Quantification of systemic o-toluidine after intrathecal administration of hyperbaric prilocaine in humans: a prospective cohort study.

Hyperbaric 2% prilocaine is increasingly used for spinal anesthesia. It is the only local anesthetic metabolized to o-toluidine, a human bladder carcinogen. Increase of o-toluidine hemoglobin adducts, a marker of o-toluidine ability to modify the DNA structure, was described following subcutaneous injection. In this prospective cohort study we aimed to assess and quantify o-toluidine hemoglobin adducts and urinary o-toluidine after a single intrathecal dose of hyperbaric prilocaine.10 patients undergoing surgery received 50 mg of hyperbaric prilocaine intrathecally. Blood and urine samples were collected before injection and up to 24 h later (Hospital Braine l'Alleud-Waterloo, Braine l'Alleud, Belgium). Urinary o-toluidine and o-toluidine hemoglobin adducts were measured by tandem mass-spectrometry after gas-chromatographic separation (Institute of the Ruhr-Universität, Bochum Germany). The trial was registered to ClinicalTrials.gov (NCT03642301; 22-08-2018)Intrathecal administration of 50 mg of hyperbaric prilocaine leads to a significant increase of o-toluidine hemoglobin adducts (0.1 ± 0.02-11.9 ± 1.9 ng/g Hb after 24 h, p = 0.001). Peak of urinary o-toluidine was observed after 8 h (0.1 ± 0.1-460.5 ± 352.8 µg/L, p = 0.001) and declined to 98 ± 66.8 µg/L after 24 h (mean ± SD)Single intrathecal administration of hyperbaric prilocaine leads to a systemic burden with o-toluidine and o-toluidine hemoglobin adducts. O-toluidine-induced modifications of DNA should be examined and intrathecal hyperbaric prilocaine should not be proposed to patients chronically exposed to o-toluidine.Clinical trial number and registry URL NCT03642301.

Novel o-Toluidine Metabolite in Rat Urine Associated with Urinary Bladder Carcinogenesis.

o-Toluidine (o-Tol), a monocyclic aromatic amine, causes bladder cancer in humans and experimental animals and is therefore classified as a Group 1 carcinogen (IARC) in which the carcinogenicity of o-Tol is involved in metabolic activation, DNA damage, and DNA adduct formation. In the DNA adduct formation mechanism, o-Tol is metabolized by N-hydroxylation, N-acetoxylation, and then deacetoxylation to produce an electrophilic nitrenium ion, which is able to bind to a DNA base, such as dG-C8. Therefore, dG-C8-o-Tol is thought to be a plausible DNA adduct of o-Tol exposure. However, direct detection of dG-C8-o-Tol in biological samples has not been reported yet. Here, we show that a novel o-Tol metabolite, 2-methyl-N1-(2-methylphenyl)benzene-1,4-diamine (MMBD), a dimer by head-to-tail binding, was identified for the first time in o-Tol-exposed rat urine. MMBD was also detected in a reaction of o-Tol and S9 mix, indicating the formation was catalyzed by an enzymatic reaction. Moreover, MMBD showed a potent stronger mutagenicity in N-acetyltransferase overexpressed Salmonella typhimurium strains,and cytotoxicity in human bladder carcinoma T24 cells and human spleen lymphoblastoid TK6 cells compared with o-Tol. Furthermore, a DNA adduct (m/z 478.1) corresponding to dG-MMBD was detected in the reaction of calf thymus DNA with rat urine containing MMBD, and also in hepatic DNA of rats treated with o-Tol. These results therefore suggested that o-Tol-induced bladder carcinogenesis could be at least partly attributed to MMBD formation. The possible dimerization of monocyclic aromatic amines should be considered in the evaluation of the risk of bladder carcinogenesis after exposure.

Biological monitoring of o-toluidine in urine pretreated by an enzymatic deconjugation method.

