Metabolomic profiling of human urine in hepatocellular carcinoma patients using gas chromatography/mass spectrometry

With the technique of metabolomics, gas chromatography/mass spectrometry (GC/MS), urine or serum metabolites can be assayed to explore disease biomarkers. In this work, we present a metabolomic method to investigate the urinary metabolic difference between hepatocellular carcinoma (HCC, n = 20) male patients and normal male subjects (n = 20). The urinary endogenous metabolome was assayed using chemical derivatization followed by GC/MS. After GC/MS analysis, 103 metabolites were detected, of which 66 were annotated as known compounds. By a two sample t-test statistics with p < 0.05, 18 metabolites were shown to be significantly different between the HCC and control groups. A diagnostic model was constructed with a combination of 18 marker metabolites or together with alphafetoprotein, using principal component analysis and receiver-operator characteristic curves. The multivariate statistics of the diagnostic model yielded a separation between the two groups with an area under the curve value of 0.9275. This non-invasive technique of identifying HCC biomarkers from urine may have clinical utility.     

On-line solid phase extraction-high performance liquid chromatography–isotope dilution–tandem mass spectrometry approach to quantify N,N-diethyl-m-toluamide and oxidative metabolites in urine

Human exposure to N,N-diethyl-m-toluamide (DEET) occurs because of the widespread use of DEET as an active ingredient in insect repellents. However, information on the extent of such exposure is rather limited. Therefore, we developed a fast on-line solid phase extraction–high performance liquid chromatography–isotope dilution tandem mass spectrometry (HPLC-MS/MS) method to measure in urine the concentrations of DEET and two of its oxidative metabolites: N,N-diethyl-3-(hydroxymethyl)benzamide and 3-(diethylcarbamoyl)benzoic acid (DCBA). To the best of our knowledge, this is the first HPLC-MS/MS method for the simultaneous quantification of DEET and its select metabolites in human urine. After enzymatic hydrolysis of the conjugated species in 0.1 mL of urine, the target analytes were retained and pre-concentrated on a monolithic column, separated from each other and from other urinary biomolecules on a reversed-phase analytical column, and detected by atmospheric pressure chemical ionization in positive ion mode. The limits of detection ranged from 0.1 ng mL⁻¹ to 1.0 ng mL⁻¹, depending on the analyte. Accuracy ranged between 90.4 and 104.9%, and precision ranged between 5.5 and 13.1% RSD, depending on the analyte and the concentration. We tested the usefulness of this method by analyzing 75 urine samples collected anonymously in the Southeastern United States in June 2012 from adults with no known exposure to DEET. Thirty eight samples (51%) tested positive for at least one of the analytes. We detected DCBA most frequently and at the highest concentrations. Our results suggest that this method can be used for the analysis of a large number of samples for epidemiological studies to assess human exposure to DEET.     

Analysis of hydroxylated polycyclic aromatic hydrocarbons in urine using comprehensive two-dimensional gas chromatography with a flame ionization detector

The determination of polycyclic aromatic hydrocarbon (PAH) metabolites in human urine is the method of choice for assessing exposure to carcinogenic compounds. The objective of this study was the development of a comprehensive two-dimensional gas chromatography (GC × GC) method using a flame ionisation detector (FID) to simultaneously determine 10 hydroxylated PAH. The method was based on enzymatic deconjugation, liquid–liquid extraction, and trimethylsilyl (TMS) derivatization of the analytes by microwave heating. Satisfactory separation was achieved. The coefficient of variance was 3.8–12.8%. LOD was 0.03–0.18 μg/L, and LOQ was 0.1–0.5 μg/L. The mean recovery was 76%. The method was applied to the analysis of urine from smokers and non-smokers.     

Determination of cyanide exposure by gas chromatography-mass spectrometry analysis of cyanide-exposed plasma proteins

Exposure to cyanide can occur in a variety of ways, including exposure to smoke from cigarettes or fires, accidental exposure during industrial processes, and exposure from the use of cyanide as a poison or chemical warfare agent. Confirmation of cyanide exposure is difficult because, in vivo, cyanide quickly breaks down by a number of pathways, including the formation of both free and protein-bound thiocyanate. A simple method was developed to confirm cyanide exposure by extraction of protein-bound thiocyanate moieties from cyanide-exposed plasma proteins. Thiocyanate was successfully extracted and subsequently derivatized with pentafluorobenzyl bromide for GC-MS analysis. Thiocyanate levels as low as 2.5 ng mL⁻¹ and cyanide exposure levels as low as 175 μg kg⁻¹ were detected. Samples analyzed from smokers and non-smokers using this method showed significantly different levels of protein-bound thiocyanate (p < 0.01). These results demonstrate the potential of this method to positively confirm chronic cyanide exposure through the analysis of protein-bound cyanide in human plasma.     

