and pH of the mobile phase. The optimized mobile phase was 0.15 M SDS and 7% 1-butanol, buffered at pH 7 with 0.01 M NaH2PO4. The chromatographic run time for simultaneous determination of selected analytes was 7.5 min. The correlation coefficient (r2) values were satisfactory between 0.998–0.999 over the linear concentration range. Limits of detection (LODs) and the limits of quantification (LOQs) for the four skin whitening agents were in the range of 0.05–0.07 μg/mg and 0.11–0.14 μg/mg, respectively. Trueness (98.4–102.7%) and precision (< 4.3%) were acceptable. The developed method was fast, cost-effective, and green which could easily analyze complex matrices (facial creams, body lotion) without any pretreatment other than filtration. The results indicated that the MLC-PDA method proved to be more suitable for the simultaneous separation of selected positional isomers.

Laser Irradiation of Organic Tattoo Pigments Releases Carcinogens with 3,3′-Dichlorobenzidine Inducing DNA Strand Breaks in Human Skin Cells

Laser Irradiation of Organic Tattoo Pigments Releases Carcinogens with 3,3′-Dichlorobenzidine Inducing DNA Strand Breaks in Human Skin Cells

Ozonation of 3,3′-dichlorobenzidine in aqueous solution: degradation efficiency and kinetics

The degradation efficiency and kinetics of 3,3′-dichlorobenzidine (DCB) in aqueous solution by ozonation was investigated in this study. In laboratory-scale experiments, the primary factors affecting the degradation of DCB were investigated. The results showed that pH strongly influenced the degradation process, with a maximum apparent rate of 0.235 min−1 obtained at pH 4. The reactivity of DCB decreased in the order (C12H10Cl2N2)2 > (C12H10Cl2N2)2·HCl > (C12H10Cl2N2)2·2HCl. The degradation rate of DCB increased with increasing ozone dosage and reaction temperature but decreased with increasing initial DCB concentration. We derived a reaction kinetics model of DCB degradation by ozonation in aqueous solution by interpreting the experimental data, and this model showed good stability. The efficiency of DCB degradation by ozonation was also evaluated. DCB could be removed efficiently by ozonation, with greater than 85% removal after 50 min at pH 4 and 16–30°C with a DCB concentration of 1.2–6.5 mg/L and an ozone dosage of 3.7–5.1 mg/min. Most of the intermediate products could not be mineralised by direct oxidation by ozone. The addition of tert-butyl alcohol or Cl− had little effect on the degradation rate of DCB, which indicated that degradation of DCB occurred via direct oxidation by ozone.

Molecular mechanisms of 3,3′‐dichlorobenzidine‐mediated toxicity in HepG2 cells

3,3'-Dichlorobenzidine (DCB) (CAS 91-94-1), a synthetic, chlorinated, primary aromatic amine, is typically used as an intermediate in the manufacturing of pigments for printing inks, textiles, paints, and plastics. In this study, we found that DCB could significantly inhibit the cell viability of HepG2 cells in a concentration-dependent manner. Flow cytometry revealed that DCB induced G2/M-phase arrest and apoptosis in HepG2 cells. DCB treatment dramatically induced the dissipation of mitochondrial membrane potential (Δψm ) and enhanced the enzymatic activities of caspase-9 and caspase-3 whilst hardly affecting caspase-8 activity. Furthermore, Western blotting indicated that DCB-induced apoptosis was accompanied by the down-regulation of Bcl-2/Bax ratio. These results suggested that DCB led to cytotoxicity involving activation of mitochondrial-dependent apoptosis through Bax/Bcl-2 pathways in HepG2 cells. Furthermore, HepG2 cells treated with DCB showed significant DNA damage as supported by the concentration-dependent increase in olive tail moments as determined by the comet assay and by concentration- and time-dependent increase in histone H2AX phosphorylation (γ-H2AX). Two-dimensional-difference gel electrophoresis (2D-DIGE), combined with mass spectrometry (MS), was used to unveil the differences in protein expression between cells exposed to 25 µM or 100 µM of DCB for 24 hr and the control cells. Twenty-seven differentially expressed proteins involved in DNA repair, unfolded protein response, metabolism, cell signaling, and apoptosis were identified. Among these, 14-3-3 theta, CGI-46, and heat-shock 70 protein 4 were confirmed using Western blot assay. Taken together, these data suggest that DCB is capable of inducing DNA damage and some cellular stress responses in HepG2 cells, thus eventually leading to cell death by apoptosis.