OBJECTIVES: To establish an enzymatic deconjugation method to separately quantify urinary o-toluidine (OT), its six metabolites, another six chemicals present in an OT-processing plant, and one metabolite of p-toluidine, and to propose optimal urinary biological monitoring items of OT exposure. METHODS: Thirty-six urine samples of an OT-processing plant's workers were obtained and pretreated by an enzymatic deconjugation method employing ß-glucuronidase/arylsulfatase for 3 hours at 37°C and measured by liquid chromatograph-mass spectrometry (LC-MS). An alkaline hydrolytic pretreatment and 1-chlorobutane extraction procedure was also examined as a widely used urinary OT measurement method. RESULTS: The 14 chemicals were separated by LC-MS condition set by us and 13 chemicals other than 2-chloroaniline showed satisfiable linearity and limits of determination. Standard substances of six OT metabolites decomposed after the alkaline heating. In the 36 urine samples, OT, N-(4-hydroxy-2-methylphenyl) acetamide (NHM), and 4-amino-m-cresol (ACR) accounted for approx. 90% of the total OT and OT metabolites, but inter-individual variation of the three substance excretion seemed to be wide. Time course of urinary excretion revealed that concentration of the three substances was higher 24 hours after the work shift's end rather than just after the work shift. CONCLUSIONS: OT and its six metabolites can each be determined with LC-MS. The alkaline method is not so optimal for exact biological monitoring. Rather, the sum of urinary OT, NHM, and ACR measured by the enzymatic method is a better index, and "end of the workweek" is a good urine-sampling time for the biological monitoring of OT exposure.

Development of a simple HPLC-MS/MS method to simultaneously determine teriflunomide and its metabolite in human plasma and urine: Application to clinical pharmacokinetic study of teriflunomide sodium and leflunomide.

A simple high-performance liquid chromatography coupled with tandem mass spectrometry method was developed and fully validated to simultaneously determine teriflunomide (TER) and its metabolite 4-trifluoro-methylaniline oxanilic acid (4-TMOA) in human plasma and urine. Merely 50 µL plasma and 20 µL urine were employed in sample preparation using protein precipitation and direct dilution method, respectively. An Agilent Zorbax eclipse plus C18 column was selected to achieve rapid separation for TER and 4-TMOA within 3 min. Electrospray ionization under multiple reaction monitoring was used to monitor the ion transitions for TER (m/z 269.0 → 159.9), 4-TMOA (m/z 231.9 → 160.0), internal standard teriflunomide-d4 (m/z 273.0 → 164.0) and 2-amino-4-trifluoromethyl benzoic acid (m/z 203.8 → 120.1), operating in the negative ion mode. This method proved to have better accuracy and precision over concentration range of 10-5000 ng/mL in plasma as well as 10-10,000 ng/mL in urine. After a full validation, this method was successfully applied in a pharmacokinetic study of teriflunomide sodium and leflunomide in Chinese healthy volunteers.

Comparison of Bladder Carcinogens in the Urine of E-cigarette Users Versus Non E-cigarette Using Controls.

Electronic cigarette (EC) use is gaining popularity as a substitute for conventional smoking due to the perception and evidence it represents a safer alternative. In contrast to the common perception amongst users that ECs represent no risk initial studies have revealed a complex composition of e-cigarette liquids. Conventional cigarette smoking is a known risk factor for developing bladder cancer and prior reports raise concern some of those causative compounds may exist in EC liquids or vapor. Urine samples were collected from 13 e-cigarette using subjects and 10 non e-cigarette using controls. Five known bladder carcinogens that are either present in conventional cigarettes, products of combustion, or solvents believed to be used in some e-cigarette formulations were quantified by liquid chromatography - mass spectrometry (LC-MS). Analysis of e-cigarette user urine revealed the presence of two carcinogenic compounds, o-toluidine and 2-naphthylamine, at a mean 2.3 and 1.3 fold higher concentration (p-value of 0.0013 and 0.014 respectively). Many of these subjects (9/13) were long term nonsmokers (>12 months). Further study is needed to clarify the safety profile of e-cigarettes and their contribution to the development of bladder cancer given the greater concentration of carcinogenic aromatic amines in the urine of e-cigarette users.

Simultaneous determination of amitraz, chlordimeform, formetanate and their main metabolites in human urine by high performance liquid chromatography-tandem mass spectrometry.

