Fast simultaneous determination of urinary 1-hydroxypyrene and 3-hydroxybenzo[a]pyrene by liquid chromatography-tandem mass spectrometry

A fast analysis method using liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry was developed for the simultaneous determination of the 1-hydroxypyrene (1-OHP) and 3-hydroxybenzo[a]pyrene (3-OHBaP) in urine. Mass transitions were monitored at m/z 219.3-200.0 for 1-OHP and m/z 269.2-252.2 for 3-OHBaP. Only 10 min was needed for the analysis. The recovery was 60% for 3-OHBaP and 91% for 1-OHP, respectively. And the method detection limits were 0.49 microg/L for 1-OHP and 1.03 microg/L for 3-OHBaP. The inter- and intra-day relative standard deviations were in the range of 2.8-8.9% for 1-OHP and 9.7-20.8% for 3-OHBaP, respectively. The developed method was successfully used to measure urinary PAH metabolites of student volunteers in a high school.     

Binding of 3-hydroxybenzo[a]pyrene to bovine hemoglobin and albumin

Previous studies examined the bioavailability and first-pass biotransformation of 3-hydroxy[(3)H]benzo[a]pyrene ([(3)H]-3-OHBaP) in an isolated perfused catfish intestinal model. This work showed that 3-OHBaP, or a metabolite formed in intestine, bound covalently to blood protein. In this study, the blood adducts were characterized in vitro by incubating bovine ferric hemoglobin or albumin with [(3)H]-3OHBaP under various conditions. Incubation of 2 microM [(3)H]-3-OHBaP with hemoglobin for 1 h resulted in 7.49 pmol bound/mg protein, while albumin binding was 1.37 pmol/mg protein. Mild acid hydrolysis released only 5% of the radioactivity from 3-OHBaP-hemoglobin adducts. After gel filtration, the 3-OHBaP-hemoglobin adducts were examined by HPLC analysis. A single peak of radioactivity was detected at the same retention time as the heme component of hemoglobin. Unbound 3-OHBaP was oxidized to BaP-3,6-dione during incubation with ferric hemoglobin. Treatment of hemoglobin with ascorbic acid decreased the formation of hemoglobin adducts by 33%, while hydrogen peroxide treatment increased adduct formation by 44%. Incubation of [(3)H]-BaP-3-beta-D-glucuronide (BaP-3G) with hemoglobin and beta-glucuronidase resulted in greater binding to hemoglobin than incubation with [(3)H]-3-OHBaP alone. The hemoglobin adduct obtained from [(3)H]-BaP-3G also co-migrated with heme. These results indicate that an oxidative process is involved in formation of the heme adduct and that 3-OHBaP or BaP-3G might be a precursor of the bound metabolite.     

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.     

Comparison of the urinary excretion time courses of pyrene-1,6-dione, pyrene-1,8-dione and 1-hydroxypyrene in rats intravenously exposed to pyrene.

The urinary excretion time courses of pyrene-1,6-dione (P16D), pyrene-1,8-dione (P18D) and 1-hydroxypyrene (1-OHP) were compared in Sprague-Dawley and Wistar rats. Groups of five male rats, of about 200 g of body weight, were injected intravenously with 0.05, 0.5, 5 and 50 micromol pyrene kg-1 of body weight. Urine was collected at 2, 4, 6, 8, 10, 12, 18, 24, 30, 42 and 48 h post-dosing. Pyrene metabolites were measured by high-performance liquid chromatography (HPLC)/fluorescence after enzymatic hydrolysis of the glucurono- and sulfo-conjugates, extraction on Sep-Pak C18 cartridges and, for the analysis of dione metabolites, derivatization to stable diacetoxypyrene molecules. Over the 48-h sampling period, on average 17.4-25.6% of the injected pyrene was excreted overall as P16D, 6.4-8.8% as P18D and 0.6-0.8% as 1-OHP in the urine of Sprague-Dawley rats. By comparison, on average 10.3-14.7% of the intravenous pyrene dose was recovered as P16D, 4.8-6.4% as P18D and 0.3-0.4% as 1-OHP in the urine of Wistar rats. In both strains of rats there was no clear effect of the dose on the 0-48-h cumulative urinary excretion of P18D and 1-OHP over the entire dose range, while the percentage of dose recovered overall as P16D in urine at the highest dose (50 micromol kg-1) was statistically lower than at the other doses. The 0-48-h cumulative percentage of pyrene dose excreted as metabolites in the urine of Sprague-Dawley rats was also significantly higher than in Wistar rats (p<0.01) exposed under identical conditions. As for the urinary excretion-time courses of the different metabolites, for a given dose and strain of rats, excretion curves of P16D, P18D and 1-OHP generally evolved in parallel. There was also no clear effect of the dose on the excretion rate, thus half-life, of pyrene metabolites, except for P16D in Sprague-Dawley rats at the highest dose where elimination tended to be slower compared with the other doses (p<0.01). The average first-order elimination half-life of P16D, P18D and 1-OHP was 4.0, 5.7 and 4.1 h, respectively, in Sprague-Dawley rats, and 5.1, 6.1 and 5.1 h, respectively, in Wistar rats (all doses combined but excluding the highest dose for P16D). This study showed the relative importance of metabolic pathways leading to diones compared with 1-OHP. These dioxygenated metabolites appear to be interesting biomarkers of pyrene exposure at environmentally and occupationally relevant doses. Their adequacy as biomarkers of human exposure has yet to be confirmed.     

