Determination of glyphosate, glufosinate and their major metabolites in urine by the UPLC-MS/MS method applicable to biomonitoring and epidemiological studies.

The preoccupation concerning glyphosate (GLYP) has rapidly grown over recent years, and the availability of genetically modified crops that are resistant to GLYP or glufosinate (GLUF) has increased the use of these herbicides. The debate surrounding the carcinogenicity of GLYP has raised interest and the desire to gain information on the level of exposure of the population. GLYP and aminomethylphosphonic acid (AMPA) are commonly simultaneously analysed. GLUF is sometimes also monitored, but its major metabolite, 3-[hydroxy(methyl)phosphinoyl]propionic acid (3MPPA), is rarely present in the method. Using a pentafluorobenzyl derivative to extract the analytes from human urine, we present a method that contains four important analytes to monitor human exposure to GLYP and GLUF. The use of the flash freeze technique speeds up the extraction process and requires less organic solvent than conventional liquid-liquid extraction. The limits of detection in the low µg/L range enable the use of this method for epidemiological studies. The results obtained for 35 volunteers from the Quebec City area are presented with the results from multiple interlaboratory comparisons (G-EQUAS, HBM4EU and OSEQAS). This methodology is currently being used in the Maternal-Infant Research on Environmental Chemicals (MIREC-ENDO) study and in the Canadian Health Measures Survey (CHMS).

New approach for the determination of ortho-phenylphenol exposure by measurement of sulfate and glucuronide conjugates in urine using liquid chromatography-tandem mass spectrometry.

Ortho-phenylphenol (OPP) has been widely used as a fungicide and preservative. Although low-dose studies have demonstrated its low toxicity in animals and humans, high-dose exposure to this contaminant has toxic effects that range from skin irritation to bladder cancer. Thus far, monitoring of OPP exposure in the general population has been performed by measuring OPP after urine hydrolysis with the ß-glucuronidase/arylsulfatase enzyme and sometimes by the use of a mineral acid. We developed a sensitive, accurate, and robust method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to specifically measure two-phase II OPP metabolites excreted in human urine, OPP sulfate (OPP-S), and OPP glucuronide (OPP-G). Comparative analysis of urine samples from 50 volunteers living in the Quebec City area using a direct method and phosphoric acid hydrolysis method previously developed in our laboratory showed no statistically significant difference (p value for paired t test = 0.701) in OPP concentrations. Moreover, a significant difference showed that underestimation (p value for paired t test = 0.025) occurs when ß-glucuronidase/arylsulfatase enzyme deconjugation is used. The LOD achieved by the direct method permits the detection of OPP-S and OPP-G metabolites in urine at the submicrogram per liter level. Graphical abstract ᅟ.

Standardized Procedure for the Simultaneous Determination of the Matrix Effect, Recovery, Process Efficiency, and Internal Standard Association.

The matrix effects (MEs) on the quantification of an analyte can be significant and should not be neglected during development and validation of an analytical method. According to this premise, we developed a standardized procedure based on a set of six tests performed on six different sample matrices to detect and characterize the effects of the matrix for single and multiple analytes methods. The link between the matrix effect, recovery, process efficiency, accuracy, precision, and calibration curve was underscored by calculations performed with peak areas, ratios of standard/internal standard peak area, and concentrations. The terms instrumental ME and global ME were introduced, and the term recovery was subdivided for clarity. The test accounts for the presence of ubiquitous and endogenous analytes through background subtraction. The results showed the necessity for using samples with an original concentration in the same range and that the concentration selected for the addition had a definite impact on the results. The use of six-sample matrices provided a standard deviation on the results, and this information could be inserted in a method performance result to show precision. The tool also allows for testing of different analytes/internal standard combinations, which helps with the selection of the association with minimum MEs. A UPLC-MS/MS method for the quantification of several phthalate metabolites in urine was developed and validated with this test. This methodology responds to a scientific need for homogeneity, clarity, and understanding of the results and facilitates the decision-making process while lowering the required costs and time.

Descriptive analysis of organophosphate ester metabolites in a pan-Canadian pregnancy cohort.