A rapid, simple and reliable high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for simultaneous determination of amitraz, chlordimeform, formetanate and their main metabolites, N-(2,4-dimethylphenyl)-N-methyl-formamidine (DMPF), 2,4-dimethylformamidine (DMF), 2,4-dimethylaniline (DMA), 4-chloro-2-methylaniline and 3-hydroxyacetanilide in human urine. The urine samples were mixed with buffer solutions (pH 8) and subsequently cleaned up by solid supported liquid/liquid extraction (SLE). The target analytes were efficiently separated with a Waters Atlantis T3 column (150mm×4.6mm, 5µm), ionized with electrospray ion source in positive mode, and quantitatively determined by tandem mass spectrometry in the multiple reaction monitoring (MRM) mode. In order to minimize matrix effects, the matrix-matched calibration curves of eight analytes were adopted with correlation coefficients (R2) above 0.99. The method were further validated by determining the limits of detection (LODs, 0.3-0.6ng/mL), the limits of quantitation (LOQs, 1.0-2.0ng/mL) and recoveries (89.1%-108.4%) with intra-day and inter-day relative standard deviation (RSD, <11%). The established method was applied and demonstrated in a real case by assaying a urine sample from a female poisoned by formetanate. The achieved results proved this method to be rapid, sensitive and accurate for simultaneous quantitation of eight analytes in human urine for intended forensic cases of human poisoning.

Biomonitoring of human exposure to arylamines.

Extensive industrial use of arylamines started in the middle of the 19th century in the dye industry. Because of the high incidence of bladder cancer, arylamines belong to the first and most intensively studied occupational and environmental carcinogens. In workers, biomonitoring of exposure to arylamines including ortho-toluidine started in the first half of the 20th century. This review highlights the many gaps in our knowledge on the human carcinogen ortho-toluidine.

Environmental determinants of the urinary concentrations of herbicides during pregnancy: the PELAGIE mother-child cohort (France).

Herbicides are generally the most extensively used of the pesticides applied to agricultural crops. However, the literature contains little evidence useful in assessing the potential sources of the general population's exposure to herbicides, including by residential proximity to crops. The objective of this study was to take advantage of data from the PELAGIE mother-child cohort to identify the main determinants of the body burden of exposure to the chloroacetanilide and triazine herbicides commonly used on corn crops in Brittany, France, before 2006. Urine samples from a randomly selected subcohort of women in the first trimester of pregnancy (n=579) were assayed for herbicide metabolites. The residential exposure resulting from proximity to corn crops was assessed with satellite-image-based scores combined with meteorological data. Data on diet, drinking tap water (from the public water supply), occupations, and household herbicide use were collected by questionnaires. Herbicides were quantified in 5.3% to 39.7% of urine samples. Alachlor and acetochlor were found most frequently in the urine of women living in rural areas. The presence of dealkylated triazine metabolites in urine samples was positively associated with residential proximity to corn crops (OR=1.38, 95% CI: 1.05-1.80). Urinary metabolites of both atrazine and dealkylated triazine were correlated with tap water consumption (OR=2.94, 1.09-7.90, and OR=1.82, 1.10-3.03, respectively); hydroxylated triazine metabolites were correlated with fish intake (OR=1.48, 1.09-1.99). This study reinforces previous results that suggest that environmental contamination resulting from agricultural activities may contribute to the general population's exposure to herbicides.

Drug peptide conjugates in human urine?

1. Once in a while, during drug metabolism studies, an unusual or unexpected pathway is unearthed. 2. Such quirky finds open a refreshing hiatus, providing a departure from the, perhaps now mundane, textbook routes. 3. This brief missive draws attention to an interesting anecdote that may be unknown to some and concerns a substituted thioxanthenone drug.

Determination of 2,5-toluylenediamine (2,5-TDA) and aromatic amines in urine after personal application of hair dyes: kinetics and doses.

The personal use of hair dye products is currently under discussion due to the potentially increased risk of bladder cancer among long-time users described in epidemiological literature. In order to investigate the dermal absorption of aromatic diamines as well as aromatic amines possibly present as contaminants in hair dye formulations, we conducted a biomonitoring study under real-life conditions and calculated kinetics and doses for the urinary excretion. Urine samples of two female subjects were collected for a time period of 48 h after personal application of a hair dye cream and analysed for aromatic diamines as well as o-toluidine and 4-aminobiphenyl using highly specific GC/MS-methods. 2,5-Toluylenediamine (2,5-TDA) as active ingredient of hair dyes is rapidly absorbed dermally. After a distribution phase of 12 h, 2,5-TDA is excreted with a half-time of 8 h. Excretion was 90% complete within 24 h after application. The doses of 2,5-TDA excreted within 48 h were 700 µg for application of a brown-reddish hair dye cream and 1.5 mg for the application of a brown-black hair dye cream. Urinary 4-aminobiphenyl as well as contaminations with other aromatic diamines were not detectable in our study. Due to the artifactual formation of o-toluidine in the presence of high concentrations of urinary 2,5-TDA, our results could not prove an increased internal exposure of humans to carcinogenic amines after personal application of hair dyes.

