Automated column-switching high-performance liquid chromatography method for the determination of 1-hydroxypyrene in human urine

An on-line sample treatment method to determine 1-hydroxypyrene (1-OHP), a metabolite of polycyclic aromatic hydrocarbons (PAHs), in human urine has been developed. The hydrolysed biological fluid was directly injected into the chromatographic system after only centrifugation. A miniature precolumn loop packed with a preparative phase and coupled on-line to a liquid chromatographic (LC) system was used for analyte enrichment. The analytes were non-selectively desorbed with the LC eluent and cleaned by means of a column-switching procedure comprising two purification columns and an analytical column. Pre-treatment and analysis were performed within 2 and 20 min, respectively. Average 1-OHP recovery reached 99% in the 1–25 μg/l range of urine, and the quantitation limit was 20 ng/l for 100 μl of injected sample. A comparison with a more time-consuming off-line method was performed by analysing 120 urine samples of PAH-exposed and expected unexposed workers; the statistical treatment indicated that both methods are in agreement.     

Trace determination of urinary 3-hydroxybenzo[a]pyrene by automated column-switching high-performance liquid chromatography

3-Hydroxybenzo[a]pyrene (3-OHB[a]P), one of the metabolites of benzo[a]pyrene (B[a]P), has been determined in human urine using an automated column-switching procedure. The hydrolysed biological sample is centrifuged just prior to being injected into a reusable precolumn loop, which is packed with a preparative phase and coupled on-line to a liquid chromatographic (LC) system. A rapid pre-treatment of the hydrolysed sample, consisting of a concentration and a crude clean-up, is performed on the precolumn. The analytes are then non-selectively desorbed with the LC eluent and the sample is cleaned again in three successive purification columns using the direct transfer or “heart-cut” technique. The pre-treatment does not exceed 3 min. and the entire analytical purification and separation procedure takes less than 30 min. Average 3-OHB[a]P recovery reaches 95% in the 1–50 ng/l range of urine, and the detection limit is 0.1 ng/l urine for a 3 ml injection of hydrolysed urine. The developed method was compared with a more time-consuming off-line method to analyse urines of B[a]P gavaged rats; the statistical treatment indicates that both methods are in agreement. The method was applied to purify and concentrate the urine samples of workers exposed and apparently unexposed to polycyclic aromatic hydrocarbons (PAHs).     

Immunodetection and immunopurification of aflatoxins using a high affinity monoclonal antibody to aflatoxin B1

A monoclonal antibody was obtained from BALB/c mice immunized with aflatoxin Bl (AFB1) conjugated to bovine serum albumin. This IgG2a antibody, ASCI, with K light chain has a high specificity for AFB1. In an indirect enzyme-linked immunosorbent assay the antibody litre in ascites fluid was 1: 6000 for 50% binding to plates coated with aflatoxin-poly-L-lysine. The assay is sensitive to 2.5 pg aflatoxin/assay. ASCI cross-reacts with closely related aflatoxin metabolites such as AFB2, AFM1 and AFG1. However, ASCI displays negligible cross-reactivity with other related aflatoxin analogues such as AFM2, AFP1, AFQ1 and aflatoxicol. An immunoabsorbent was prepared by coupling ASCI antibody to Ultrogel AcA 22. This immunomatrix was used to purify aflatoxins at 0–1 ng/ml levels from contaminated body fluids such as bovine milk. The antibody affinity column was regenerated and re-used several times. Owing to its high specificity for AFB1 and AFM1, ASCI will be of value in immunodetection and immunopurification of these toxins in various foodstuffs.     

Aflatoxin Binders II: Reduction of aflatoxin M1 in milk by sequestering agents of cows consuming aflatoxin in feed