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.     

Use of Physiologically-Based Pharmacokinetic Modeling to Simulate the Profiles of 3-Hydroxybenzo(a)pyrene in Workers Exposed to Polycyclic Aromatic Hydrocarbons

Biomathematical modeling has become an important tool to assess xenobiotic exposure in humans. In the present study, we have used a human physiologically-based pharmacokinetic (PBPK) model and an simple compartmental toxicokinetic model of benzo(a)pyrene (BaP) kinetics and its 3-hydroxybenzo(a)pyrene (3-OHBaP) metabolite to reproduce the time-course of this biomarker of exposure in the urine of industrially exposed workers and in turn predict the most plausible exposure scenarios. The models were constructed from in vivo experimental data in rats and then extrapolated from animals to humans after assessing and adjusting the most sensitive model parameters as well as species specific physiological parameters. Repeated urinary voids from workers exposed to polycyclic aromatic hydrocarbons (PAHs) have been collected over the course of a typical workweek and during subsequent days off work; urinary concentrations of 3-OHBaP were then determined. Based on the information obtained for each worker (BaP air concentration, daily shift hours, tasks, protective equipment), the time courses of 3-OHBaP in the urine of the different workers have been simulated using the PBPK and toxicokinetic models, considering the various possible exposure routes, oral, dermal and inhalation. Both models were equally able to closely reproduce the observed time course of 3-OHBaP in the urine of workers and predicted similar exposure scenarios. Simulations of various scenarios suggest that the workers under study were exposed mainly by the dermal route. Comparison of measured air concentration levels of BaP with simulated values needed to obtain a good approximation of observed time course further pointed out that inhalation was not the main route of exposure for most of the studied workers. Both kinetic models appear as a useful tool to interpret biomonitoring data of PAH exposure on the basis of 3-OHBaP levels.     

Comparison of the urinary excretion time courses of pyrene-1,6-dione,pyrene-1,8-dione and 1-hydroxypyrene in rats intravenously exposed to pyrene

