Guide for Combining Primary Tumors for Statistical Analysis in Rodent Carcinogenicity Studies.

The Tumor Combination Guide was created at the request of the U. S. Food and Drug Administration (FDA) by a Working Group of biopharmaceutical experts from international societies of toxicologic pathology, the Food and Drug Administration (FDA), and members of the Standard for Exchange of Nonclinical Data (SEND) initiative, to assist pharmacology/toxicology reviewers and biostatisticians in statistical analysis of nonclinical tumor data. The guide will also be useful to study and peer review pathologists in interpreting the tumor data. This guide provides a higher-level hierarchy of tumor types or categories correlating the tumor names from the International Harmonization of Nomenclature and Diagnostic Criteria (INHAND) publications with those available in the NEOPLASM controlled terminology (CT) code list in SEND. The version of CT used in a study should be referenced in the nonclinical study data reviewer's guide (SDRG) (section 3.1) of electronic submissions to the FDA. The tumor combination guide instructions and examples are in a tabular format to make informed decisions for combining tumor data for statistical analysis. The strategy for combining tumor types for statistical analysis is based on scientific criteria gleaned from the current scientific literature; as SEND and INHAND terminology and information evolve, this guide will be updated.

Inhibition of both sclerostin and DKK1 results in novel skull findings in the rat and non-human primate that is not observed with inhibition of sclerostin alone.

Sclerostin is an extracellular inhibitor of canonical Wnt signaling that inhibits bone formation and stimulates bone resorption. Anti-sclerostin antibodies (Scl-Ab) have been developed as bone-building agents. DKK1, another extracellular inhibitor of the pathway, is upregulated in osteocytes in response to sclerostin inhibition. To further enhance bone-forming effects, a bispecific antibody inhibiting both sclerostin and DKK1 was created (AMG 147). In nonclinical safety studies, AMG 147 resulted in novel skull findings. In the rat, there was increased thickness of skull bones of neural crest origin due to increased subperiosteal compact lamellar and intramembranous woven bone. Externally, subperiosteal fibroblastic/osteoblastic stromal cell proliferation with woven bone and hemorrhage was also observed. Scl-Ab alone resulted in increased skull thickness in the rat, like AMG 147, but without the stromal cell proliferation/woven bone formation. In contrast to embryonic flat bone development, intramembranous bone formed similar to plexiform bone. In the monkey, AMG 147 resulted in macroscopic skull thickening due to a diffuse increase in appositional lamellar bone and increased intramembranous bone on both periosteal surfaces of all skull bones. These data demonstrate that dual inhibition of sclerostin and DDK1 results in unique effects on the skull not observed with sclerostin inhibition alone.

New sensitive LC-MS/MS method for the simultaneous determination of 13 phenolic and carboxylic acid pesticide biomarkers in human urine, including dicamba.

The commercialization in 2016 of genetically engineered seeds tolerant to dicamba and/or 2,4-dichlorophenoxyacetic acid (2,4-D) has caused a rapid increase in the use of these herbicides. New questions about the reproductive and chronic health effects of long-term exposure to these herbicides have been raised. To assess exposure to dicamba and other pesticides of interest in the Heartland Study, a birth cohort study based in the United States, a new analytical method was needed. The present study describes the development and validation of this new solid phase extraction and liquid chromatography-tandem mass spectrometry method that detects simultaneously 13 pesticides or their metabolites in 250 µL of urine. More specifically, the method allows the analysis of dicamba, 2,4-D and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), which are herbicides, of malathion dicarboxylic acid (MDA), para-nitrophenol (PNP), 3,5,6-trichloro-2-pyridinol (TCPy), 2-diethylamino-6-methylpyrimidin-4-ol (DEAMPY) and 2-isopropyl-6-methyl-4-pyrimidinol (IMPY), which are metabolites of organophosphate insecticides, and finally of cis-3-(2,2-Dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (cis-DCCA), trans-3-(2,2-Dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (trans-DCCA), 3-Phenoxybenzoic acid (3-PBA), 4-Fluoro-3-phenoxybenzoic acid (4-F-3-PBA) and cis-3-(2,2-Dibromovinyl)-2,2-dimethylcyclopropane carboxylic acid (cis-DBCA), which are metabolites of synthetic pyrethroids insecticides. The method was validated under ISO/IEC 17025 guidance. The limit of detection (LOD) in urine samples was 0.10 µg/L for dicamba, while the LOD for other analytes ranged between 0.0038 µg/L and 0.091 µg/L. Accuracy was evaluated by analyzing samples from two External Quality Assessment Schemes, namely G-EQUAS and OSEQAS. Preliminary results obtained following the analysis of 91 urine samples taken from pregnant women enrolled in the Heartland Study are presented here. This method is suitable for human biomonitoring studies.