Ultra-trace level determinations of benzidine and dichlorobenzidine residues in surface water by liquid chromatography-electrospray ionization tandem mass spectrometry

A liquid chromatography-electrospray ionization tandem mass spectrometric method (LC-ESI-MS/MS) has been developed for the determination of benzidine (BZ) and 3, 3′-dichlorobenzidine (DCBZ) from surface water. Parent ion of DCBZ gave strong signal with ESI by dissolving with 15 mM ammonium formate, which is also used as mobile phase. Deuterated BZ (d8-BZ) was chosen as the internal standard (IS). BZ and DCBZ were extracted by solid-phase extraction on Oasis hydrophilic-lipophilic balance (HLB) from water at pH 8.0. The coefficients of variation of BZ and DCBZ were less than 13.1% and the detection limits were 0.004 ng L−1 for BZ and 0.7 ng L−1 for DCBZ using 0.5 L surface water. The detection limit shows fair lower value than the water quality criteria for BZ and DCBZ established by the US EPA. When the proposed method was used to analyze the target compounds in sixteen surface water samples, BZ was detected in a concentration range of 0.15–2.33 ng L−1 in four of sixteen surface water samples

Development of a Strategy for Biological Monitoring in a Chemical Plant Producing 3,3′-Dichlorobenzidine Dihydrochloride

In a chemical plant in Germany producing 3,3′-dichlorobenzidine dihydrochloride for the manufacture of colorants, blood and urine samples were taken for biological monitoring. 3,3′-Dichlorobenzidine (DBZ) was analyzed in urine by thin-layer chromatography and subsequently further combined with analysis of adducts of 3,3′-DBZ in hemoglobin. Data highlight current ranges of industrial exposure to 3,3′-DBZ in Germany and demonstrate the applicability of biological monitoring to minimize this exposure. Effective biological monitoring was achieved by a combination of monitoring hemoglobin adducts with spot samplings of urinary 3,3′-DBZ excretion in cases of reported exposure periods. Data presented might help to identify biological guidance values (BGV/BAR) for 3,3′-DBZ-exposed individuals.

Identification of a new mutagen, 4,4′-diamino-3,3′-dichloro-5-nitrobiphenyl, in river water flowing through an industrial area in Wakayama, Japan

3,3′-Dichlorobenzidine (DCB), which has been assigned as a possible carcinogen to humans (Group 2B) by IARC, is produced as a raw material in the manufacture of polymers and dye intermediates. In our previous paper, we identified DCB as an indirect-acting mutagenic constituent in the water concentrates from the Waka River, which flows through an industrial area in Wakayama, Japan. In this study, we have identified a novel mutagen in the water samples from the Waka River. Organic chemicals in the river water were adsorbed to blue rayon at the site where effluents from chemical plants and a sewage plant were discharged into the river. The adsorbate was highly mutagenic in Salmonella YG1024 in the presence and absence of S9 mix, inducing 440,000 and 170,000 revertants/g blue rayon equivalent, respectively. Two mutagenic fractions, which accounted for 18% and 12% of the total mutagenicity of the water concentrate in YG1024 with S9 mix, were separated by HPLC with a reversed-phase column following Sephadex LH20 column chromatography. Both fractions were further separated by HPLC using reversed-phase columns. On the basis of spectral analysis and co-chromatography using an authentic chemical standard, one mutagen in the former fraction was identified as DCB and one mutagen in the latter fraction was deduced to be a novel chemical, a 5-nitro derivative of DCB (5-nitro-DCB; 4,4′-diamino-3,3′-dichloro-5-nitrobiphenyl). 5-Nitro-DCB showed strong mutagenicity in YG1024 especially with S9 mix, inducing 24,200 revertants/μg. 5-Nitro-DCB was detected in water concentrates in the range from less than detection limit to 6.9 μg/g of blue rayon. DCB was also detected in the range from 13.2 to 104 μg/g of blue rayon. These results demonstrate that Waka River water might be continually contaminated with the indirect-acting mutagens DCB and 5-nitro-DCB as major mutagenic constituents of the river water.