Sensitive and rapid method for the determination of urinary cotinine in non-smokers: an application for studies assessing exposures to second hand smoke (SHS)

We describe a sensitive and rapid method to assay urinary cotinine levels among non-smokers using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) and its application in studies assessing exposures to second hand smoke (SHS). Cotinine was initially extracted from 1 ml of urine with methylene chloride by using a liquid-liquid extraction Chem Elut™ column. The extracted sample was further separated by using a BetaBasic C18 column (1 mm × 150 mm, 3 μm) with isocratic elution (60:40 acetonitrile and 5 mM ammonium acetate at pH 5), and then examined using a triple quadrupole mass spectrometer with an electrospray ionization (ESI) source in multiple-reaction-monitoring (MRM) mode. The elution of cotinine from the LC column took approximately 2.3 min and the detection of cotinine by ESI/MS/MS provided a limit of detection (LOD) of 0.3 ng/ml. The ESI/MS/MS detection was able to easily distinguish between cotinine and nicotine. This method, validated using a cotinine concentration range from 0.8 to 102.4 ng/ml, was successfully applied in a cross-sectional study examining differences in levels and sources of second hand smoke (SHS) exposure among non-smokers. Self-reported measures of SHS exposure were significantly associated with urinary cotinine levels. This urinary cotinine assay using LC-ESI/MS/MS provides a robust, high throughput and very sensitive method for the evaluation of SHS exposure for use in epidemiologic and clinical research studies.     

Needle Trap Device-GC-MS for Characterization of Lung Diseases Based on Breath VOC Profiles

Volatile organic compounds (VOCs) have been assessed in breath samples as possible indicators of diseases. The present study aimed to quantify 29 VOCs (previously reported as potential biomarkers of lung diseases) in breath samples collected from controls and individuals with lung cancer, chronic obstructive pulmonary disease and asthma. Besides that, global VOC profiles were investigated. A needle trap device (NTD) was used as pre-concentration technique, associated to gas chromatography-mass spectrometry (GC-MS) analysis. Univariate and multivariate approaches were applied to assess VOC distributions according to the studied diseases. Limits of quantitation ranged from 0.003 to 6.21 ppbv and calculated relative standard deviations did not exceed 10%. At least 15 of the quantified targets presented themselves as discriminating features. A random forest (RF) method was performed in order to classify enrolled conditions according to VOCs’ latent patterns, considering VOCs responses in global profiles. The developed model was based on 12 discriminating features and provided overall balanced accuracy of 85.7%. Ultimately, multinomial logistic regression (MLR) analysis was conducted using the concentration of the nine most discriminative targets (2-propanol, 3-methylpentane, (E)-ocimene, limonene, m-cymene, benzonitrile, undecane, terpineol, phenol) as input and provided an average overall accuracy of 95.5% for multiclass prediction.     

Comprehensive profiling of mercapturic acid metabolites from dietary acrylamide as short-term exposure biomarkers for evaluation of toxicokinetics in rats and daily internal exposure in humans using isotope dilution ultra-high performance liquid chromatography tandem mass spectrometry

Mercapturic acid metabolites from dietary acrylamide are important short-term exposure biomarkers for evaluating the in vivo toxicity of acrylamide. Most of studies have focused on the measurement of two metabolites, N-acetyl-S-(2-carbamoylethyl)-l-cysteine (AAMA) and N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-l-cysteine (GAMA). Thus, the comprehensive profile of acrylamide urinary metabolites cannot be fully understood. We developed an isotope dilution ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous determination of all four mercapturic acid adducts of acrylamide and its primary metabolite glycidamide under the electroscopy ionization negative (ESI-) mode in the present study. The limit of detection (LOD) and limit of quantification (LOQ) of the analytes ranged 0.1–0.3 ng/mL and 0.4–1.0 ng/mL, respectively. The recovery rates with low, intermediate and high spiking levels were calculated as 95.5%–105.4%, 98.2%–114.0% and 92.2%–108.9%, respectively. Acceptable within-laboratory reproducibility (RSD < 7.0%) substantially supported the use of current method for robust analysis. Rapid pretreatment procedures and short run time (8 min per sample) ensured good efficiency of metabolism profiling, indicating a wide application for investigating short-term internal exposure of dietary acrylamide. Our proposed UHPLC-MS/MS method was successfully applied to the toxicokinetic study of acrylamide in rats. Meanwhile, results of human urine analysis indicated that the levels of N-acetyl-S-(2-carbamoylethyl)-l-cysteine-sulfoxide (AAMA-sul), which did not appear in the mercapturic acid metabolites in rodents, were more than the sum of GAMA and N-acetyl-S-(1-carbamoyl-2-hydroxyethyl)-l-cysteine (iso-GAMA). Thus, AAMA-sul may alternatively become a specific biomarker for investigating the acrylamide exposure in humans. Current proposed method provides a substantial methodology support for comprehensive profiling of toxicokinetics and daily internal exposure evaluations of acrylamide in vivo.     