Urinary concentrations and determinants of glyphosate and glufosinate in pregnant Canadian participants in the MIREC study.

BACKGROUND: Glyphosate is the most widely applied herbicide in agriculture. Glufosinate is a broad spectrum herbicide used to manage glyphosate-resistant weeds. Despite the widespread use of these herbicides, biomonitoring data - which inform risk assessment and management - are sparse. OBJECTIVES: To identify determinants of urinary concentrations of these herbicides and their metabolites in pregnancy. METHODS: We measured urinary concentrations of glyphosate, glufosinate, and their primary metabolites aminomethylphosphonic acid (AMPA) and 3-methylphosphinicopropionic acid (3-MPPA) in a single spot urine specimen collected during the first trimester of pregnancy from the Maternal-Infant Research on Environmental Chemicals (MIREC) study. MIREC recruited about 2000 pregnant women from 10 Canadian cities between 2008 and 2011. We used UItra-Performance Liquid Chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) with sensitive limits of detection to quantify analyte concentrations. We examined urinary concentrations according to maternal sociodemographics, sample collection characteristics, reported pesticide use, and consumption of fruits, vegetables, legumes, and grain products. We used ANOVA models with specific gravity-standardized chemical concentrations as the dependent variable to determine associations with maternal and sample determinants. RESULTS: Among women with biobanked urine samples (n = 1829-1854), 74% and 72% had detectable concentrations of glyphosate and AMPA, respectively. In contrast, one and six percent of women had detectable concentrations of glufosinate and 3-MPPA, respectively. The specific gravity-standardized geometric mean (95% CI) concentrations of glyphosate and AMPA were 0.112 (0.099-0.127) µg/L and 0.159 (0.147-0.172) µg/L, respectively. We observed a dose-response relationship between consumption of whole grain bread and higher urinary glyphosate concentrations. Season of urine collection and self-reported pesticide use were not associated with increased concentrations of any analyte. CONCLUSIONS: We detected glyphosate and AMPA in the majority of pregnant women from this predominantly urban Canadian cohort. Diet was a probable route of exposure.

A rare case of bilateral antero-internal shoulder dislocation in a judo player: a case study and review of the literature.

Pure anterior bilateral shoulder dislocations are rare clinical features, especially in traumatic forms. They are most often posterior, occurring during an epileptic seizure. Few cases are described in the literature, and the mechanism varies from case to case. We report a specific case of pure bilateral anterior shoulder dislocation in a 29-year-old judo player following an accident during his training and discuss the circumstances, mechanism, treatment, and prognosis.

A rare case of bilateral antero-internal shoulder dislocation in a judo player: a case study and review of the literature

Pure anterior bilateral shoulder dislocations are rare clinical features, especially in traumatic forms. They are most often posterior, occurring during an epileptic seizure. Few cases are described in the literature, and the mechanism varies from case to case. We report a specific case of pure bilateral anterior shoulder dislocation in a 29-year-old judo player following an accident during his training and discuss the circumstances, mechanism, treatment, and prognosis.

Automatic Quantification of Atherosclerosis in Contrast-Enhanced MicroCT Scans of Mouse Aortas Ex Vivo.

OBJECTIVE: While microCT evaluation of atherosclerotic lesions in mice has been formally validated, existing image processing methods remain undisclosed. We aimed to develop and validate a reproducible image processing workflow based on phosphotungstic acid-enhanced microCT scans for the volumetric quantification of atherosclerotic lesions in entire mouse aortas. Approach and Results. 42 WT and 42 apolipoprotein E knockout mouse aortas were scanned. The walls, lumen, and plaque objects were segmented using dual-threshold algorithms. Aortic and plaque volumes were computed by voxel counting and lesion surface by triangulation. The results were validated against manual and histological evaluations. Knockout mice had a significant increase in plaque volume compared to wild types with a plaque to aorta volume ratio of 0.3%, 2.8%, and 9.8% at weeks 13, 18, and 26, respectively. Automatic segmentation correlated with manual (r 2 ≥ 0.89; p < .001) and histological evaluations (r 2 > 0.96; p < .001). CONCLUSIONS: The semiautomatic workflow enabled rapid quantification of atherosclerotic plaques in mice with minimal manual work.