Sequestering agents bind dietary aflatoxin B1 (AFB1) and reduce absorption from an animal's gastrointestinal tract. As a result, they protect an animal from the toxic effects of AFB1 and reduce transfer of the metabolite, aflatoxin M1 (AFM1), into milk. Three experiments, using late-lactation Holstein cows fed AFB1-contaminated feed, were conducted to evaluate several potential sequestering agents for their abilities to prevent or reduce the transmission of AFM1 into milk. Six agents previously tested in our laboratory for AFB1 binding in vitro were evaluated in these experiments. These were: SA-20, an activated carbon (AC-A); Astra-Ben-20, a sodium bentonite (AB-20); MTB-100, an esterified glucomannan (MTB-100); Red Crown, a calcium bentonite (RC); Flow Guard, a sodium bentonite (FG); and Mycrosorb, a sodium bentonite (MS). Five of the six sequestering agents significantly (P < 0.01) reduced AFM1 contamination of milk (AB-20, 61%; FG, 65%; MS, 50%; MTB-100, 59%; and RC, 31%); whereas, AC-A, activated carbon, had no effect on AFM1 transmission at 0.25% of feed. By the first milking (1 day after cows consumed contaminated feed), AFM1 appeared in milk, then reached maximum levels after three days, and was absent from milk within four days after AFB1 was removed from the feed. Sodium bentonites at 1.2% of feed showed good potential as AFB1 binders; MTB-100, a yeast cell wall product, was equally effective at 0.05% in feed. Potential AFB1 binding agents should be evaluated experimentally to demonstrate efficacy. Our data show that sequestering agents can reduce AFM1 in milk of cows fed AFB1-contaminated feed.     

Highly sensitive and rapid determination of theophylline, theobromine and caffeine in human plasma and urine by gradient capillary high-performance liquid chromatography-frit-fast atom bombardment mass spectrometry

A sensitive and reliable analytical procedure has been established for the detection of theophylline (TH), theobromine (TB) and caffeine (CA) in human plasma and urine by gradient capillary high-performance liquid chromatography (HPLC)-frit-fast atom bombardment mass spectrometry (FAB-MS) (LC-frit-FAB-MS). Two capillary columns and a column-switching valve were used in this LC system to allow all of the sample injected to be introduced into the MS system. 7-Ethyltheophylline was used as the internal standard (I.S.). The xanthines in the specimen were extracted with an Extrelut column. The lowest detected amount was ca. 5 ng/ml using this method.     

Determination of 3-methoxy-4-hydroxyphenylethylene glycol in urine using reversed-phase liquid chromatography with column switching and electrochemical detection

A column-switching method was developed for the determination of total 3-methoxy-4-hydroxy-phenylethyleneglycol (MHPG) in urine. This was performed by first treating samples with β-glucuronidase, followed by extraction with ethyl acetate. The reconstituted extracts with injected onto an HPLC system containing an amperometric detector and tandem Nucleosil C₁₈ and C₈ reversed-phase columns connected by a switching valve. The total analysis time for MHPG was 12 min. The limit of detection was 0.18 ng, or 9 μg/l for 20-μl injections of a 1.0-ml reconstituted extract prepared from 1.0 ml of urine. The linear range extended up to 80 mg/l. The within-day precision for a urine sample containing 170 μg/l total MHPG was ±6% and the day-to-day precision was ±15%. The average levels determined by this method for total MHPG in normal subjects showed good agreement with previous literature values. This approach could be modified for the determination of free MHPG by using only ethyl acetate extraction for sample pretreatment.     

Carryover of aflatoxin from feed to milk in dairy cows with low or high somatic cell counts

Aflatoxin M1 (AFM1) residues in milk are regulated in many parts of the world and can cost dairy farmers significantly due to lost milk sales. Additionally, due to the carcinogenicity of this compound contaminated milk can be a major public health concern. Thirty-four lactating dairy cows were utilised to investigate the relationship between somatic cell counts (SCC), milk yield and conversion of dietary aflatoxin B1 (AFB1) into milk AFM1 (carryover (CO)). The AFM1 in milk increased as soon as the first milking after animal ingestion with a pattern of increment up to the observed plateau (between 7th and 12th days of AFB1 ingestion). There was a significant (P < 0.01) effect of the milk yield whereas no effect could be attributed to the SCC levels or to the milk yield × SCC interaction. Similarly, the main effect of milk yield was also observed (P < 0.01) on the total amount of AFM1 excreted during the ingestion period. Although the plasma concentration of gamma-glutamyl transferase was significantly affected by aflatoxin administration, levels of this liver enzyme were within the normal range for lactating dairy cows. The current data suggest that milk yield is the major factor affecting the total excretion of AFM1 and that SCC as an indicator of mammary gland permeability was not related to an increase in AFM1 CO.     