Organophosphate esters (OPEs) are widely used in numerous consumer products for their flame retardant and plasticizing properties. Despite potential widespread exposure, biomonitoring data during critical windows of development are scarce and limited to the most widely studied metabolites. We quantified urinary concentrations of multiple OPE metabolites in a vulnerable Canadian population. Using data and biobanked specimens from the Maternal-Infant Research on Environmental Chemicals (MIREC) study (2008-2011), we measured first trimester urinary concentrations of 15 OPE metabolites as well as one flame retardant metabolite and quantified associations with sociodemographic and sample collection characteristics in 1865 pregnant participants. We applied 2 different analytical methods to quantify OPEs, one using UItra-Performance Liquid Chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) and the other using Atmospheric Pressure Gas Chromatography coupled to mass spectrometry (APGC-MS/MS) with sensitive limits of detection (0.008-0.1 µg/L). We modelled associations between sociodemographic and sample collection characteristics and specific gravity-standardized chemical concentrations. Six OPE metabolites were detected in the majority (68.1-97.4 %) of participants. Bis-(2-chloroethyl) hydrogen phosphate had the highest detection rate (97.4 %). Diphenyl phosphate had the highest geometric mean concentration (0.657 µg/L). Metabolites of tricresyl phosphate were detected in few participants. Associations between sociodemographic characteristics varied according to each OPE metabolite. Pre-pregnancy body mass index tended to be positively associated with OPE metabolite concentrations whereas age tended to be inversely associated with OPE concentrations. OPE concentrations were, on average, higher in urine samples collected in the summer than other seasons the winter. We present the largest biomonitoring study of OPE metabolites in pregnant people to date. These findings demonstrate widespread exposure to OPEs and their metabolites and identify subpopulations who may experience heightened exposure.

Measurement of 24 phthalate metabolites in 1st trimester urine samples: The MIREC study.

Phthalates are non-persistent chemicals measured as metabolites in urine. Over time, new metabolites have been identified. In the original Maternal-Infant Research on Environmental Chemicals (MIREC) study (2008-2011), we measured 11 phthalate metabolites in first trimester urine samples. The goal of the present study was to develop a method to measure new metabolites, to increase the sensitivity for some previously measured metabolites, and to measure these new metabolites in biobanked urine samples from MIREC participants. Using Ultra Performance Liquid Chromatography with a tandem mass spectrometer, we developed a method to measure 24 metabolites from 10 different parent phthalates. Chromatographic interpretation of some of the di-iso-decyl phthalate metabolites (mono-(2-propyl-6oxoheptyl) phthalate (MOiDP), mono-(2,7-methyl-7-carboxyheptyl) phthalate (MCiNP), mono-(2-propyl-6-hydroxy-heptyl) phthalate (MHiDP)) and di-iso-nonyl phthalate metabolites (mono(oxo-isononyl) phthalate (MOiNP), mono(carboxy-isooctyl) phthalate (MCiOP), mono(hydroxy-isononyl) phthalate (MHiNP) and mono-isononyl phthalate (MiNP)) was challenging as these are complex isomeric mixtures. To validate and confirm our quantitation peaks, an assay using a high-resolution detection technique was developed on a Quadrupole Time-of-Flight (QToF) system. This system has a mass resolution of at least 0.005 amu, compared to 0.5 amu for the MS/MS detector. Using the QToF system, the distinction between an isomer and possible interference was achieved with the use of the exact mass. In about 1800 MIREC samples, mono-cyclo-hexyl phthalate (MCHP), mono-(7-carboxy-n-heptyl) phthalate (MCHpP), mono-iso-decyl phthalate (MiDP), and mono-n-octyl phthalate (MnOP) were rarely detected, while detection of MMP was improved. MCiOP, MiNP and MCiNP had to be reported semi-quantitatively. Given the complexity of isomeric mixtures of some phthalates, researchers must be careful in their determination of the analytes and the approach used in their quantification when generating biomonitoring data. This study produced biomonitoring data for a large population of pregnant people that can be used in risk assessment of phthalates. Future work will examine associations with birth and child outcomes.