Monoarylamines in the general population--a cross-sectional population-based study including 1004 Bavarian subjects.

AIM: The population-based cross-sectional study including 1004 Bavarian volunteers aged 3 up to 84 years (median: 42 years) was aimed to quantify the internal burden of monocyclic arylamines in the general population and to yield reference values. MATERIAL AND METHODS: Participants were asked to complete a questionnaire, to give a venous blood sample and a urinary sample. The selected monoarylamines (aniline, o-anisidine, all isomers of toluidine, single and double chlorinated anilines) represent main sources of potential environmental exposure. The venous blood sample was taken to determine the smoking-specific acrylonitrile-adduct N-cyanoethylvaline. RESULTS: Detectable levels of aniline were found in the urine of 93.9% of the participants, whereas 3-chloroaniline was only detected in 16% of the samples. The influence of smoking on the urinary arylamine concentration was weak. Only for o-toluidine, m-toluidine and o-anisidine values were significantly higher in smokers. Therefore, while the 95th percentile based on the total sample (n=1004) is the best reference value for all other arylamines (i.e. p-toluidine, 3-chloroaniline, 4-chloroaniline, 3,4-dichloroaniline) we suggest separate reference values for smokers and non-smokers for the former three compounds. A statistically significant difference in urinary arylamine concentration between men and women was observed for 3,5-dichloroaniline, o-anisidine and aniline (p<0.001). Therefore we suggest gender-specific reference values for dichloroaniline and aniline; for o-anisidine we suggest gender- and smoking-specific reference values. The observation of o-toluidine in 178 urinary samples in concentration above the limit of quantification raises concern regarding human carcinogenicity. CONCLUSION: This study supports the notion of further relevant sources of o-toluidine exposure except smoking and occupation. Compared to other environmental risk factors (e.g. environmental tobacco smoke) the risk of o-toluidine-induced cancer seems to be extremely low for the general population.

Identification of human urinary metabolites of acetochlor in exposed herbicide applicators by high-performance liquid chromatography-tandem mass spectrometry.

Acetochlor is a preemergent chloroacetanilide herbicide used to control annual grasses and small-seeded broadleaf weeds. It is the second most abundantly applied herbicide on corn crops in the United States; however, human metabolites associated with known exposure to acetochlor have not been positively identified and confirmed. We positively identified acetochlor mercapturate (ACM) as a metabolite of acetochlor in urine samples collected during a 24-h period from custom (commercial) applicators who had applied acetochlor on either the day of or the day before urine collection. Concentrations in applicator urine samples ranged from 0.5 to 449 microg/l (0.3-121 microg/g creatinine). We found that ACM accounted for as much as 42% of the total acetochlor-derived metabolites; however, as the exposure level decreased (based on total acetochlor metabolite level), ACM became a less abundant metabolite of acetochlor (<17%). Unmetabolized acetochlor was also measured in the urine samples analyzed. At high exposures (classified as >100 microg/l), acetochlor accounted for about 0.8% of the total excreted acetochlor metabolites (approximately 2% of the ACM concentrations). At lower exposures (classified as ACM<10 microg/l), ACM and acetochlor concentrations were similar. Additionally, we tentatively identified another acetochlor metabolite that appeared to be important at low levels of exposure.

Identification of urinary metabolites of orally administered N,N-dimethyl-p-toluidine in male F344 rats.

The metabolism of orally administered N,N-dimethyl-p-toluidine (DMPT) in male F344 rats was investigated. The rat urinary metabolite profile was determined by analytical reverse-phase high performance liquid chromatography (HPLC). Four radiolabeled peaks were observed, isolated, and purified by solid-phase extraction (SPE) and preparative HPLC methods. The 4 peaks were identified as p-(N-acetylhydroxyamino)hippuric acid (M1), DMPT N-oxide (M2), N-methyl-p-toluidine (M3), and parent DMPT. Metabolites M1 and M2 were identified by spectrometric and spectroscopic methods, including mass fragmentation pattern identification from both liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry, and from chemical analysis of nuclear magnetic resonance spectra. Structural confirmation of metabolite M2 was accomplished by comparison with a synthetic standard. Peaks M3 and the peak suspected to be DMPT were identified by comparison of their HPLC retention times and mass fragmentation patterns with authentic standards of N-methyl-p-toluidine and DMPT, respectively. DMPT metabolism is similar to that reported for N,N-dimethylaniline.