Evaluating the toxicity of escalating dose of oral picolinic acid in Sprague-Dawley rats.

Picolinic acid (PIC) is a byproduct of tryptophan catabolism through the kynurenine pathway, with anabolic effects on bone in vivo and in vitro. Hence, PIC has been nominated as a possible candidate to treat and/or prevent osteoporosis. However, the effective dose and toxicity of PIC are not known yet. To test the effect of escalating and very high doses of oral PIC, male Sprague-Dawley rats were gavaged PIC: Group 1 (n = 3) received incremental doses of 125, 250 and 500 mg/kg/day PIC on days 1, 3 and 5. Group 2 (n = 3) received 500 mg/kg BID (8 h apart; i.e. 1000 mg/kg/day) PIC on Day 1. Group 3 (n = 3) received 125 mg/kg/day PIC for seven consecutive days. Group 4 (n = 3) received 250 mg/kg/day PIC for seven consecutive days. Groups 1, 3 and 4 rats were euthanized on Day 8. Group 5 (n = 6) received 500 mg/kg/day PIC for two consecutive days and then once a week dose (Days 9, 16 and 23) of 500 mg/kg/dose PIC, until euthanasia (Day 30). Blood and cerebrospinal fluid (CSF) were sampled at euthanasia, and tissues showing abnormalities at necropsy underwent histopathology evaluation. All rats displayed some degree of mild hypercalcemia and hyperkalemia. Rats receiving high doses (500 or 1000 mg/kg/day) of PIC died or were euthanized on humane grounds within the first week after showing clinical neurological signs, with animals later revealed to have brain necrosis and hemorrhage at histopathology. Rats receiving lower doses (125 or 250 mg/kg/day) of PIC completed treatment course without apparent clinical adverse events. In summary, very high doses of PIC (≥500 mg/kg/day) were vascular-neurotoxic. Possible future experiments must consider significantly lower doses.

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).

International Harmonization of Nomenclature and Diagnostic Criteria (INHAND): Nonproliferative and Proliferative Lesions of the Dog.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions) Project (www.toxpath.org/inhand.asp) is a joint initiative of the societies of toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying lesions observed in most tissues and organs from the dog used in nonclinical safety studies. Some of the lesions are illustrated by color photomicrographs. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions, lesions induced by exposure to test materials, and relevant infectious and parasitic lesions. A widely accepted and utilized international harmonization of nomenclature for lesions in laboratory animals will provide a common language among regulatory and scientific research organizations in different countries and increase and enrich international exchanges of information among toxicologists and pathologists.

Large Differences in Urinary Benzene Metabolite S-Phenylmercapturic Acid Quantitation: A Comparison of Five LC-MS-MS Methods.

Benzene is a known genotoxic carcinogen linked to many hematological abnormalities. S-phenylmercapturic acid (PHMA, N-acetyl-S-(phenyl)-L-cysteine, CAS# 4775-80-8) is a urinary metabolite of benzene and is used as a biomarker to assess benzene exposure. Pre-S-phenylmercapturic acid (pre-PHMA) is a PHMA precursor that dehydrates to PHMA at acidic pH. Published analytical methods that measure urinary PHMA adjust urine samples to a wide range of pH values using several types of acid, potentially leading to highly variable results depending on the concentration of pre-PHMA in a sample. Information is lacking on the variation in sample preparation among laboratories regularly measuring PHMA and the effect of those differences on PHMA quantitation in human urine samples. To investigate the differences in PHMA quantitation, we conducted an inter-laboratory comparison that included the analysis of 50 anonymous human urine samples (25 self-identified smokers and 25 self-identified non-smokers), quality control samples and commercially available reference samples in five laboratories using different analytical methods. Observed urinary PHMA concentrations were proportionally higher at lower pH, and results for anonymous urine samples varied widely among the methods. The method with the neutral preparation pH yielded results about 60% lower than the method using the most acidic conditions. Samples spiked with PHMA showed little variation, suggesting that the variability in results in human urine samples across methods is driven by the acid-mediated conversion of pre-PHMA to PHMA.