Vaccination of lactating dairy cows for the prevention of aflatoxin B1 carry over in milk

The potential of anaflatoxin B(1) (AnAFB(1)) conjugated to keyhole limpet hemocyanin (KLH) as a vaccine (AnAFB(1)-KLH) in controlling the carry over of the aflatoxin B(1) (AFB(1)) metabolite aflatoxin M(1) (AFM(1)) in cow milk is reported. AFB(1) is the most carcinogenic compound in food and foodstuffs amongst aflatoxins (AFs). AnAFB(1) is AFB(1) chemically modified as AFB(1)-1(O-carboxymethyl) oxime. In comparison to AFB(1), AnAFB(1) has proven to be non-toxic in vitro to human hepatocarcinoma cells and non mutagenic to Salmonella typhimurium strains. AnAFB(1)-KLH was used for immunization of cows proving to induce a long lasting titer of anti-AFB(1) IgG antibodies (Abs) which were cross reactive with AFB(1), AFG(1), and AFG(2). The elicited anti-AFB(1) Abs were able to hinder the secretion of AFM(1) into the milk of cows continuously fed with AFB(1). Vaccination of lactating animals with conjugated AnAFB(1) may represent a solution to the public hazard constituted by milk and cheese contaminated with AFs.     

Aflatoxin M1 contamination in commercial pasteurized milk from local markets in Fariman, Iran

Contamination of milk and dairy products with aflatoxin M₁ (AFM₁) presents a risk for human health. The aim of this study was to investigate the presence of AFM₁ in pasteurized milk samples in Fariman, located in the province of Khorasan Razavi, Iran, by enzyme-linked immunosorbent assay (ELISA). Forty-five samples of pasteurized milk from different supermarkets were collected during 3 months in summer (July to September, 2012). AFM₁ contamination was detected in all of milk samples. The mean concentration of aflatoxin M₁ was 27.2 ng/l. The range of AFM₁ content was 8.8–64 ng/l. Thirteen (28.8 %) of the samples had AFM₁ levels exceeding the maximum levels (50 ng/l) accepted by the European Union. Due to the fact that milk is used by all the age groups including infants and children in Fariman city, it is necessary to minimize the health risk from AFM1 contamination in milk. For this reason, the level of its precursor, aflatoxin B₁ (AFB₁), in dairy feeds must be reduced, requiring constant aflatoxin monitoring of relevant agricultural commodities.     

Determination of 1-hydroxypyrene in children urine using column-switching liquid chromatography and fluorescence detection

This study developed an acid hydrolysis method instead of using enzyme extraction, equipped with column-switching system for the pretreatment of samples, in the determination of 1-hydroxypyrene in the urine from children and pyrene in airborne particulates. We collected both types of samples from areas near a petrochemical industry and rural areas as reference. Samples were first treated with acid hydrolysis and followed by solvent extraction prior to being injected into the separation system for the determination with high performance liquid chromatography and fluorescence. A column-switching system was on-line with a C18 separation column to remove matrix interference and obtain a stable baseline of the chromatogram. The eluent used to separate the 1-hydroxypyrene was 60% (v/v) aqueous acetonitrile solution. A fluorescence detector was used to monitor 1-hydroxypyrene at lambdaex = 348 nm and lambdaem = 388 nm, and pyrene at lambdaex = 331 nm and lambdaem = 390 nm. Both calibration graphs were linear with very good correlation coefficients (r > 0.999) and the detection limits were ca. 2pg (5ng/l). Results showed that there was a significant association between 1-hydroxypyrene levels in urine specimens and pyrene levels in airborne particulate samples (r = 0.68, P < 0.05). The average levels of pyrene in the particulates (0.18 versus 0.09ng/m3) and of 1-hydroxypyrene in urine specimens (155.9 versus 110.2ng/g creatinine) were higher for the petrochemical area than for the rural area. This method is stable and sensitive for measuring polycyclic aromatic hydrocarbons in environmental samples.     

Comparison of urinary aflatoxin M1 and aflatoxin albumin adducts as biomarkers for assessing aflatoxin exposure in Tanzanian children

Purpose: To determine levels of urinary aflatoxin M1 (AFM1) in children and correlate the concentrations with previously reported aflatoxin albumin adduct (AF-alb) levels in these children.

Materials and methods: Matched urine and blood samples were collected from 84 Tanzanian children aged 6–14 months old. From 31 children in one village (Kigwa), samples were collected at three time points six months apart. Samples were collected from 31 and 22 children from two different regions at the second time point only. Urinary AFM1 was measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit with a modified protocol to improve sensitivity. AF-alb was measured using an established ELISA method.