Quantification of a potent mutagenic 4-amino-3,3′-dichloro-5,4′-dinitrobiphenyl (ADDB) and the related chemicals in water from the Waka River in Wakayama, Japan

4-Amino-3,3′-dichloro-5,4′-dinitrobiphenyl (ADDB) is a novel chemical exerting strong mutagenicity, especially in the absence of metabolic activation. In addition to mutagenicity, ADDB may also disrupt the endocrine system in vitro. ADDB may be discharged from chemical plants near the Waka River and could be unintentionally formed via post-emission modification of drainage water containing 3,3′-dichlorobenzidine (DCB), which is a precursor in the manufacture of polymers and dye intermediates in chemical plants. The main purpose of this study was to make a comprehensive survey of the behaviour and levels of ADDB and suspected starting material or intermediates of ADDB, i.e., DCB, 3,3′-dichloro-4,4′-dinitrobiphenyl (DDB), and 4-amino-3,3′-dichloro-4′-nitrobipheny (ADNB) in Waka River water samples. We also postulated the formation pathway of ADDB. Water samples were collected at five sampling sites from the Waka River four times between March 2003 and December 2004. Samples were passed through Supelpak2 columns, and adsorbed materials were then extracted with methanol. Extracts were used for quantification of ADDB and the related chemicals by HPLC on reverse-phase columns; mutagenicity was evaluated in the Salmonella assay using the O-acetyltransferase-overexpressing strain YG1024. High levels of ADDB, DCB, DDB, and ADNB (12.0, 20,400, 134.8, and 149.4 ng/L-equivalent) were detected in the samples collected at the site where wastewater was discharged from chemical plants into the river. These water samples also showed stronger mutagenicity in YG1024 both with and without S9 mix than the other water samples collected from upstream and downstream sites. The results suggest that ADDB is unintentionally formed from DCB via ADNB in the process of wastewater treatment of drainage water containing DCB from chemical plants.

Determination of nucleic acids at nanogram levels using the resonance light scattering technique with 3,3′-dichlorobenzidine

A novel method for the determination of nucleic acid at nanogram levels was developed based on the measurement of resonance light scattering (RLS) signals of 3,3′-dichlorobenzidine (DCB). In the Britton-Robinson buffer (pH 2.21), the weak light scattering of DCB was greatly enhanced by addition of calf thymus DNA (ctDNA), the maximum RLS peak is at 346 nm and the enhanced intensity of RLS is in proportion to the concentration of ctDNA. The linear range is 0.05–5 µg mL−1 for ctDNA, and the detection limit is 14 ng mL−1 (3σ). DNA in synthetic samples was analyzed with satisfactory results.

Analysis of Benzidine and Dichlorobenzidine at Trace Levels in Water by Silylation and Gas Chromatography–Mass Spectrometry

Conditions for silylation of benzidine (BZ) and 3,3′-dichlorobenzidine (DCBZ) have been optimized. Reactivity, repeatability, and derivative stability were compared for the silylating reagents N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and N-Methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBDMSTFA) and the catalysts 3% trimethylsilylimidazole (TMS-I) and 0.3% NH4-I–dithioerythritol. The results showed that derivatization with MTBDMSTFA/NH4-I containing 0.1 mg dithioerythritol was superior to other methods. The silylation conditions selected were reaction with (MTBDMSTFA)–NH4I, 1000:3, with catalysis by dithioerythritol, at 80 °C for 80 min. The TBDMS derivatives of BZ and DCBZ had very good chromatographic properties and very sensitive detection was achieved by gas chromatography with electron-impact ionization mass spectrometry (GC-EIMS). Simultaneous determination of BZ and DCBZ in water was developed on the basis of the TBDMS derivatives. Deuterated BZ (d8-BZ) was chosen as internal standard (IS) for analysis of water samples. BZ and DCBZ were extracted from water at pH 8.5 with dichloromethane and the extract was then dried and silylated. Recoveries of BZ and DCBZ were approximately 102 and 103% at a concentration of 2.0 ng mL−1. The coefficients of variation for BZ and DCBZ were less than 9 and 4% at concentrations of 0.2 and 0.5 ng mL−1, respectively. The method detection limits for 200 mL water were 0.004 ng mL−1 for BZ and 0.02 ng mL−1 for DCBZ.