Bacteria detection based on the evolution of enzyme-generated volatile organic compounds: Determination of Listeria monocytogenes in milk samples

The rapid detection of Listeria monocytogenes contamination in food is essential to prevent food-borne illness in humans. The aim of this study was to differentiate non-contaminated milk from milk contaminated with L. monocytogenes using enzyme substrates coupled with the analysis of volatile organic compounds (VOCs). The method is based on the activity of β-glucosidase and hippuricase enzymes and the detection of a specific VOC i.e. 2-nitrophenol and 3-fluoroaniline, respectively. VOCs were extracted, separated and detected by headspace-solid phase microextraction coupled to gas chromatography–mass spectrometry (HS-SPME GC–MS). This approach required the inclusion of the selective agent's cycloheximide, nalidixic acid and acriflavine HCl in the growth medium to inhibit interfering bacteria. The VOCs were liberated by L. monocytogenes provided that samples contained at least 1–1.5 × 10² CFU ml⁻¹ of milk prior to overnight incubation. This approach shows potential for future development as a rapid method for the detection of L. monocytogenes contaminated milk.     

A passive sampling-based analytical strategy for the determination of volatile organic compounds in the air of working areas

An analytical methodology based on the use of a polyethylene layflat tube filled with activated carbon and Florisil (ACFL-VERAM) was employed for the passive sampling of volatile organic compounds (VOCs) in the air of working areas of packing industries. VOCs amount in the ACFL-VERAM sampler was directly determined through head-space-gas chromatography-mass spectrometry (HS-GC-MS) allowing a direct determination in only 20 min without the need of any previous treatment. Uptake parameters, like sampling rate (R_S) and sampler-air partition coefficient (K_SA), were determined for every studied VOC from adsorption isotherm data. Additionally, experimental equations have been proposed to predict R_S and K_SA from the octanol-air partition coefficients reported in the literature. The proposed methodology reaches method detection levels from 0.007 to 0.2 mg m⁻³ for the studied VOCs.     

Urinary metabolic profiling of volatile organic compounds in acute exposed volunteers after an oil spill in Republic of Korea

The Herbei Spirit oil spill occurred in western Korea. A large number of people who participated in the cleanup tasks of the contaminated area were exposed to crude oil component. We developed a method to monitor volatile organic compound (VOC) metabolites in urine, and evaluate the acute exposure caused by the oil spill in exposed volunteer workers (n = 100, 20.7 ± 2.1 years, mean ± SD). Acidified urine samples were extracted by SPE, derivatized with trimethylsilyl, and analyzed using gas chromatography–mass spectrometry. Calibration curves were found to be linear from 3 to 1000 ng/mL (r² > 0.993). Accuracy was over 82.4%, and precision was less than 24.8%. Using this method, the VOC metabolites, except hippuric acid, were present at higher levels in the urine samples of volunteers after cleanup work. The levels of mandelic acid (MA) and trans,trans‐muconic acid (t,t‐MU) were increased significantly (p < 0.001). The exposure effect was greater in women than in men. The effect of smoking was analyzed in all exposed and non‐exposed groups, with non‐smokers showing increased MA and t,t‐MU levels related to exposure. The present method was reliable to determine VOC metabolites in urine and could be useful for biomonitoring of acute exposure effects of VOCs. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of volatile organic compounds in urban and industrial air from Tarragona by thermal desorption and gas chromatography-mass spectrometry