Results: The relative ranking of the three villages for exposure to aflatoxin based on either AFM₁ or AF-alb biomarker measurements was the same. In Kigwa village, both AFM1 and AF-alb levels were higher at six months post-harvest compared to baseline. However, at the next visit, the AFM1 levels dropped from a GM (interquartile range) of 71.0 (44.7, 112.6) at visit two to 49.3 (31.5, 77.3) pg/ml urine, whereas AF-alb levels increased from 47.3 (29.7, 75.2) to 52.7 (35.4, 78.3) pg/mg albumin between these two visits, reflecting the fact that AFM1 measures short-term exposure, whereas AF-alb measures longer term exposure. There was a correlation between AFB1 intake and AFM1 excretion (r=?0.442, p?≤?0.001).

Conclusions: Urinary AFM1 is a good biomarker for AFB1 exposure in Tanzanian children, reflecting geographical and temporal variations in exposure to this foodborne toxin.     

Detection of trace aflatoxin M1 in human urine using a commercial ELISA followed by HPLC

Aflatoxin is a liver carcinogen, and rapid, inexpensive methods to detect its urinary biomarkers are needed. We used a commercial enzyme-linked immuno-sorbent assay (ELISA) for aflatoxin M1 in urine (Helica Biosystems) to test 52 Haitian samples. Using this ELISA, we detected traces above the limit of detection (0.2?ng/ml urine) but below the limit of quantitation (0.4?ng/ml) in 14 samples. Liquid chromatography of all 52 Haitian urine samples revealed that only 11 had quantifiable AFM1 (mean: 29.5?pg/ml, standard error: 10.8, range: 2.94–96.5?pg/ml). The Helica ELISA may have detected forms of aflatoxin other than AFM1 in the Haitian samples, or matrix enhancement may have affected results at low AFM1 concentrations. This ELISA may serve as an initial, qualitative indicator of aflatoxin exposure for epidemiological purposes. But this method’s utility as a precise and specific indicator of AFM1 concentrations will require additional refinement and validation.     

Monitoring of individual human exposure to aflatoxins (AF) and N-nitrosamines (NNO) by immunoassays

Highly sensitive immunoassays have been used to quantitate aflatoxins (AF) and N-nitrosamines (NNO) in human body fluids and tissues, respectively. This approach was taken in order to quantitate environmental exposure to these agents at an individual level to facilitate the investigation of their role in the etiology of human cancer. In order to analyse AF in human urine, an immunopurification step has been developed by using AF-specific antibody bound to AH-Sepharose 4B gel in a small (4-ml gel volume) affinity column prior to enzyme-linked immunosorbent assay (ELISA). The ELISA can be used to quantitate aflatoxin B1 (AFB1) over the range 0.01 ng/ml to 10 ng/ml and the assay system has been validated by using human urine samples spiked with AFB1 over this concentration range. In addition, 29 urine samples from the Philippines have been analysed and found to contain a range of levels from zero to 4.25 ng/ml AFB1 equivalent with a mean of 0.875 ng/ml. This compared with a mean of 0.066 ng/ml AFB1 equivalent in samples from France. Radioimmunoassay of O6-methyldeoxyguanosine (O6-medG) has been performed on human oesophageal and cardiac stomach mucosal DNA from tissue samples obtained during surgery in Linxian County, People's Republic of China, an area of high risk for both oesophageal and stomach cancer. Using the methodology described and having 1 mg of hydrolyzed DNA allows the detection of approximately 25 fmol O6medG per mg DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oral platelet aggregation inhibitor Ro 48-3657: Determination of the active metabolite and its prodrug in plasma and urine by high-performance liquid chromatography using automated column switching

A sensitive and highly automated high-performance liquid chromatography (HPLC) column-switching method has been developed for the simultaneous determination of the active metabolite III and its prodrug II, both derivatives of the oral platelet inhibitor Ro 48-3657 (I), in plasma and urine of man and dog. Plasma samples were deproteinated with perchloric acid (0.5 M), while urine samples could be processed directly after dilution with phosphate buffer. The prepared samples were injected onto a pre-column of a HPLC column switching system. Polar plasma or urine components were removed by flushing the precolumn with phosphate buffer (0.1 M, pH 3.5). Retained compounds (including II and III) were backflushed onto the analytical column, separated by gradient elution and detected by means of UV detection at 240 nm. The limit of quantification for both compounds was 1 ng/ml (500 μl of plasma) and 25 ng/ml (50 μl of urine) for plasma and urine, respectively. The practicability of the new method was demonstrated by the analysis of about 6000 plasma and 1300 urine samples from various toxicokinetic studies in dogs and phase 1 studies in man.     