Abstract

The urinary excretion time courses of pyrene-1,6-dione (P16D), pyrene-1,8-dione (P18D) and 1-hydroxypyrene (1-OHP) were compared in Sprague–Dawley and Wistar rats. Groups of five male rats, of about 200 g of body weight, were injected intravenously with 0.05, 0.5, 5 and 50 µmol pyrene kg^?1 of body weight. Urine was collected at 2, 4, 6, 8, 10, 12, 18, 24, 30, 42 and 48 h post-dosing. Pyrene metabolites were measured by high-performance liquid chromatography (HPLC)/fluorescence after enzymatic hydrolysis of the glucurono- and sulfo-conjugates, extraction on Sep-Pak C18 cartridges and, for the analysis of dione metabolites, derivatization to stable diacetoxypyrene molecules. Over the 48-h sampling period, on average 17.4–25.6% of the injected pyrene was excreted overall as P16D, 6.4–8.8% as P18D and 0.6–0.8% as 1-OHP in the urine of Sprague–Dawley rats. By comparison, on average 10.3–14.7% of the intravenous pyrene dose was recovered as P16D, 4.8–6.4% as P18D and 0.3–0.4% as 1-OHP in the urine of Wistar rats. In both strains of rats there was no clear effect of the dose on the 0–48-h cumulative urinary excretion of P18D and 1-OHP over the entire dose range, while the percentage of dose recovered overall as P16D in urine at the highest dose (50 µmol kg^?1) was statistically lower than at the other doses. The 0–48-h cumulative percentage of pyrene dose excreted as metabolites in the urine of Sprague–Dawley rats was also significantly higher than in Wistar rats (p<0.01) exposed under identical conditions. As for the urinary excretion-time courses of the different metabolites, for a given dose and strain of rats, excretion curves of P16D, P18D and 1-OHP generally evolved in parallel. There was also no clear effect of the dose on the excretion rate, thus half-life, of pyrene metabolites, except for P16D in Sprague–Dawley rats at the highest dose where elimination tended to be slower compared with the other doses (p<0.01). The average first-order elimination half-life of P16D, P18D and 1-OHP was 4.0, 5.7 and 4.1 h, respectively, in Sprague–Dawley rats, and 5.1, 6.1 and 5.1 h, respectively, in Wistar rats (all doses combined but excluding the highest dose for P16D). This study showed the relative importance of metabolic pathways leading to diones compared with 1-OHP. These dioxygenated metabolites appear to be interesting biomarkers of pyrene exposure at environmentally and occupationally relevant doses. Their adequacy as biomarkers of human exposure has yet to be confirmed.     

Evaluation of a battery of Salmonella typhimurium tester strains for biomonitoring of mutagenic polycyclic aromatic hydrocarbons, nitroarenes and aromatic amines

Various combinations of Salmonella typhimurium tester strains and S9 mix for bioactivation (TA98+S9 mix, TA98S; YG1041+S9 mix, YG1041S) and strain YG1041 in the absence of S9 mix (YG1041) were used to evaluate the mutagenic activity of eight polycyclic aromatic hydrocarbons (PAHs), seven nitroarenes (NAs) and seven aromatic amines (AAs). Three cigarette smoke extracts and two extracts of smokers' urine (SUE) were also included. Urinary mutagenicity was then determined on 31 individuals, potentially exposed to PAHs, for 0 h, 7 h, 12 h and 24 h. Concentrations of urinary 1-hydroxypyrene (1OHP) and 3-hydroxybenzo[a]pyrene (3OHBaP), the levels of atmospheric pyrene (Py) and benzo[a]pyrene (BaP), and particulate concentrations in air (AP) were also measured. PAHs could be detected by TA98S and YG1041S, with TA98S being more sensitive than YG1041S. While NAs could be detected by all combinations, YG1041 and YG1041S were more sensitive than TA98S. Although both YG1041S and TA98S could detect AAs, YG1041S was more sensitive than TA98S. Cigarette smoke extract contained mutagenic AAs and NAs, but AAs were the only mutagenic compounds detected in the extracts of smokers' urine. The concentrations of 1OHP (7 h and 12 h) were significantly higher than those at 0 h, but no difference could be detected with 3OHBaP. Correlations were found between Py and 1OHP (7 h and 24 h) and between BaP and 3OHBaP concentrations (7 h, 12 h and 24 h). A significantly elevated urinary mutagenicity was detected with YG1041S at 7h in the group of smokers. A good correlation was determined between AP and the test results with TA98S (7 h) and with YG1041 (0 h and 7 h). Urinary 1OHP correlated with the test results with YG1041S (0 h, 7 h and 12 h) while 3OHBaP correlated with those obtained with YG1041S (7 h). Overall, 21/31 individuals were occupationally exposed to AAs, 15/31 individuals were exposed to NAs, and 2/31 were exposed to PAHs as indicated by the Salmonella mutagenicity assay. The urine mutagenicity test was not effective at monitoring occupational exposure to PAHs. However, the correlation with AP implied the presence of unknown mutagenic atmospheric substances that could modulate the urinary mutagenicity.     

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.     

Sensitive biomarker of polycyclic aromatic hydrocarbons (PAHs): urinary 1-hydroxyprene glucuronide in relation to smoking and low ambient levels of exposure.