This study describes the optimisation of an analytical method to determine 54 volatile organic compounds (VOCs) in air samples by active collection on multisorbent tubes, followed by thermal desorption and gas chromatography-mass spectrometry. Two multisorbent beds, Carbograph 1/Carboxen 1000 and Tenax/Carbograph 1TD, were tested. The latter gave better results, mainly in terms of the peaks that appeared in blank chromatograms. Temperatures, times and flow desorption were optimised. Recoveries were higher than 98.9%, except methylene dichloride, for which the recovery was 74.9%. The method's detection limits were between 0.01 and 1.25 μg m⁻³ for a volume sample of 1200 ml, and the repeatability on analysis of 100 ng of VOCs, expressed as relative standard deviation for n = 3, was lower than 4% for all compounds. Urban and industrial air samples from the Tarragona region were analysed. Benzene, toluene, ethylbenzene and xylenes (BTEX) were found to be the most abundant VOCs in urban air. Total VOCs in urban samples ranged between 18 and 307 μg m⁻³. Methylene chloride, 1,4-dichlorobenzene, chloroform and styrene were the most abundant VOCs in industrial samples, and total VOCs ranged between 19 and 85 μg m⁻³.     

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the determination of phenolic polycyclic aromatic hydrocarbons (OH-PAH) in urine of non-smokers and smokers

Polycyclic aromatic hydrocarbons (PAH) are products of the incomplete combustion of organic materials and, therefore, occur ubiquitously in the environment and also in tobacco smoke. Since some PAH have been classified as carcinogens, it is important to have access to suitable analytical methods for biomarkers of exposure to this class of compounds. Past experience has shown that measuring a profile of PAH metabolites is more informative than metabolites of a single PAH. Assessment of environmental and smoking-related exposure levels requires analytical methods with high sensitivity and specificity. In addition, these methods should be fast enough to allow high throughput. With these pre-conditions in mind, we developed and validated a high-performance liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the determination of phenolic metabolites of naphthalene, fluorene, phenanthrene and pyrene in urine of smokers and non-smokers. Sample work-up comprised enzymatic hydrolysis of urinary conjugates and solid-phase extraction on C18 cartridges. The method showed good specificity, sensitivity, and accuracy for the intended purpose and was also sufficiently rapid with a sample throughput of about 350 per week. Application to urine samples of 100 smokers and 50 non-smokers showed significant differences between both groups for all measured PAH metabolites, and strong correlations with markers of daily smoke exposure in smoker urine. Urinary levels were in good agreement with previously reported data using different methodologies. In conclusion, the developed LC-MS/MS method is suitable for the quantification of phenolic PAH metabolites of naphthalene, fluorene, phenanthrene, and pyrene in smoker and non-smoker urine.     

3,4-Methylenedioxypyrovalerone (MDPV) and metabolites quantification in human and rat plasma by liquid chromatography–high resolution mass spectrometry

Synthetic cathinones are recreational drugs that mimic the effects of illicit stimulants like cocaine, amphetamine or Ecstasy. Among the available synthetic cathinones in the United States, 3,4-methylenedioxypyrovalerone (MDPV) is commonly abused and associated with dangerous side effects. MDPV is a dopamine transporter blocker 10-fold more potent than cocaine as a locomotor stimulant in rats. Previous in vitro and in vivo studies examining MDPV metabolism reported 3,4-dihydroxypyrovalerone (3,4-catechol-PV) and 4-hydroxy-3-methoxypyrovalerone (4-OH-3-MeO-PV) as the two primary metabolites. We developed and validated a liquid chromatography–high resolution mass spectrometry method to quantify MDPV and its primary metabolites in 100 μL human and rat plasma. Plasma hydrolysis was followed by protein precipitation before analysis. Limits of detection were 0.1 μg L⁻¹, with linear ranges from 0.25 to 1000 μg L⁻¹. Process efficiency, matrix effect, total imprecision (%CV) and accuracy (%target) were 36–93%, from −8 to 12%, 2.1 to 7.3% and 86 to 109%, respectively. MDPV and metabolites were stable at room temperature for 24 h, 4 °C for 72 h and after 3 freeze-thaw cycles with less than 10% variability. Human-rat plasma cross validation demonstrated that rat plasma could be accurately quantified against a human plasma calibration curve. As proof of this method, rat plasma specimens were analyzed after intraperitoneal and subcutaneous dosing with MDPV (0.5 mg kg⁻¹). MDPV, 3,4-catechol-PV and 4-OH-3-MeO-PV concentrations ranged from not detected to 107.5 μg L⁻¹ prior to and up to 8 h after dosing. This method provides a simultaneous quantification of MDPV and two metabolites in plasma with good selectivity and sensitivity.     