In vitro metabolism of aflatoxin B1 by larvae of navel orangeworm, Amyelois transitella (Walker) (Insecta, Lepidoptera, Pyralidae) and codling moth, Cydia pomonella (L.) (Insecta, Lepidoptera, Tortricidae)

Larvae of the navel orangeworm (NOW), Amyelois transitella (Walker), a major pest of almonds and pistachios, and the codling moth (CM), Cydia pomonella (L.), the principal pest of walnuts and pome fruits, are commonly found in tree nut kernels that can be contaminated with aflatoxin, a potent carcinogen. The ability of larvae of these insects to metabolize aflatoxin B1 (AFB1) was examined. A field strain of NOW produced three AFB1 biotransformation products, chiefly aflatoxicol (AFL), and minor amounts of aflatoxin B2a (AFB2a) and aflatoxin M1 (AFM1). With AFL as a substrate, NOW larvae produced AFB1 and aflatoxicol M1 (AFLM1). A lab strain of CM larvae produced no detectable levels of AFB1 biotransformation products in comparison to a field strain which produced trace amounts of only AFL. Neither NOW nor CM produced AFB1-8,9-epoxide (AFBO), the principal carcinogenic metabolite of AFB1. In comparison, metabolism of AFB1 by chicken liver yielded mainly AFL, whereas mouse liver produced mostly AFM1 at a rate eightfold greater than AFL. Mouse liver also produced AFBO. The relatively high production of AFL by NOW compared to CM may reflect an adaptation to detoxify AFB1. NOW larvae frequently inhabit environments highly contaminated with fungi and, hence, aflatoxin. Only low amounts, if any, of this mycotoxin occur in the chief CM hosts, walnuts, and pome fruits. Characterizations of enzymes and co-factors involved in biotransformation of AFB1 are discussed.     

Simultaneous determination of methamphetamine and its metabolite, amphetamine, in urine using a high performance liquid chromatography column-switching method

We describe here a simple, precise, and highly sensitive method for the simultaneous determination of methamphetamine (MA) and amphetamine (AM) in urine using a high performance liquid chromatography (HPLC) column-switching method. A PK-2A (Shodex) column was used for extraction and deproteinization, and a CAPCELL PAK SCX semi-micro, polymer-coated cation-exchange column was employed for separation. The urine sample was mixed with an equal volume of borate buffer (0.1M, pH 9.4), and then 100 microl of the mixture was injected into the HPLC column. The column was switched for 6 min, and then 10 min later detection was performed at 210 nm. Recovery yields of the MA and AM spiked in the urine were 93.0-100.4% with a coefficient of variation of less than 1%. The calibration curves of MA and AM were in the range of 0.1-10 microg/ml with good linearity (r(2)=0.999), with the limit of qualification being 0.005 microg/ml. This method of using HPLC with column-switching can be used for both qualification and quantification of MA and its metabolite, AM, in urine, especially in forensic cases.     

2(3)-tert-butyl-4-hydroxyanisole inhibits oxidative metabolism of aflatoxin B1 in isolated rat hepatocytes