Percutaneous absorption of crotamiton in man following single and multiple dosing.

PURPOSE: Crotamiton is a topical drug used in the treatment of scabies and pruritus. We determined its percutaneous absorption following single and multiple dosing in normal skin. METHODS: We used in vivo measurement of percutaneous absorption of [14C] crotamiton in a multidose regimen by measuring urinary excretion and liquid scintillation counting in three groups of four healthy volunteers. The Feldmann urinary excretion method was utilized to ascertain percutaneous absorption. Our results showed that tape stripping does not increase percutaneous absorption of crotamiton; upon repeated application.

Determination of three carcinogenic aromatic amines in urine of smokers and nonsmokers.

Aromatic amines (arylamines) such as o-toluidine, 2-aminonaphthalene, and 4-aminobiphenyl occur in the environment and are constituents of tobacco smoke. Human exposure to these aromatic amines has long been associated with an elevated risk of bladder cancer. A validated, specific, and sensitive method for measuring o-toluidine, 2-aminonaphthalene, and 4-aminobiphenyl in cigarette smokers and nonsmokers was developed. The method uses acid hydrolysis of the arylamine conjugates in urine, extraction with n-hexane, derivatization with pentafluoropropionic anhydride, and subsequent analysis with gas chromatography combined with mass spectrometry using negative ion chemical ionization. The limits of detection were 4 ng/L for o-toluidine and 1 ng/L for 2-aminonaphthalene and 4-aminobiphenyl. Smokers (N = 10) excreted significantly higher amounts of o-toluidine (204 versus 104 ng/24 h), 2-aminonaphthalene (20.8 versus 10.7 ng/24 h), and 4-aminobiphenyl (15.3 versus 9.6 ng/24 h) than nonsmokers (N = 10). Urinary arylamine excretion in smokers was associated with the extent of smoking as assessed by daily cigarette consumption, urinary excretion of nicotine equivalents (nicotine plus its five major metabolites), cotinine in saliva, and carbon monoxide in exhaled breath. All nonsmokers investigated had quantifiable amounts of o-toluidine, 2-aminonaphthalene, and 4-aminobiphenyl in their urine, confirming that other environmental sources of exposure to these compounds also occur. In conclusion, the analytical method is suitable for measuring short-term exposure to arylamines in urine of non-occupationally exposed smokers and nonsmokers.

Quantification of six herbicide metabolites in human urine.

We developed a sensitive, selective and precise method for measuring herbicide metabolites in human urine. Our method uses automated liquid delivery of internal standards and acetate buffer and a mixed polarity polymeric phase solid phase extraction of a 2 mL urine sample. The concentrated eluate is analyzed using high-performance liquid chromatography-tandem mass spectrometry. Isotope dilution calibration is used for quantification of all analytes. The limits of detection of our method range from 0.036 to 0.075 ng/mL. The within- and between-day variation in pooled quality control samples range from 2.5 to 9.0% and from 3.2 to 16%, respectively, for all analytes at concentrations ranging from 0.6 to 12 ng/mL. Precision was similar with samples fortified with 0.1 and 0.25 ng/mL that were analyzed in each run. We validated our selective method against a less selective method used previously in our laboratory by analyzing human specimens using both methods. The methods produced results that were in agreement, with no significant bias observed.

Effect of dietary fat on selected parameters of toxicity following 1- or 3-month exposure of rats to toluidine isomers.