Detection of 3,3′-Dichlorobenzidine in Water from the Waka River in Wakayama, Japan

In a previous study, we found that water concentrate from the Waka River, which flows through an industrial area in Wakayama, Japan, showed significant mutagenicity in Salmonella typhimurium YG1024 without a mammalian metabolic system (S9 mix), and 4-amino-3,3′-dichloro-5,4′-dinitrobiphenyl (ADDB) was identified as a major direct-acting mutagenic constituent in the water concentrate. ADDB induced 428,000 revertants/μg in YG1024 without S9 mix, and this activity was 48 times as high as that with S9 mix. In this study, to clarify whether other mutagenic contaminants were present in the river, water concentrates were collected at three sites along the Waka River and examined for mutagenicity by the Ames test using YG1024 and YG1029 with and without S9 mix. All water concentrates showed potent mutagenicity in YG1024 with and/or without S9 mix. ADDB was detected in water concentrate that was extremely mutagenic without S9 mix. An indirect-acting mutagen, which accounted for more than 30% of the total mutagenicity of the water concentrates in YG1024 with S9 mix, was isolated from two extremely mutagenic water concentrates by HPLC separation. On the basis of spectral data and co-chromatography using authentic chemicals, this mutagen was identified as 3,3′-dichlorobenzidine (DCB). DCB showed strong mutagenicity in YG1024 with S9 mix, inducing 5,186 revertants/μg. DCB was detected in water concentrates collected on the other three sampling dates in the range from 15.8 to 28.5 μg/g of blue rayon. These results suggest that Waka River water might be continually contaminated with mutagens, and DCB was thought to be the major mutagenic constituent of the river water.

Sorption of benzidine and 3,3′‐dichlorobenzidine to lake sediments. 1. conceptualization and development of a multiparameter model

Aromatic amines, such as benzidine and 3,3'-dichlorobenzidine (DCB), are part of the dyes and pigments manufacturing process. The prolonged use of these carcinogenic chemicals in the past generation has introduced a significant amount of contamination to the environment. Their persistency in several mediums has sparked a number of studies in an attempt to develop predictive tools of their fate and transport in the environment. In this study, benzidine and DCB batch isotherms were developed and evaluated. The sediment samples were variable in composition, ranging from sandy to silty-clay sediment samples. The batch isotherms were then analyzed using high-performance liquid chromatography. Subsequently, a multiparameter model (MPM) that accounted for partitioning, covalent bonding, and cation exchange was developed and tested in an effort to understand the various mechanisms. Results proved the proposed model to be effective in predicting sorption of aromatic amines to lake sediments. The findings suggest that the MPM can provide a better understanding of the sorption process of aromatic amines than more conventional models.

Concentrations and distribution of 3,3′-dichlorobenzidine and its congeners in environmental samples from Lake Macatawa

Release of 3,3′-dichlorobenzidine (DCB), an intermediate in dye manufacturing processes, is of environmental concern due to its carcinogenic nature. An 11-year field study has been conducted to elucidate the fate and behavior of DCB and its congeners in the Lake Macatawa (Holland, MI, USA) sediment–water system. The sediments were variable in composition, ranging from sandy sediments with 1–8.7% total organic carbon (OC) to silty-clay sediments with 7.5–20.6% total OC. The pH ranged from 6.3 to 7.4. The findings from this field study confirmed that DCB and its congener, benzidine, are transported over long distances. The concentration of DCB in the water phase was found to be from non-detectable to approximately 1300 times greater than the water quality criteria established for DCB (0.021 μg/l). Its congener, benzidine, which is substantially more toxic for humans than its parent compound has been found up to approximately 12 300 times higher levels than the EPA guidelines suggest (0.000086 μg/l). Both DCB and benzidine were found in the sediment phase. DCB was observed at almost 70 mg/kg in a sample collected in 1993. The concentrations in both phases have declined in recent years. To assess the threat of the released pollutants, it is necessary to understand how pollutants tend to behave in various media. Therefore, it is crucial to identify and quantify all sources of the chemical and its congeners in order to meaningfully predict the fate (and transport) of a hydrophobic organic compound (HOC) in the environment.

