Quantification of urinary mono-hydroxylated metabolites of polycyclic aromatic hydrocarbons by on-line solid phase extraction-high performance liquid chromatography-tandem mass spectrometry.

Human exposure to polycyclic aromatic hydrocarbons (PAHs) can be assessed through monitoring of urinary mono-hydroxylated PAHs (OH-PAHs). Gas chromatography (GC) has been widely used to separate OH-PAHs before quantification by mass spectrometry in biomonitoring studies. However, because GC requires derivatization, it can be time consuming. We developed an on-line solid phase extraction coupled to isotope dilution-high performance liquid chromatography-tandem mass spectrometry (on-line-SPE-HPLC-MS/MS) method for the quantification in urine of 1-OH-naphthalene, 2-OH-naphthalene, 2-OH-fluorene, 3-OH-fluorene, 1-OH-phenanthrene, the sum of 2-OH and 3-OH-phenanthrene, 4-OH-phenanthrene, and 1-OH-pyrene. The method, which employed a 96-well plate platform and on-line SPE, showed good sensitivity (i.e., limits of detection ranged from 0.007 to 0.09 ng/mL) and used only 100 µL of urine. Accuracy, calculated from the recovery percentage at three spiking levels, varied from 94 to 113 %, depending on the analyte. The inter- and intra-day precision, calculated from 20 repeated measurements of two quality control materials, varied from 5.2 to 16.7 %. Adequate method performance was also confirmed by acceptable recovery (83-102 %) of two NIST standard reference materials (3672 and 3673). This high-throughput on-line-SPE-HPLC-MS/MS method can be applied in large-scale epidemiological studies. Graphical abstract Example LC-MS chromatogram of urinary mono-hydroxylated PAH metabolites.

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 (13)C-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.

Excretion profiles and half-lives of ten urinary polycyclic aromatic hydrocarbon metabolites after dietary exposure.

Human exposure to polycyclic aromatic hydrocarbons (PAHs) can be assessed by biomonitoring of their urinary monohydroxylated metabolites (OH-PAHs). Limited information exists on the human pharmacokinetics of OH-PAHs. This study aimed to investigate the excretion half-life of 1-hydroxypyrene (1-PYR), the most used biomarker for PAH exposure, and 9 other OH-PAHs following a dietary exposure in 9 nonsmoking volunteers with no occupational exposure to PAHs. Each person avoided food with known high PAH-content during the study period, except for a high PAH-containing lunch (barbecued chicken) on the first day. Individual urine samples (n = 217) were collected from 15 h before to 60 h following the dietary exposure. Levels of all OH-PAHs in all subjects increased rapidly by 9-141-fold after the exposure, followed by a decrease consistent with first-order kinetics, and returned to background levels 24-48 h after the exposure. The average time to reach maximal concentration ranged from 3.1 h (1-naphthol) to 5.5 h (1-PYR). Creatinine-adjusted urine concentrations for each metabolite were analyzed using a nonlinear mixed effects model including a term to estimate background exposure. The background-adjusted half-life estimate was 3.9 h for 1-PYR and ranged 2.5-6.1 h for the other 9 OH-PAHs, which in general, were shorter than those previously reported. The maximum concentrations after barbecued chicken consumption were comparable to the levels found in reported occupational settings with known high PAH exposures. It is essential to consider the relatively short half-life, the timing of samples relative to exposures, and the effect of diet when conducting PAH exposure biomonitoring studies.

Determination of 43 polycyclic aromatic hydrocarbons in air particulate matter by use of direct elution and isotope dilution gas chromatography/mass spectrometry.

We are reporting a method for measuring 43 polycyclic aromatic hydrocarbons (PAH) and their methylated derivatives (Me-PAHs) in air particulate matter (PM) samples using isotope dilution gas chromatography/high-resolution mass spectrometry (GC/HRMS). In this method, PM samples were spiked with internal standards, loaded into solid phase extraction cartridges, and eluted by dichloromethane. The extracts were concentrated, spiked with a recovery standard, and analyzed by GC/HRMS at 10,000 resolution. Sixteen (13)C-labeled PAHs and two deuterated Me-PAHs were used as internal standards to account for instrument variability and losses during sample preparation. Recovery of labeled internal standards was in the range of 86-115%. The proposed method is less time-consuming than commonly used extraction methods, such as sonication and accelerated solvent extraction (ASE), and it eliminates the need for a filtration step required after the sonication extraction method. Limits of detection ranged from 41 to 332 pg/sample for the 43 analytes. This method was used to analyze reference materials from the National Institute of Standards and Technology. The results were consistent with those from ASE and sonication extraction, and these results were also in good agreement with the certified or reference concentrations. The proposed method was then used to measure PAHs on PM(2.5) samples collected at three sites (urban, suburban, and rural) in Atlanta, GA. The results showed distinct seasonal and spatial variation and were consistent with an earlier study measuring PM(2.5) samples using an ASE method, further demonstrating the compatibility of this method and the commonly used ASE method.

Assessment of non-occupational exposure to polycyclic aromatic hydrocarbons through personal air sampling and urinary biomonitoring.