Previous studies indicate that dietary administration of phenolic antioxidants, 2(3)-tert-butyl-4-hydroxyanisole (BHA) and 3,5-di-tert-butyl-4-hydroxytoluene, inhibits the carcinogenic effect of a number of chemical carcinogens including aflatoxin B1 (AFB1). Induction of hepatic enzymes, such as glutathione S-transferase, UDP-glucuronyltransferase, and epoxide hydrolase, has been shown to be responsible for the reduction of AFB1 cytotoxic and carcinogenic effects. The effect of BHA on AFB1 activation was examined in vitro utilizing isolated rat hepatocytes and liver microsomes. In hepatocytes, the total AFB1 content and bound form of AFB1 were 3.4 and 1.4 pmol/10(6) cells, respectively. In the cell-free microsomal activating system, 2.2 pmol were activated per mg of microsomal protein during 60 min of incubation. BHA (0.1-0.5 mM) inhibited AFB1 activation and binding in both systems in a dose-dependent manner; in hepatocytes, 90% inhibition was observed at 0.5 mM. Analyzing various AFB1 adducts, BHA (0.25 mM)-treated hepatocytes contained a significantly reduced amount of AFB1 macromolecular adducts. The antioxidant neither stimulated nor inhibited the cytosolic glutathione S-transferase and microsomal UDP-glucuronyltransferase activities. Analysis of various hydroxylated (aflatoxins M1 and Q1 (AFM1 and AFQ1] and demethylated (aflatoxin P1 (AFP1] metabolites of AFB1 in both the conjugated and unconjugated form indicated that there was a 30-50% reduction of unconjugated AFP1, AFQ1, and AFM1, whereas AFB1 was increased 3-fold. There was no significant change of conjugated metabolites. The effect of BHA on AFB1 activation in hepatocytes was compared with that of other cytochrome P-450 inhibitors; the ED50 values of SKF 525A, BHA, and metyrapone were 9 microM, 40 microM, and 280 microM, respectively. In the cell-free microsomal system, biotransformation of AFB1 to AFP1, AFM1, and AFQ1 was also inhibited. Kinetic analysis of p-nitroanisole O-demethylase activity of rat liver microsomes demonstrated that BHA inhibited noncompetitively with an apparent Ki of 90 microM. In the absence of enzyme induction, the phenolic antioxidant, BHA, blocks the oxidative biotransformation of AFB1 in isolated hepatocytes.     

High-performance liquid chromatographic determination of finasteride in human plasma using direct injection with column switching

A fully automated column-switching high-performance liquid chromatographic (HPLC) method was developed for the quantification of finasteride [N-(1,1-dimethylethyl)-3-oxo-4-aza-5α-androst-1-ene-17β-carboxamide] in human plasma. Plasma samples were diluted with an equal volume of ethylene glycol-water (40:60, v/v), then the diluted sample (150 μl) was injected into the HPLC system without clean-up. The analyte was retained on a pretreatment column, whereas plasma proteins and other endogenous components were washed out to waste. The analyte was transferred to the analytical column in the heart-cut mode and then detected at 210 nm. A quantification limit of 1 ng/ml was attained. There was a linear relationship between peak height and drug concentration in plasma in the range 1–50 ng/ml. This method was validated and applied to the assay of plasma samples to characterize pharmacokinetic parameters in clinical studies.     

On-line solid-phase extraction of ceftazidime in serum and determination by high-performance liquid chromatography

A column-switching high-performance liquid chromatographic assay is described for the determination of ceftazidime (a third-generation cephalosporin) in human serum. The method does not require prior sample pretreatment. Serum is directly injected in a first chromatographic column for sample clean-up and extraction. Thereafter, using an on-line column-switching system, the drug is quantitatively transferred and separated on a second, analytical column followed by determination using ultraviolet absorption at 258 nm. The technique allows direct, rapid, precise, and simple determination of ceftazidime in serum over the range of 1–250 μg/ml using 12.5 μl of serum. This method was applied to study the pharmacokinetics of the drug in patients undergoing vascular surgery.     

A column-switching liquid chromatography-tandem mass spectrometry method for quantitation of 2-cyanoethylmercapturic acid and 2-hydroxyethylmercapturic acid in Chinese smokers

The acrylonitrile metabolites 2-cyanoethylmercapturic acid (CEMA) and 2-hydroxyethylmercapturic acid (HEMA) have been determined in human urine using an automated column-switching procedure. A diluted sample was centrifuged just prior to being injected into a reusable precolumn packed with a restricted access material and coupled to a liquid chromatography-tandem mass spectrometry system. This method achieved satisfactory reproducibility and accuracy. Average intra- and interday variations (% relative standard deviations) ranged from 2.4 to 3.8% for CEMA and from 2.7 to 10.5% for HEMA. The limits of quantification were 0.003 and 0.099ng/ml for CEMA and HEMA, respectively. It was used to study the uptake of acrylonitrile from smoke constituents by both nonsmokers and smokers of different tar yield cigarettes under ISO 3308 smoking condition. Metabolite concentrations in smoker urine samples were approximately 12 times higher compared with those in nonsmokers for CEMA and 3 times higher for HEMA. Urinary CEMA levels show a clear dose-response relationship with daily cigarette consumption and urinary cotinine. CEMA can also discriminate between smokers of different ISO cigarettes. Because HEMA is not specific, it is only slightly related to smoking and acrylonitrile exposure. The validated biomarker CEMA will continue to be useful for studies of acrylonitrile uptake by smokers.