Combined micro-electrochemical and biological process for treatment of 3,3?-dichlorobenzidine (DCB) industrial wastewater

Combined micro-electrochemical and biological process for treatment of 3,3?-dichlorobenzidine (DCB) industrial wastewater

Biodegradation of 3,3′-dichlorobenzidine in freshwater lake sediments

3,3′-Dichlorobenzidine (DCB) and its degradation products, 3-chlorobenzidine (MCB) and benzidine, are of environmental concern due to their toxic and carcinogenic nature. Laboratory experiments have been conducted to elucidate the biodegradation behavior of DCB in Lake Macatawa (Holland, Michigan) sediment–water systems at 4, 24, and 30 °C incubation temperatures. Sediment samples varied in pH (6.45–7.41), total organic carbon (OC) content (1.4–17.1), and particle size distribution (silty-clay to sandy). Biodegradation of DCB was observed in non-autoclaved samples that were incubated at 4, 24, and 30 °C for approximately 12 months. The dechlorination rate as a function of OC content was essentially found constant at 4 °C and 24 °C, but demonstrated a roughly linear increase at 30 °C, thereby suggesting that OC content might be correlated to dechlorination rate at 30 °C incubation temperature. The rate as a function of particle size (e.g., &lt;74 µm) did not demonstrate a clearly defined relationship. Key words: 3,3′-dichlorobenzidine, 3-chlorobenzidine, benzidine, biodegradation.

Determination of 3,3'-Dichlorobenzidine in Industrial Wastewaters

Abstract A solid-phase extraction, chemical derivatization, and gas chromatography/mass spectrometry (GC/MS) method has been developed for the determination of trace 3,3'-dichlorobenzidine (DCB) in industrial wastewater samples. Instead of an octadecylsilane (ODS) cartridge, a Porapak RDX cartridge was used since the latter could be optimized for the extraction of DCB. After the pre-concentration, interfering coextractives in the sample were selectively removed from the cartridge by elution with a 1:1 mixture of acetonitrile and water. DCB was then eluted with acetonitrile and derivatized with pentafluoropropionic acid anhydride. The product was subsequently analyzed by GC/MS in either the electron impact or negative ion chemical ionization mode. Recoveries of DCB and the surrogate, DCB-d6, were better than 90% at spiking levels of 10, 1, and 0.1 µg/L. Based on a concentration factor of 100, the detection limits for DCB in wastewater samples were determined to be 0.1 µg/L by electron impact GC/MS detection, and 0.01 µg/L by negative ion chemical ionization GC/MS detection, respectively. This method has been successfully applied to wastewater samples collected in the Toronto area.

Urinary Monitoring Method of 3,3'-Dichlorobenzidine(DCB) and Its Metabolites, N-Acetyl-DCB and N,N'-Diacetyl-DCB.

3,3'-dichlorobenzidine (DCB) is suspected to be arcinogenic in experimental animal and human. Several studies have investigated excretion of metabolites in urine, hemoglobin adduction and cancer incidence among workers exposed occupationally to DCB. In these researches, metabolites of DCB had a very important role. The purpose of this study was to develop the urinary monitoring method of its metabolites from rats exposed with DCB, by easily synthesizing them in the laboratory. N,N'-diacetyl-DCB was easily synthesized with DCB in pyridine by adding sufficient acetyl chloride or acetic anhydride. N-acetyl-DCB was isolated from the supernatant, which made by adding 21/microl acetyl chloride (more 3 times than DCB in moles) to 32 mg DCB in 2 ml pyridine and 0.3 ml acetic acid. Gas chromatography/mass spectrometry (GC/MS) was used for the identification, gas chromatography nitrogen phosphorous detection (GC-NPD) for the quantification and gas chromatography flame ionization detection (GC-FID) for the determination of purity. The base peak of DCB, N-acetyl-DCB and N,N'-diacetyl-DCB was 252 m/z. The other main peaks were 294 m/z for N-acetyl-DCB, and 294 and 336 m/z for N,N'-diacetyl-DCB. The purities of N-acetyl-DCB and N,N'-diacetyl-DCB were identified as 98.82 and 98.72% by GC-FID, respectively. After treatment orally to rats with 20 mg DCB/kg body weight for 2 weeks, the urinary excretion amount of DCB was nearly constant at range of 0.11-0.18 mg/L for 2 weeks. But excretion of N-acetyl-DCB was continually increased from 1.30 mg/L on 1st day to 4.15 mg/L on 14th day. And level of N,N'-diacetyl-DCB in urine was sharply increased from 2.13 mg/L on 1st day to 11.00 mg/L on 14th day.