Non-occupational inhalation and ingestion exposure to polycyclic aromatic hydrocarbons (PAHs) has been studied in 8 non-smoking volunteers through personal air sampling and urinary biomonitoring. The study period was divided into 4 segments (2 days/segment), including weekdays with regular commute and weekends with limited traffic related exposures; each segment had a high or low PAH diet. Personal air samples were collected continuously from the subjects while at home, at work, and while commuting to and from work. All urine excretions were collected as individual samples during the study. In personal air samples, 28 PAHs were measured, and in urine samples 9 mono-hydroxylated metabolites (OH-PAHs) from 4 parent PAHs (naphthalene, fluorene, phenanthrene and pyrene) were measured. Naphthalene was found at higher concentrations in air samples collected at the subjects' residences, whereas PAHs with four or more aromatic rings were found at higher levels in samples taken while commuting. Urinary OH-PAH biomarker levels increased following reported high inhalation and/or dietary exposure. On days with a low PAH diet, the total amount of inhaled naphthalene during each 24-hour period was well correlated with the amount of excreted naphthols, as was, to a lesser extent, fluorene with its urinary metabolites. During days with a high dietary intake, only naphthalene was significantly correlated with its excreted metabolite. These findings suggest that this group of non-occupational subjects were exposed to naphthalene primarily through indoor air inhalation, and exposed to other PAHs such as pyrene mainly through ingestion.

Urinary concentrations of monohydroxylated polycyclic aromatic hydrocarbons in adults from the U.S. Population Assessment of Tobacco and Health (PATH) Study Wave 1 (2013-2014).

BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants formed from incomplete combustion of organic matter; some PAHs are carcinogens. Smoking, diet, and other activities contribute to exposure to PAHs. Exposure data to PAHs among combustible tobacco product users (e.g. cigarette smokers) exist; however, among non-combustible tobacco products users (e.g., e-cigarette users), such data are rather limited. OBJECTIVES: We sought to evaluate exposure to PAHs among participants in Wave 1 (2013-2014) of the Population Assessment of Tobacco and Health (PATH) Study based on the type of tobacco product (combustible vs non-combustible), and frequency and intensity of product use. METHODS: We quantified seven PAH urinary biomarkers in 11,519 PATH Study participants. From self-reported information, we categorized 8327 participants based on their use of tobacco products as never-tobacco user (never user, n = 1700), exclusive current established combustible products user (combustible products user, n = 5767), and exclusive current established non-combustible products user (non-combustible products user, n = 860). We further classified tobacco users as exclusive cigarette user (cigarette user, n = 3964), exclusive smokeless product user (SLT user, n = 509), and exclusive e-cigarette user (e-cigarette user, n = 280). Last, we categorized frequency of product use (everyday vs some days) and time since use (last hour, within 3 days, over 3 days). We calculated geometric mean (GM) concentrations, and evaluated associations between tobacco product user categories and PAH biomarkers concentrations. RESULTS: Combustible products users had significantly higher GMs of all biomarkers than non-combustible products users and never users; non-combustible products users had significantly higher GMs than never users for four of seven biomarkers. For all biomarkers examined, cigarette users had the highest GMs compared to other tobacco-product users. Interestingly, GMs of 2-hydroxyfluorene, 3-hydroxyfluorene and ∑2,3-hydroxyphenanthrene were significantly higher in SLT users than in e-cigarette users; 3-hydroxyfluorene and 1-hydroxypyrene were also significantly higher in e-cigarette and SLT users than in never users. Everyday cigarette and SLT users had significantly higher GMs for most biomarkers than some days' users; cigarette and SLT users who used the product in the last hour had significantly higher GMs of most biomarkers than other occasional cigarette or SLT users respectively. By contrast, everyday e-cigarette users' GMs of most biomarkers did not differ significantly from those in some days' e-cigarette users; we did not observe clear trends by time of last use among e-cigarette users. CONCLUSIONS: Users of tobacco products had higher PAH urinary biomarker concentrations compared to never users, and concentrations differed by type and frequency of tobacco product use.

Urinary polycyclic aromatic hydrocarbon metabolites as biomarkers to woodsmoke exposure - results from a controlled exposure study.

Woodsmoke contains harmful components - such as fine particulate matter (PM2.5) and polycyclic aromatic hydrocarbons (PAHs) - and impacts more than half of the global population. We investigated urinary hydroxylated PAH metabolites (OH-PAHs) as woodsmoke exposure biomarkers in nine non-smoking volunteers experimentally exposed to a wood fire. Individual urine samples were collected from 24-h before to 48-h after the exposure and personal PM2.5 samples were collected during the 2-h woodsmoke exposure. Concentrations of nine OH-PAHs increased by 1.8-7.2 times within 2.3-19.3 h, and returned to baseline approximately 24 h after the exposure. 2-Naphthol (2-NAP) had the largest post-exposure increase and exhibited a clear excretion pattern in all participants. The level of urinary OH-PAHs, except 1-hydroxypyrene (1-PYR), correlated with those of PM2.5, levoglucosan and PAHs in personal PM2.5 samples. This finding suggests that several urinary OH-PAHs, especially 2-NAP, are potential exposure biomarkers to woodsmoke; by contrast, 1-PYR may not be a suitable biomarker. Compared with levoglucosan and methoxyphenols - two other urinary woodsmoke biomarkers that were measured in the same study and reported previously - OH-PAHs might be better biomarkers based on sensitivity, robustness and stability, particularly under suboptimal sampling and storage conditions, like in epidemiological studies carried out in less developed areas.