The study was conducted in a Chinese population with occupational or environmental exposures to polycyclic aromatic hydrocarbons (PAHs). A total of 106 subjects were recruited from coke-oven workers (workers), residents in a metropolitan area (residents) and suburban gardeners (gardeners). All subjects were monitored twice for their personal exposures to PAHs. The biological samples were collected for measurements of 1-hydroxypyrene (1-OHP) and cotinine in urine. The geometric means of personal exposure levels of pyrene, benz(a)anthracene (BaA) and benzo(a)pyrene (BaP) in workers were 1.470, 0.978 and 0.805 microg m-3, respectively. The corresponding levels in residents were 0.050, 0.034 and 0.025 microg m-3; and those in gardeners were 0.011, 0.020 and 0.008 microg m-3, respectively. The conjugate of 1-OHP with glucuronide (1-OHP-G) is the predominant form of pyrene metabolite in urine and it showed strong associations with exposures not only to pyrene, but also to BaA, BaP and total PAHs. Most importantly, a significant difference in 1-OHP-G was even detected between the subgroups with exposures to BaP at < 0.010 and > 0.010 but < 0.020 microg m-3, suggesting that 1-OHP-G is a good marker that can be used for the risk assessment of BaP exposure at levels currently encountered in ambient air. Furthermore, multiple regression analyses of 1-OHP-G on PAHs exposure indicated that cigarette smoke was a major confounding factor and should be considered and adjusted for while using 1-OHP to estimate PAHs exposure.     

Fast and sensitive determination of urinary 1-hydroxypyrene by packed capillary column switching liquid chromatography coupled to micro-electrospray time-of-flight mass spectrometry

The present work reports capillary liquid chromatographic column switching methodology tailored for fast, sensitive and selective determination of 1-hydroxypyrene (1-OHP) in human urine using micro-electrospray ionization time-of-flight mass spectrometric detection. Samples (100 microl) of deconjugated, water diluted and filtered urine samples were loaded onto a 150 microm I.D.x 30 mm 10 microm Kromasil C(18) pre-column, providing on-line sample clean-up and analyte enrichment, prior to back flushed elution onto a 150 microm I.D.x 100 mm 3.5 microm Kromasil C(18) analytical column. Loading flow rates up to 100 microl/min in addition to the use of isocratic elution by a mobile phase composition of acetonitrile/water (70/30, v/v) containing 5 mM ammonium acetate provided elution of 1-OHP within 5.5 min and a total analysis time of less than 15 min with manual operation. Ionization was performed in the negative mode and 1-OHP was observed as [M-H](-) at m/z 217.08. The method was validated over the concentration range 0.2-40 ng/ml 1-OHP in pre-treated urine, yielding a coefficient of correlation of 0.997. The within-assay (n=6) and between-assay (n=6) precisions were in the range 6.4-7.3 and 7.0-8.1%, respectively, and the recoveries were in the range 96.2-97.5 within the investigated concentration range. The method mass limit of detection was 2 pg, corresponding to a 1-OHP concentration limit of detection of 20 pg/ml (0.09 nmol/l) diluted urine or 0.3 ng/ml (1.35 nmol/l) urine.     

Urinary 1-hydroxypyrene as a biomarker of exposure to polycyclic aromatic hydrocarbons: biological monitoring strategies and methodology for determining biological exposure indices for various work environments