Direct LC-MS/MS and indirect GC-MS/MS methods for measuring urinary bisphenol A concentrations are comparable.

BACKGROUND: Bisphenol A (BPA) is typically measured in urine using an indirect method that involves enzymatic deconjugation and extraction. In contrast, the direct method measures free and conjugated BPA concurrently and sums them to estimate urinary BPA concentrations. Statistical comparison of total BPA results using the direct and indirect methods is necessary to accurately interpret biomonitoring data for risk assessments. OBJECTIVES: To compare urinary BPA concentrations estimated from the indirect and direct methods in duplicate first trimester urine samples collected from 1879 pregnant women from the MIREC Study. METHODS: For the indirect method, we measured urinary BPA concentrations using GC-MS/MS. For the direct method, we summed free and conjugated BPA concentrations measured using LC-MS/MS. We evaluated deviation between the two methods using the Bland-Altman analysis in the total sample and stratified (1) by specific gravity and (2) at the limit of quantification (LOQ). RESULTS: Median urinary BPA concentrations for the direct and indirect methods were 0.89 µg BPA equivalents/L and 0.81 µg/L respectively. Concentrations from the direct method were, on average, 8.6% (95% CI: 6.7%, 10.5%) higher than the indirect method in a Bland-Altman analysis. The percent differences between the two methods was 4.0% in urines with specific gravities < 1.02 (n = 1348, 72%) and 20.3% in urine with specific gravity ≥ 1.02. In values below the LOQ (n = 663, 35%), we observed smaller average percent deviation (4.8%) between the two methods but wider limits of agreement. DISCUSSION: Results from this study, based on the largest statistically rigorous comparison of the direct and indirect methods of BPA measurement, contrast previous findings reporting that the indirect method underestimates total BPA exposure. The difference in urinary BPA concentrations we observed with the indirect and direct methods is unlikely to alter the interpretation of health outcome data.

Maternal and early life exposure to phthalates: The Plastics and Personal-care Products use in Pregnancy (P4) study.

Phthalates are a group of chemicals found in a number of consumer products; some of these phthalates have been shown to possess estrogenic activity and display anti-androgenic effects. While a number of biomonitoring studies of phthalates in pregnant women and infants have been published, there is a paucity of data based on both multiple sampling periods and in different matrices. Phthalate metabolites were measured in 80 pregnant women and their infants in Ottawa Canada (2009-2010) in urine, meconium and breast milk collected at various time periods pre- and post-parturition. At least 50% of the women had at least one urine sample greater than the limit of detection (LOD) for the various phthalate metabolites, with the exception of mono-n-octyl phthalate (MnOP), mono-isononyl phthalate (MiNP) and mono(carboxy-isooctyl) phthalate (MCiOP). Four major clusters of maternal urinary metabolites were identified. Among infants (n=61), the following metabolites were rarely (< 10%) detected: mono-cyclohexyl phthalate (MCHP), mono-isononyl phthalate (MiNP), mono-methyl phthalate (MMP), and mono-n-octyl phthalate (MnOP). While mono-benzyl phthalate (MBzP), mono-3-carboxypropyl phthalate (MCPP), MEHHP, and MEOHP were frequently detected in maternal urines at any time point, these metabolites were rarely detected in breast milk. Maternal urinary concentrations of MEP and the DEHP metabolites were higher in samples collected during pregnancy than postnatally. No statistically significant differences were observed in infant's urinary phthalate concentrations between breast-fed and bottle-fed infants. Significant correlations were observed between maternal urinary MEHHP (r=0.35), MEOHP (r=0.35) and MEP (r=0.37) collected at <20weeks gestation with levels in meconium and between MBzP (r=0.78) and MEP (r=0.56) in maternal and infant urine collected 2-3months after birth. These results suggest at least some maternal-fetal-infant transfer of phthalates and that meconium may be a useful matrix for measuring in utero exposure to phthalates.

Bisphenol A and phthalate metabolite urinary concentrations: Daily and across pregnancy variability.