Individual back-calculated size-at-age based on otoliths from Pacific coral reef fish species.

Somatic growth is a critical biological trait for organismal, population, and ecosystem-level processes. Due to its direct link with energetic demands, growth also represents an important parameter to estimate energy and nutrient fluxes. For marine fishes, growth rate information is most frequently derived from sagittal otoliths, and most of the available data stems from studies on temperate species that are targeted by commercial fisheries. Although the analysis of otoliths is a powerful tool to estimate individual growth, the time-consuming nature of otolith processing is one barrier for collection of comprehensive datasets across multiple species. This is especially true for coral reef fishes, which are extremely diverse. Here, we provide back-calculated size-at-age estimates (including measures of uncertainty) based on sagittal otoliths from 710 individuals belonging to 45 coral reef fish species from French Polynesia. In addition, we provide Von Bertalanffy growth parameters which are useful to predict community level biomass production.

Nonclinical cardiovascular safety evaluation of romosozumab, an inhibitor of sclerostin for the treatment of osteoporosis in postmenopausal women at high risk of fracture.

Romosozumab (EVENITY™ [romosozumab-aqqg in the US]) is a humanized monoclonal antibody that inhibits sclerostin and has been approved in several countries for the treatment of osteoporosis in postmenopausal women at high risk of fracture. Sclerostin is expressed in bone and aortic vascular smooth muscle (AVSM). Its function in AVSM is unclear but it has been proposed to inhibit vascular calcification, atheroprogression, and inflammation. An increased incidence of positively adjudicated serious cardiovascular adverse events driven by an increase in myocardial infarction and stroke was observed in romosozumab-treated subjects in a clinical trial comparing alendronate with romosozumab (ARCH; NCT01631214) but not in a placebo-controlled trial (FRAME; NCT01575834). To investigate the effects of sclerostin inhibition with sclerostin antibody on the cardiovascular system, a comprehensive nonclinical toxicology package with additional cardiovascular studies was conducted. Although pharmacodynamic effects were observed in the bone, there were no functional, morphological, or transcriptional effects on the cardiovascular system in animal models in the presence or absence of atherosclerosis. These nonclinical studies did not identify evidence that proves the association between sclerostin inhibition and adverse cardiovascular function, increased cardiovascular calcification, and atheroprogression.

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.

A Diagnostic Approach for Rodent Progressive Cardiomyopathy and Like Lesions in Toxicology Studies up to 28 Days in the Sprague Dawley Rat (Part 2 of 2).

To test the diagnostic approach described in part 1 of this article, 2 exercises were completed by pathologists from multiple companies/agencies. Pathologist's examination of whole slide image (WSI) heart sections from rats using personal diagnostic approaches (exercise #1) corroborated conclusions from study #1. Using the diagnostic approach described in part 1, these pathologists examined the same WSI heart sections (exercise #2) to determine whether that approach increased consistency of diagnosis of rodent progressive cardiomyopathy (PCM) lesions. In exercise #2, there was improved consistency of categorization of small borderline morphologies and mild lesions, but a decrement in consistency of categorizing minimal lesions. Exercises 1 and 2 suggest the described diagnostic approach is representative of that in use by the majority of toxicologic pathologists across companies/agencies and that application by all may improve diagnostic consistency of PCM/like lesions. Additionally, a criterion of approximately 5% heart section involvement is suggested for separating mild from moderate or greater severity. While evidence is not absolute, until further investigation shows otherwise, microscopic changes resembling PCM, but located in the epicardial and subepicardial region of the right ventricle, may be considered as part of the spectrum of PCM.

A Diagnostic Approach for Rodent Progressive Cardiomyopathy and Like Lesions in Toxicology Studies up to 28 Days in the Sprague Dawley Rat (Part 1 of 2).

