Direct measurement of the glucuronide conjugate of 1-hydroxypyrene in human urine by using liquid chromatography with tandem mass spectrometry (original) (raw)
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Toxicology letters, 1999
Urinary pyrene metabolites, 1-OHP and 1-OHPG, have been used as biomarkers for the assessment of occupational and environmental exposure to PAHs. This study compares the sensitivity and applicability of the different analytical methods of 1-OHPG for human biomonitoring of low level exposure to PAHs. Three analytical methods were compared: (1) HPLC method from that reported by Singh et al. (Singh, R., Tucek, M., Maxa, K., Tenglerova, J., Weyand, E.H., 1995. A rapid and simple method for the analysis of 1-hydroxypyrene glucuronide: a potential biomarker for polycyclic aromatic hydrocarbon exposure. Carcinogenesis 16, 2909-2915); (2) IAC-SFS method: the rapid and simple assay using IAC purification using monoclonal antibody specific for PAH-DNA adduct and PAH metabolites and SFS quantitation; and (3) IAC-HPLC method: IAC and HPLC separation and quantitation. The correlation between the IAC-SFS method, HPLC method, and the IAC-SFS method was determined in 20 first year-grade junior high...
Rapid Communications in Mass Spectrometry, 2005
This article presents an analytical approach that used chemical derivatization to enhance mass spectrometric (MS) response in electrospray ionization (ESI) mode of 1-hydroxypyrene (1-OHP), a commonly used biomarker to monitor human exposure to polycyclic aromatic hydrocarbons (PAHs). The enhancement successfully enabled the desired detection of 50 pg/mL in human urine. The introduction of an MS-friendly dansyl group to 1-OHP enhanced both ionization efficiency in the ESI source and collision-activated dissociation (CAD) in the collision cell. The response increase was estimated to be at least 200-fold, and enabled the reduction of sample size to only 100 mL. The selective MS detection also facilitated a fast (run time 3 min) liquid chromatography (LC) method which successfully resolved the analyte and interferences. The sample processing procedure included enzymatic hydrolysis of glucuronide and sulfate conjugates, liquid-liquid extraction, derivatization with dansyl chloride and a final liquid-liquid extraction to generate clean extracts for LC/MS/MS analysis. This approach has been validated as sensitive, linear (50-1000 pg/mL), accurate and precise for the quantitation of 1-OHP in human urine. This is the first report of using chemical derivatization to enhance MS/MS detection with fast chromatography in the determination of 1-OHP in human urine.
2006
Urinary 1-hydroxypuren (1-OHP) is commonly used as a major metabolite and biological indicator of the overall exposure to polycyclic aromatic hydrocarbons (PAHs). For evaluation of human exposure to such compounds, biological monitoring is an essential process, in which, preparation of samples is one of the most time-consuming and error-prone aspects prior to chromatographic techniques. In this study, non classic form of liquid-liquid extraction (LLE) was optimized with regard to solvent type, solvent volume, extraction temperature, mixing type, and mixing duration. Through the extraction process, a mild temperature was used to keep the compound of interest as stable as possible. In this study, a high performance liquid chromatography, using reverse-phase column was used. The isocratic run was done at a constant flow rate of 0.8 ml/min, the mobile phase was methanol/water and a fluorescence detector was used, setting at 242 nm and 388 nm. At thedeveloped, the extraction recovery was exceeded 87.3%, achieving detection limit of 0.2 µg/l. The factors were evaluated statically the procedure was validated with three different pools of spiked urine samples and showed a good reproducibility over six consecutive days as well as experiments. It was concluded that, this optimized method could simplify sample preparation for trace residue analysis of PAHs metabolites.
Journal of Analytical Toxicology, 2012
An analytical method using a liquid chromatograph combined with a simple online column switching sample pre-treatment system was developed for the determination of 1-hydroxypyrene (1-HP) in urine. This compound is the metabolite of pyrene and is used to assess the exposure of workers to polycyclic aromatic hydrocarbons (PAHs). After enzymatic hydrolysis, a urine sample was directly injected into a high-performance liquid chromatograph (HPLC) where it automatically underwent a sample cleanup using a column switching device. The procedure is simpler than previous methods because it uses only one switching valve, one extraction column and one HPLC pump. The analyte was retained on a short extraction column and after interferences were eluted to waste, was subsequently switched onto the analytical column. This allowed a short analysis time of 15 min. The calibration graph was found to be linear within the concentration range of 0.5 to 20 mg/L with a coefficient of determination exceeding r 2 5 0.99. Recoveries were found to be greater than 96% in the range 1 to 10 mg/L with intermediate precision of 2.5 to 5.8% relative standard deviation. This online method was verified by a comparison with an existing manual method by the analysis of 81 urine samples from workers exposed to PAHs and showed that the test results from both methods were in agreement with a probability obtained from the paired Student's t-test of P > 0.76. The proposed online method was found to be simple, fast and suited to routine analyses of 1-HP in urine for the assessment of occupational exposure to PAHs.