Phthalates and bisphenol A (BPA) are high production volume and ubiquitous chemicals that are quickly metabolized in the body. Traditionally, studies have relied on single spot urine analyses to assess exposure; ignoring variability in concentrations throughout a day or over a longer period of time. We compared BPA and phthalate metabolite results from urine samples collected at five different time points. Participants (n=80) were asked to collect all voids in a 24 h period on a weekday and then again on a weekend before 20 weeks of pregnancy. During the second and third trimesters and in the postpartum period, single spot urines were collected. Variability over time in urinary concentrations was assessed using intraclass correlation coefficients (ICCs) and the sensitivity to correctly classify a single sample as high or low versus the geometric mean (GM) of all samples was calculated. We found low reproducibility and sensitivity of BPA and all phthalate metabolites throughout pregnancy and into the postpartum period but much higher reproducibility within a day. Time of day when the urine was collected was a significant predictor of specific gravity adjusted exposure levels. We concluded that, if the interest is in average exposures across pregnancy, maternal/fetal exposure estimation may be more accurate if multiple measurements, collected across the course of the entire pregnancy, rather than a single spot measure, are performed.

Exposure to free and conjugated forms of bisphenol A and triclosan among pregnant women in the MIREC cohort.

BACKGROUND: Bisphenol A (BPA) and triclosan (TCS) are two nonpersistent chemicals that have been frequently measured in spot urine samples from the general population but less so in pregnant women; however, data are limited on the free (bioactive) and conjugated forms of these phenols. OBJECTIVES: The Maternal-Infant Research on Environmental Chemicals (MIREC) Study addressed these data gaps by utilizing stored maternal urine samples from a large multicenter cohort study of Canadian pregnant women. METHODS: Concentrations of free and conjugated forms of BPA and TCS were measured in about 1,890 first-trimester urine samples by ultra performance liquid chromatography-tandem mass spectrometry using isotope dilution. RESULTS: The glucuronides of BPA and TCS were the predominant forms of these chemicals measured (detected in 95% and 99% of samples, respectively), whereas the free forms were detected in 43% and 80% of samples, respectively. The geometric mean urinary concentrations for glucuronides of BPA and TCS were 0.80 µg/L (95% CI: 0.75, 0.85) and 12.30 µg/L (95% CI: 11.08, 13.65), respectively. Significant predictors of BPA included maternal age < 25 vs. ≥ 35 years, current smoking, low vs. high household income, and low vs. high education. For TCS, urinary concentrations were significantly higher in women ≥ 25 years of age, never vs. current smokers, and women with high household income and high education. CONCLUSIONS: The results from this study represent the largest national-level data on urinary concentrations of free and conjugated forms of BPA and TCS in pregnant women and suggest that maternal characteristics predicting elevated urinary concentrations of these phenols largely act in opposite directions.

Determination of bisphenol A, triclosan and their metabolites in human urine using isotope-dilution liquid chromatography-tandem mass spectrometry.

Bisphenol A (BPA) and triclosan (TCS) are ubiquitous environmental phenols exhibiting endocrine disrupting activities that may be involved in various health disorders in humans. There is a need to measure separately free forms and conjugated metabolites because only the former are biologically active. We have developed sensitive methods using isotope-dilution liquid chromatography-tandem mass spectrometry for individual measurements of free BPA and TCS as well as their metabolites, BPA glucuronide (BPAG), BPA monosulfate (BPAS), BPA disulfate (BPADS), TCS glucuronide (TCSG) and TCS sulfate (TCSS) in urine. Comparative analyses of urine samples from 46 volunteers living in the Quebec City area using the new methods and a GC-MS/MS method previously used in our laboratory revealed very strong correlations for total BPA (Spearman's rs=0.862, p<0.0001) and total TCS concentrations (rs=0.942, p<0.0001). Glucuronide metabolites were the most abundant BPA and TCS species in urine samples (>94% of total urinary concentrations). Unconjugated TCS concentrations represented a small proportion of total TCS species (median=1.6%) but its concentration was likely underestimated due to losses by adsorption to the surface of polypropylene tubes used for sample storage. To our knowledge, we are the first to report levels of free, sulfated and glucuronidated TCS levels in human urine.