Quantification of 21 metabolites of methylnaphthalenes and polycyclic aromatic hydrocarbons in human urine

Polycyclic aromatic hydrocarbons (PAHs) and their alkylated derivatives, such as methylnaphthalenes (MeNs), are harmful pollutants ubiquitously present in the environment. Exposure to PAHs has been linked to a variety of adverse health effects and outcomes, including cancer. Alkyl PAHs have been proposed as petrogenic source indicators because of their relatively high abundance in unburned petroleum products. We report a method to quantify 11 urinary methylnaphthols (Me-OHNs), metabolites of 1- and 2-methylnaphthalenes, and 10 monohydroxy PAH metabolites (OH-PAHs), using automated liquid-liquid extraction and isotope dilution gas chromatography tandem mass spectrometry (GC-MS/MS). After spiking urine (1 mL) with ¹³C-labeled internal standards, the conjugated target analytes were hydrolyzed enzymatically in the presence of ascorbic acid. Then, their free species were preconcentrated into 20 % toluene in pentane, derivatized and quantified by GC-MS/MS. The 11 Me-OHNs eluted as 6 distinct chromatographic peaks, each representing 1???3 isomers. Method detection limits were 1.0? 41 pg/mL and the coefficients of variation in quality control materials were 4.7???19 %. The method was used to analyze two National Institute of Standards and Technology’s Standard Reference Materials® and samples from 30 smokers and 30 non-smokers. Geometric mean concentrations were on average 37 (Me-OHNs) and 9.0 (OH-PAHs) fold higher in smokers than in non-smokers. These findings support the usefulness of Me-OHNs as potential biomarkers of non-occupational exposure to MeNs and sources containing MeNs.

Validation of an online dual-loop cleanup device with an electrospray ionization tandem mass spectrometry-based system for simultaneous quantitative analysis of urinary benzene exposure biomarkers trans, trans-muconic acid and S-phenylmercapturic acid

The aim of this study is to validate isotope-dilution electrospray ionization tandem mass spectrometry (ESI-MS-MS) method with a dual-loop cleanup device for simultaneous quantitation of two benzene metabolites, trans, trans-muconic acid (ttMA) and S-phenylmercapturic acid (SPMA), in human urine. In this study, a pooled blank urine matrix from rural residents was adopted for validation of the analytical method. The calibration curve, detection limit, recovery, precision, accuracy and the stability of sample storage for the system have been characterized. Calibration plots of ttMA and SPMA standards spiked into two kinds of urine matrixes over a wide concentration range, 1/32-8-fold biological exposure indices (BEIs) values, showed good linearity (R > 0.9992). The detection limits in pooled urine matrix for ttMA and SPMA were 1.27 and 0.042 μg g⁻¹ creatinine, respectively. For both of ttMA and SPMA, the intra- and inter-day precision values were considered acceptable well below 25% at the various spiked concentrations. The intra- and inter-day apparent recovery values were also considered acceptable (apparent recovery >90%). The ttMA accuracy was estimated by urinary standard reference material (SRM). The accuracy reported in terms of relative error (RE) was 5.0 ± 2.0% (n = 3). The stability of sample storage at 4 or −20 °C were assessed. Urinary ttMA and SPMA were found to be stable for at least 8 weeks when stored at 4 or −20 °C. In addition, urine samples from different benzene exposure groups were collected and measured in this system. Without tedious manual sample preparation procedure, the analytical system was able to quantify simultaneously ttMA and SPMA in less than 20 min.     

Analysis of volatile organic compounds released from the decay of surrogate human models simulating victims of collapsed buildings by thermal desorption–comprehensive two-dimensional gas chromatography–time of flight mass spectrometry

Field experiments were devised to mimic the entrapment conditions under the rubble of collapsed buildings aiming to investigate the evolution of volatile organic compounds (VOCs) during the early dead body decomposition stage. Three pig carcasses were placed inside concrete tunnels of a search and rescue (SAR) operational field terrain for simulating the entrapment environment after a building collapse. The experimental campaign employed both laboratory and on-site analytical methods running in parallel. The current work focuses only on the results of the laboratory method using thermal desorption coupled to comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (TD-GC × GC-TOF MS). The flow-modulated TD-GC × GC-TOF MS provided enhanced separation of the VOC profile and served as a reference method for the evaluation of the on-site analytical methods in the current experimental campaign. Bespoke software was used to deconvolve the VOC profile to extract as much information as possible into peak lists. In total, 288 unique VOCs were identified (i.e., not found in blank samples). The majority were aliphatics (172), aromatics (25) and nitrogen compounds (19), followed by ketones (17), esters (13), alcohols (12), aldehydes (11), sulfur (9), miscellaneous (8) and acid compounds (2). The TD-GC × GC-TOF MS proved to be a sensitive and powerful system for resolving the chemical puzzle of above-ground “scent of death”.     