Transport behavior of 3,3′‐dichlorobenzidine in a freshwater estuary

Like many hydrophobic organic compounds, 3,3'-dichlorobenzidine (DCB) partitions preferentially to (sediment) particles in lake systems. As such, the behavior of DCB in these systems is substantially affected by the movement of sediments. A field investigation of DCB distribution in sediments of Lake Macatawa (Holland, MI, USA) was initiated. The pattern of DCB distribution within the lake was found to display an oscillatory pattern that was consistent with a wind-driven mechanism of sediment transport. Numerical modeling of seiching behavior supported the hypothesized importance of this mechanism of sediment transport and redistribution. The dynamic behavior of sediment-associated DCB within Lake Macatawa seems to be strongly influenced by phenomena that are common to many freshwater estuaries. As such, the behavior of this system is expected to represent a reasonable model of the dynamic behavior of hydrophobic contaminants in other freshwater estuaries.

TRANSPORT BEHAVIOR OF 3,3′-DICHLOROBENZIDINE IN A FRESHWATER ESTUARY

Like many hydrophobic organic compounds, 3,3′-dichlorobenzidine (DCB) partitions preferentially to (sediment) particles in lake systems. As such, the behavior of DCB in these systems is substantially affected by the movement of sediments. A field investigation of DCB distribution in sediments of Lake Macatawa (Holland, MI, USA) was initiated. The pattern of DCB distribution within the lake was found to display an oscillatory pattern that was consistent with a wind-driven mechanism of sediment transport. Numerical modeling of seiching behavior supported the hypothesized importance of this mechanism of sediment transport and redistribution. The dynamic behavior of sediment-associated DCB within Lake Macatawa seems to be strongly influenced by phenomena that are common to many freshwater estuaries. As such, the behavior of this system is expected to represent a reasonable model of the dynamic behavior of hydrophobic contaminants in other freshwater estuaries.

Determination of hemoglobin adducts formed in rats exposed orally with 3,3'-dichlorobenzidine by GC/MS-SIM.

3,3'-dichlorobenzidine (DCB) can be metabolically N-acetylated and/or N-oxidized, and can form hemoglobin adducts. Gas chromatography/mass spectrometry-selected ion monitoring detection mode (GC/MS-SIM) could be a good analytical method to detect them. 4-Aminobiphenyl and phenanthrene-d10 were used as internal standards, and standard metabolites of DCB were synthesized from DCB. Pyridine is a promoter and acetic acid is a controller in the acetylation of DCB during titrating with acetyl chloride. After washing with acetone, the purity of N-acetyl DCB and N,N'-diacetyl DCB were 98.72% and 98.82%, respectively. The maximum detection limits (MDLs) by GC/MS-SIM were 0.5 microg/L in DNA adduct and 1.0 ng/g in hemoglobin for DCB and N-acetyl DCB. The base peaks of their fragmentation pattern were 252 m/z at the peak of DCB, 252 m/z and 294 m/z at the peak of N-acetyl DCB, and 252 m/z, 294 m/z, and 336 m/z at the peak of N,N'-diacetyl DCB. This analytical method was applied to determine hemoglobin adducts formed in young female Sprague-Dawley rats orally exposed with 20, 30, and 40 mg DCB/kg/day for three weeks. Two adducts were detectable by GC/MS-SIM after alkaline hydrolysis of hemoglobin samples and extraction. The structure of these adducts could be assigned to DCB and N-acetyl DCB by co-chromatography with the synthetic standards. After the first week of treatment, the total amount of hemoglobin adducts determined was 837.5 approximately 2501 ng/g hemoglobin. The adduct levels were increased up to 1203.3 approximately 2605.4 ng/g after the second week, and slightly decreased after the third week. The ratio of DCB and N-acetyl DCB was nearly similar in all treatment groups at the third week, such as 4.28 approximately 4.78. Three different treatments (20, 30, and 40 mg DCB/kg) of rats resulted in dose-proportional increases in the total and DCB amount of hemoglobin adducted formed for three weeks. The relative contribution of DCB and N-acetyl DCB to the total hemoglobin adduct level was strongly dose dependent. The results show that GC/ MS-SIM is suitable for the biological monitoring of humans exposed to DCB or DCB-containing products.