The aim of the study was to investigate the effect of the dietary fat on selected parameters of toluidines toxicity in rats during subchronic exposure. Three isomers of toluidine (ortho, meta, and para) were administered to rats in the diet for 1 and 3 months at levels 40, 80, 160 mg/kg/day in two kinds of diet containing either 4% or 14% fat. All doses of toluidine isomers produced a 1.5- to 9.8-fold increase in methemoglobin (MetHb) level during both treatment periods. A distinct dose-response relationship was observed, especially for o- and m-toluidine; the effect was generally greater in rats fed high-fat diet. Reduced glutathione level in liver was increased in all treated groups, 1.5- to 5.1-fold, irrespective of the kind of diet. An increase in hepatic lipid peroxidation (thiobarbituric acid reactive substances; TBARS), 1.5- to 4.5-fold, was noticed in the majority of the treated groups. Generally, there was no consistent effect of diet except for p-toluidine where the level of hepatic TBARS was lower in rats fed high-fat diet. Blood urea nitrogen (BUN) level in animals treated with all doses of o- and m-toluidine was 1.3- to 5.0-fold higher in comparison with respective controls. No clear relationship between BUN level and the kind of diet was found. No effect of toluidines on the activity of serum aspartate aminotransferase (AST) and sorbitol dehydrogenase (SDH) were observed. In the majority of groups treated for 30 and 90 days the amount of toluidines in 24-h urine was lower in rats fed high-fat diet. Final body weight gain in rats treated with o- and p-toluidine (80 and 160 mg/kg body weight [b.w.]) was lower as compared to controls. In summary the high-fat diet stimulated methemoglobin formation in rats treated with o- and m-toluidine and cause the decrease in the amount of toluidines in 24-h urine. The high content of fat did not affect consistently the other parameters tested.

Development of a method for the detection and confirmation of the alpha-2 agonist amitraz and its major metabolite in horse urine.

Amitraz (N'-(2,4-dimethylphenyl)-N-[[(2,4-dimethylphenyl)imino]methyl]-N-methyl-methanimidamide) is an alpha-2 adrenergic agonist used in veterinary medicine primarily as a scabicide- or acaricide-type insecticide. As an alpha-2 adrenergic agonist, it also has sedative/tranquilizing properties and is, therefore, listed as an Association of Racing Commissioners International Class 3 Foreign Substance, indicating its potential to influence the outcome of horse races. We identified the principal equine metabolite of amitraz as N-2,4-dimethylphenyl-N'-methylformamidine by electrospray ionization(+)-mass spectrometry and developed a gas chromatographic-mass spectrometric (GC-MS) method for its detection, quantitation, and confirmation in performance horse regulation. The GC-MS method involves derivatization with t-butyldimethylsilyl groups; selected ion monitoring (SIM) of m/z 205 (quantifier ion), 278, 261, and 219 (qualifier ions); and elaboration of a calibration curve based on ion area ratios involving simultaneous SIM acquisition of an internal standard m/z 208 quantifier ion based on an in-house synthesized d(6) deuterated metabolite. The limit of detection of the method is approximately 5 ng/mL in urine and is sufficiently sensitive to detect the peak urinary metabolite at 1 h post dose, following administration of amitraz at a 75-mg/horse intravenous dose.

Biological monitoring of phenmedipham: determination of m-toluidine in urine.

Phenmedipham [methyl-3-(3-methylphenylcarbamoyloxy)carbamate] is used as a herbicide, especially in the growing of sugar beet and strawberries. During metabolism of the substance in rats, the two carbamate moieties of phenmedipham are cleaved and the metabolites methyl-N-(3-hydroxyphenyl)-carbamate, m-aminophenol and hydroxyacetanilide are formed. These compounds and their conjugates are excreted in urine. Additionally, it has been suggested that m-toluidine is formed during metabolism. For the first time it has been possible to detect this metabolite in the urine of workers after agricultural use of phenmedipham. The concentrations of m-toluidine in urine were significantly higher in persons occupationally exposed than in controls. The median values for each group were 0.36 microg/l and 0.16 microg/l, respectively. This means that persons not exposed to phenmedipham also excrete m-toluidine, possibly as a result of the uptake of pesticides like phenmedipham from the diet.

Gas chromatographic method for the determination of toluidines in spiked urine samples.

A capillary gas-chromatographic method was developed for the analysis of a mixture of toluidines in urine. The method is based on the extraction of toluidines with toluene and derivatisation with heptafluorobutyric anhydride to form a product for electron capture detection. The procedure gave a linear response at concentrations of 0.02-0.20 microg/ml with sufficient reproducibility. The method is simple, requires little sample pretreatment and is being considered for biomonitoring workers exposed to toluidines.