High-throughput intracellular pteridinic profiling by liquid chromatography–quadrupole time-of-flight mass spectrometry

Pteridines are a diverse family of endogenous metabolites that may serve as useful diagnostic biomarkers for disease. While many preparative and analytical techniques have been described for analysis of selected pteridines in biological fluids, broad intracellular pteridine detection remains a significant analytical challenge. In this study, a novel, specific and sensitive extraction and high performance liquid chromatography–quadrupole time-of-flight mass spectrometry (HPLC–QTOF MS) method was developed to simultaneously quantify seven intracellular pteridines and monitor 18 additional, naturally-occurring intracellular pteridines. The newly developed method was validated through evaluation of spiked recoveries (84.5–109.4%), reproducibility (2.1–5.4% RSD), method detection limits (0.1–3.0 μg L⁻¹) and limits of quantitation (0.1–1 μg L⁻¹), and finally application to non-small cell lung cancer A549 cells. Twenty-three pteridine derivatives were successfully detected from cell lysates with an average RSD of 12% among culture replicates. Quantified intracellular pteridine levels ranged from 1 to 1000 nM in good agreement with previous studies. Finally, this technique may be applied to cellular studies to generate new biological hypotheses concerning pteridine physiological and pathological functions as well as to discovery new pteridine-based biomarkers.     

Determination of volatile organic compounds in contaminated air using semipermeable membrane devices

Semipermeable membrane devices (SPMDs) were evaluated as passive samplers for the determination of 26 volatile organic compounds (VOCs) in contaminated air of occupational environments. A direct methodology based on the use of head-space-gas chromatography-mass spectrometry (HS-GC-MS) was developed for VOCs determinations in SPMDs, without any sample pre-treatment and avoiding the use of solvents. A desorption temperature of 150 °C for 10 min was sufficient for a sensitive VOCs determination providing limits of detection in the range of 15 ng SPMD⁻¹ for 21 of 26 studied compounds. Linear and equilibrium uptake models were established for each VOC from compound isotherms. Highly volatile compounds were slightly absorbed and moderately volatile compounds were strongly absorbed by SPMDs. This study is the first precedent of the use of SPMDs for the simultaneous sampling of a wide number of VOCs. The use of SPMDs is a simple and low cost alternative to ordinary sampling devices such as Radiello^® diffusive samplers or badge-type solid-phase supports.     

Accurate quantification of mercapturic acids of styrene (PHEMAs) in human urine with direct sample injection using automated column-switching high-performance liquid chromatography coupled with tandem mass spectrometry

Styrene is one of the most important industrial chemicals, with an enormously high production volume worldwide. The urinary mercapturic acids of its metabolite styrene-7,8-oxide, namely N-acetyl-S-(2-hydroxy-1-phenylethyl)-l-cysteine (PHEMA 1) and N-acetyl-S-(2-hydroxy-2-phenylethyl)-l-cysteine (PHEMA 2), are specific biomarkers for the determination of individual internal exposure to this highly reactive intermediate of styrene. We have developed and validated a fast, specific and very sensitive method for the accurate determination of the sum of phenylhydroxyethyl mercapturic acids (PHEMAs) in human urine with an automated multidimensional liquid chromatography–tandem mass spectrometry method using ¹³C₆-labelled PHEMAs as internal standards. Analytes were stripped from the urinary matrix by online extraction on a restricted access material, transferred to the analytical column and subsequently determined by tandem mass spectrometry. The limit of quantification (LOQ) for the sum of PHEMAs was 0.3 μg/L urine and allowed us to quantify the background exposure of the (smoking) general population. Precision within series and between series ranged from 1.5 to 6.8% at three concentrations ranging from 3 to 30 μg/L urine; the mean accuracy was between 104 and 110%. We applied the method to spot urine samples from 40 subjects of the general population with no known occupational exposure to styrene. The median levels (range) for the sum of PHEMAs in urine of non-smokers (n = 22) were less than 0.3 μg/L (less than 0.3 to 1.1 μg/L), whereas in urine of smokers (n = 18), the median levels were 0.46 μg/L (less than 0.3 to 2.8 μg/L). Smokers showed a significantly higher excretion of the sum of PHEMAs (p = 0.02). Owing to its automation and high sensitivity, our method is well suited for application in occupational or environmental studies.