Spontaneous rodent progressive cardiomyopathy (PCM) in the Sprague Dawley rat may confound identification and/or interpretation of potential test article (TA)-related cardiotoxicity. Pathologists apply diagnostic term(s) and thresholds for diagnosing and assigning severity grades for PCM and/or PCM-like (PCM/like) lesions consistently within a study, which is necessary to identify and interpret TA-related findings. Due to differences in training and/or experiences, diagnostic terms and thresholds may vary between pathologists. Harmonized terminology and thresholds across studies will generate better historical control data, will likely enhance interpretation of study data, and may further enhance our understanding of the spontaneous change. An assessment of the diagnostic approaches of a group of 37 pathologists identified an approach that is relatively easily applied; and if adopted, it could enhance diagnostic consistency across studies. This approach uses the single "slash" term "necrosis/inflammatory cell infiltrate (NICI)" as the diagnosis for the spectrum of lesions seen in younger rats, uses no threshold for diagnosis (e.g., diagnose all lesions clearly identifiable as PCM/like), and uses aggregate lesion size of approximately ≥45% of the field of view (FOV) using a 10×/22 eyepiece and the 40× objective or approximately ≥100% of the FOV using the 60× objective as the criterion separating minimal from mild severities.

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.

Quantitative analysis of amino acids and acylcarnitines combined with untargeted metabolomics using ultra-high performance liquid chromatography and quadrupole time-of-flight mass spectrometry.

Metabolomics is an "omic" technique being increasingly used in epidemiological and clinical studies. We developed a method combining untargeted metabolomics with the quantitative determination of eight amino acids (AA) and eight acylcarnitines (AC) in plasma using ultra-high pressure liquid chromatography (UHPLC), electrospray ionization (ESI) and quadrupole time-of-flight mass spectrometry (QTOFMS). Separation of metabolites is performed by ion-pair reverse phase UHPLC using a HSS T3 column (2.1×100mm, 100Å, 1.8µm particle size) and formic acid-ammonium acetate-heptafluorobutyric acid in water and formic acid-ammonium acetate in methanol as mobile phases. Metabolite identification and quantification are achieved using a QTOFMS operating in ESI-positive and full-scan mode along with MS(E) acquisition of fragmentation patterns. Targeted metabolites are quantified using the appropriate labeled standards and include branched-chain AA (leucine, isoleucine, valine), aromatic AA (phenylalanine, tyrosine) as well as acetylcarnitine and propionylcarnitine, which have been identified as biomarkers of future cardiometabolic disease risk. The inter-day precision (relative standard deviation) for the targeted method was <15% for all but one metabolite and accuracy (bias) of amino acids ranged from 0.5% to 13.9% using SRM 1950 as the external standard. Untargeted metabolomics in 30 plasma samples from the general Canadian population revealed 5018 features, of which 48 metabolites were identified using the MZmine 2.19 software including 23 by our in-house library that comprises 671 annotated metabolites. SRM 1950 analysis revealed 11,684 features, among which 154 metabolites were identified. Our method is currently applied in several epidemiological studies to better characterize cardiometabolic diseases and identify new biomarkers for disease prevention.

Stability issues in the determination of 19 urinary (free and conjugated) monohydroxy polycyclic aromatic hydrocarbons.

Data on the stability of monohydroxy polycyclic aromatic hydrocarbons (OH-PAHs; metabolites of PAHs) in urine are needed in order to effectively study the effects of PAHs in the body, but the relevant data are not available in the literature. Therefore, in this work, we investigated the stability of OH-PAHs in urine. For each OH-PAH studied, the free form (as opposed to the conjugated form) comprised <10 % of the total OH-PAH in urine samples obtained from a normal population, except for 9-OH-phenanthrene (where the free form represented 22.2 % of the total 9-OH-phenanthrene). 1-Naphthol and 9-OH-phenanthrene were found to be less stable in their free forms in urine than in their conjugated forms when the urine samples were stored at 4 °C or room temperature. Free 3-OH-fluoranthene was also very unstable at 4 °C or room temperature. The conjugated forms of the OH-PAHs were more stable than their corresponding free forms. However, the free and conjugated forms of all the OH-PAHs were stable in urine at -20 °C and -80 °C. A freeze and thaw assay also revealed that freezing and thawing had minimal impact on the stability of the OH-PAHs in urine. For the derivatized extracts, storing the samples under an argon atmosphere at 4 °C was found to maintain sample integrity. In order to measure the stabilities of 19 hydroxylated metabolites of PAHs in urine, we developed a method with sensitivity in the low pg/mL range using nine labeled internal standards. This method combined enzymatic deconjugation with liquid-liquid extraction, derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), and gas chromatography/tandem mass spectrometry (GC-MS/MS). Graphical abstract Stability of the conjugated forms of the OH-PAHs versus free forms (e.g. 1-naphthol).

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.