This article reviews the published studies on urinary 1-hydroxypyrene (1-OHP) as a biomarker of exposure to polycyclic aromatic hydrocarbons (PAHs) in work environments. Sampling and analysis strategies as well as a methodology for determining biological exposure indices (BEIs) of 1-OHP in urine for different work environments are proposed for the biological monitoring of occupational exposure to PAHs. Owing to the kinetics of absorption of pyrene by different exposure routes and excretion of 1-OHP in urine, in general, 1-OHP urinary excretion levels increase during the course of a workday, reaching maximum values 3-9 h after the end of work. When the contribution of dermal exposure is important, post-shift 1-OHP excretion can however be lower than pre-shift levels in the case where a worker has been exposed occupationally to PAHs on the day prior to sampling. In addition, 1-OHP excretion levels in either pre-shift, post-shift or evening samples increase during the course of a work-week, levelling off after three consecutive days of work. Consequently, ideally, for a first characterization of a work environment and for an indication of the major exposure route, considering a 5-day work-week (Monday to Friday), the best sampling strategy would be to collect all micturitions over 24 h starting on Monday morning. Alternatively, collection of pre-shift, post-shift and evening urine samples on the first day of the work-week and at the end of the work-week is recommended. For routine monitoring, pre-shift samples on Monday and post-shift samples on Friday should be collected when pulmonary exposure is the main route of exposure. On the other hand, pre-shift samples on Monday and Friday should be collected when the contribution of skin uptake is important. The difference between beginning and end of work-week excretion will give an indication of the average exposure over the workweek. Pre-shift samples on the first day of the work-week will indicate background values, and, hence, reflect general environment exposure and body burden of pyrene and/or its metabolites. On the other hand, since PAH profile can vary substantially in different work sites, a single BEI cannot apply to all workplaces. A simple equation was therefore developed to establish BEIs for workers exposed to PAHs in different work environments by using a BEI already established for a given work environment and by introducing a correction factor corresponding to the ratio of the airborne concentration of the sum of benzo(a)pyrene (BaP) equivalent to that of pyrene. The sum of BaP equivalent concentrations represents the sum of carcinogenic PAH concentrations expressed as BaP using toxic equivalent factors. Based on a previously estimated BEI of 2.3 μmol 1-OHP mol^-1 creatinine for coke-oven workers, BEIs of 4.4, 8.0 and 9.8 μmol 1-OHP mol^-1 creatinine were respectively calculated for vertical pin Söderberg workers, anode workers and pre-bake workers of aluminium plants and a BEI of 1.2 μmol 1-OHP mol^-1 creatinine was estimated for iron foundry workers. This approach will allow the potential risk of cancer in individuals occupationally exposed to PAHs to be assessed better.     

Automated, fast and sensitive quantification of 17 alpha-hydroxy-progesterone, androstenedione and testosterone by tandem mass spectrometry with on-line extraction

Plasma 17 alpha-hydroxyprogesterone (17-OHP), androstenedione and testosterone measurements are important for the diagnosis and monitoring of hyperandrogenic disorders, most importantly for congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. The reliability of immunoassays has proved questionable especially for newborns and children. In order to reduce the analytical interferences due to cross-reactivity or matrix effects, to improve accuracy and shorten the analysis time, we have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with atmospheric pressure chemical ionization (APCI) for simultaneous measurement. An on-line extraction cartridge with column-switching technique and liquid chromatography over a Chromolith RP 18 e column allow a rapid and easy quantification. The lowest limit of detection was 0.03-0.06 microg/L. Our method has proved linear up to 250 microg/L (r=0.999). Recoveries (S.D.) of 17-OHP, androstenedione and testosterone in plasma were 100% (5), 102% (2) and 92% (4), respectively. The regression equation for the LC-MS/MS (x) and immunoassay (y) methods for 17-OHP (excluding neonate samples) was y=1.942 x+0.255 nmol/L (r=0.695; n=97). In comparison to our values, the immunoassay generally overestimates steroid concentration. The regression equation for the LC-MS/MS (x) and immunoassay (y) methods for testosterone was y=0.963 x+0.035 nmol/L (r=0.955; n=107). Preliminary reference intervals for children were determined as a function of age and sex. The sensitivity and specificity of the LC-MS/MS method offer advantages over routine immunoassays due to the elimination of interferences especially for newborns, high throughput and short chromatographic run time.     

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).     

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.     

Determination of polycyclic aromatic hydrocarbons in ambient particulate matter PM2.5 and 1-hydroxypyrene in children from an area near an oil refinery in Northeast Mexico

Exposure to polycyclic aromatic hydrocarbons (PAHs) has been associated with carcinogenic and mutagenic effects. Urban areas characteristically have high concentrations of PAHs associated with vehicle traffic and industry; among these oil refining stands out. One of the six refineries in Mexico has operated in the Metropolitan Area of Monterrey since 1979. The objective of this study was to evaluate exposure to PAHs by determining 1-hydroxypyrene (1-OHP) concentration in children living in an urban area near an oil refinery as well as to determine the relative health risk by exposure to PAHs in this population. This study included 90 children of both sexes aged 5–12 years. Two urine samples were collected, one in summer and one in winter to determine urinary 1-OHP. At the same time, the concentrations of PAHs associated with PM_2.5 were collected in the study area. The mean concentration of 1-OHP was 0.14 μmol/mol of creatinine. Twelve PAHs were quantified, seven are potential carcinogenics and one (benzo[a]pyrene) is carcinogenic for humans, and used as an indicator of cancer relative risk from exposure to PAHs. The annual average concentration of benzo[a]pyrene suggest an excess lifetime cancer risk of 1/100,000 habitants, in the study area.     