Rapid Communications in Mass Spectrometry, 2006
1-Hydroxypyrene is a metabolite of pyrene, a member of the class of polycyclic aromatic hydrocarbons (PAHs) whose toxic properties in some cases include carcinogenicity. The determination of 1hydroxypyrene in human urine is used as a biological indicator for exposure to PAHs, which is related to the combustion of organic materials, like smoking, living in urban environments, and eating grilled or smoked food. The determination of 1-hydroxypyrene by high-performance liquid chromatography (HPLC) with fluorescence detection has very good sensitivity but it is not highly specific: this can reduce accuracy in the quantitative determination of low levels of analyte in a complex matrix like urine. An HPLC method that uses triple quadrupole mass detection has been validated with the objective both to improve the signal-to-noise (S/N) ratio and to achieve the maximum specificity for the analyte in those urine samples that are richer in possible inteferents. The calibration range for 1-hydroxypyrene is from 0.005-0.1 mg/L in the urine of non-smoking healthy volunteers. After solid-phase extraction, samples were analyzed by HPLC/tandem mass spectrometry (MS/MS) in the multiple reaction monitoring (MRM) mode. In order to obtain reliable results quantitative analysis must be performed by means of the internal standard method (we used deuterium-labelled 1-hydroxypyrene): the method accuracy is not less than 85%. The S/N ratio at a concentration of 0.1 mg/L is about 10, and therefore this can be considered the lowest limit of quantitation. The method performance does not change if urine samples are measured using a calibration curve prepared in methanol, thus reducing the time of analysis and costs.
Urinary 1-Hydroxypyrene as a Biomarker to Carcinogenic Polycyclic Aromatic Hydrocarbon Exposure
Biomarkers in Cancer, 2012
In order to capture the extent of exposure to polycyclic aromatic hydrocarbons (PAHs), various biomarkers have been employed. The biomarkers employed for PAHs include PAHs genetoxic end points in lymphocytes, urinary metabolites, PAH-DNA adducts, and PAH-Protein adducts. Of these, excretory 1-hydroxypyene, a metabolite of pyrene, has been used extensively as a biological monitoring indicator of exposure to PAHs. This study attempts to assess the level of this biomarker in the body fluid of 68 exposed subjects using high performance liquid chromatography HPLC. The subjects screened included auto mechanics, drivers, and fuel attendants. 1-hydroxypyrene was extracted from the urine of the subjects using solid phase extraction method. The HPLC analysis was done in isocratic mode using water:methanol (12:88 v/v) mobile phase. The stationary phase was XBridge C18 (150 × 4.6 mm) 5 µm column. The wavelength was 250 nm at a flow rate of 1.2 mL/min. The oven temperature was 30 °C and the injection volume was 20 µL. The run time was 3 minutes. The level of urinary 1-hydroxypyrene detected varied for the different categories of occupation studied. About 27% of sampled fuel attendants and 22% of auto mechanics had detectable 1-hydroxypyrene in their urine samples. There was no detectable 1-hydroxypyene in the urine samples of commercial drivers or in the urine samples of students used as controls. The results of this study showed that fuel attendants and auto mechanics have significant exposures to PAHs. So far, there is no established benchmark for level of PAHs in urine, but our findings indicate the possibility of future cancer cases in this population as a result of their occupational exposure. The study was not able to link the level of 1-hydroxypyene with the smoking habits of the subjects.
Analytica Chimica Acta, 1999
Measurement of urinary metabolites constitutes a non-invasive method to assess exposures resulting from all routes. An immunochemical assay (enzyme-linked immunosorbent assay) was applied for the detection of metabolites excreted in urine as the result of exposure to polycyclic aromatic hydrocarbons (PAHs). Ten male subjects potentially exposed to PAHs were employed in road bituminization. Same number of referents came from university staff. The metabolites were analyzed in an indirect competitive ELISA, using a polyclonal antiserum that has been raised against pyrenebutyric acid coupled to thyroglobulin and 1-hydroxypyrene as a calibrator. Antiserum specificity was tested with several PAH metabolites. Binding was highest for 1-hydroxypyrene (100%), was acceptable for 1-hydroxypyreneglucuronide (22%) and the phenanthrols (6-32%), but was low (<1%) for 1-hydroxypyrenesulfate, 1-naphthol, and 3-hydroxybenzo(a)pyrene. No binding was observed with 9,10-dihydroxy-9,10-dihydrophenanthrene. Results given as 1-hydroxypyrene equivalents were compared to the 1-hydroxypyrene concentration as determined by off-line solid phase extraction/HPLC analysis and the sum of 1-, 2-, 3-, 4-, 9-hydroxyphenanthrenes and 1-hydroxypyrene from coupled-column HPLC analysis. All metabolite concentrations were corrected for creatinine. PAH metabolites were detected in all of the urine samples. Results obtained with the ELISA in most samples were higher than corresponding 1-hydroxypyrene concentrations and lower than the sum of phenanthrols and 1-hydroxypyrene as measured by HPLC. With both HPLC and the ELISA no significant difference in PAH metabolite excretion of exposed subjects and referents was found, whereas urinary PAH excretion was significantly higher in smokers compared to non-smokers. It is concluded that the ELISA has proved to be a useful tool for biomonitoring studies that allows an estimation of PAH urinary excretion after a simple sample dilution and without any time-consuming preliminary enzymatic hydrolysis or derivatization.
Analytical and bioanalytical chemistry, 2017
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 %, depe...