Automated column-switching high-performance liquid chromatography for the determination of aflatoxin M1

An extractionless method for determining aflatoxin M1 (AFM1), a major metabolite of aflatoxin B1 (AFB1), in human urine was developed. The biological fluid is injected directly into the chromatographic system after simple dilution and centrifugation. A pre-column, packed with a cation-exchange phase and coupled on-line to a column-switching liquid chromatography (LC) system, is used for sample pre-treatment and concentration. The analytes are non-selectively desorbed with the LC eluent and cleaned by means of a column-switching procedure. Pre-treatment and analysis were performed within 40 min. Average AFM1 recovery reached 97% in the 10–100 ng/l range of urine. The detection limit of AFM1 in urine and milk was 2.5 ng/l for 1 ml of injected sample. A comparison with an immunoaffinity column clean-up and LC method was performed. The method was applied to determine AFM1 in the urine of AFB1 gavaged rats, and in the urine of both potentially exposed and supposedly unexposed workers. The method was also extended to milk.     

Simple determination of mycophenolic acid in human serum by column-switching high-performance liquid chromatography

A column-switching high-performance liquid chromatographic analysis was established to monitor the serum concentration of mycophenolic acid, the active metabolite from mycophenolate mofetil administered for the prophylaxis of acute organ rejection in renal transplantation. The system consisted of two pumps for solvent delivery, a column-switching valve, a precolumn, and a reversed-phase analytical column. The present method enabled us to determine MPA by injecting serum samples directly into HPLC without any pretreatment. The mobile phases with different amounts of organic solvent were delivered to the precolumn and analytical column by separate lines, and samples were applied to the precolumn. The column switching valves were switched automatically following the processes for the elimination of protein and the drug analysis. The peak heights of MPA were linearly related to the concentrations (r=0.999) in the range of 0.1-20 micro g/ml, and the limit of quantification was 0.1 micro g/ml (S/N ratio=3). This method was accurate and reproducible on the basis of the results of recovery (94.0-98.0%) and small coefficient of variations of intra and inter-assay (less than 8.3%).     

Automated determination of orotic acid, uracil and pseudouridine in urine by high-performance liquid chromatography with column switching

A column-switching high-performance liquid chromatographic method, requiring no sample preparation apart from filtration, is described for quantification of urinary orotic acid, uracil and pseudouridine. The analyses were carried out using a reversed-phase octadecylsilane-bonded column for sample clean-up and a cation-exchange column for separation; 5–20 ]sml samples of urine were directly analysed, and more than 100 samples could be analysed consecutively. Each sample required only 30 min. Detection limits of these compounds were 5 pmol. Creatinine-related urinary uracil excretion was lowest in the newborn period (17.3 ± 14.4 μmol/g of creatinine). A patient with partial ornithine transcarbamylase deficiency and his mother usually excreted a high level of uracil during the period of normal orotic acid excretion and normal serum ammonia level.     

Enantioselective Separation and Simultaneous Determination of Tolperisone and Eperisone in Rat Plasma by LC‐MS/MS

Tolperisone and eperisone used as muscle relaxants possess one chiral center each and exist as two optical isomers for each drug. Therefore, enantioselective assays to measure each enantiomer in biological matrices are of great importance. In the present study a simple and complete reverse‐phase liquid chromatography tandem mass spectrometric method for separation and enantioselective determination of tolperisone and eperisone in rat plasma was developed. The analytes were extracted from rat plasma by a simple protein precipitation method with acetonitrile as the extraction solvent. The enantioselective separation of analytes was achieved on a Cellulose Tris (4‐chloro‐3‐methylphenylcarbamate) chiral column with a mobile phase of acetonitrile: 10 mM ammonium acetate in an isocratic mode of elution and mass spectrometric detection. The calibration curve for each enantiomer was found to be linear over 0.2 to 20 ng/mL for each enantiomer. The proposed method exhibited good intra‐ and interday precision (% CV) ranged between 0.95–6.05% and 1.11–8.21%, respectively. The intra‐ and interday accuracy for the proposed assay method ranged between 94.0–100.5% and 92.7–102.1%, respectively. The proposed method was validated as per regulatory guidelines. Chirality 25:622–627, 2013. © 2013 Wiley Periodicals, Inc.