Dansyl chloride

Participants. The Maternal-Infant Research on Environmental Chemicals (MIREC) Study is a national-level pregnancy cohort of approximately 2,000 women recruited in the first trimester of pregnancy from 10 cities across Canada between 2008 and 2011 (Arbuckle et al. 2013 (link)). The protocol was approved by ethics committees at Health Canada and the Sainte-Justine University Hospital Center; study subjects gave written informed consent.
Data collection. We collected detailed information on demographic and lifestyle factors from questionnaires administered at recruitment in the first trimester. The date and time of the urine collection, as well as the time since last urine void, were also noted.
Urine collection and field blanks. During the first-trimester clinic visit, a spot urine sample was collected in polypropylene cups, aliquoted into 30-mL Nalgene® tubes, frozen at –20°C, and shipped on dry ice to the MIREC Biobank. Of the 2,001 participants, 43 did not consent to the Biobank, 18 women subsequently withdrew from the study, 47 urine samples were not collected, and 3 samples were insufficient, leaving a total of 1,890 urine samples for analysis.
We included field blanks to assess the potential contamination from the material used for collection and storage of urine samples as well as from the environment of collection sites. Water (Steril.O reagent-grade deionized distilled water) was used as a surrogate matrix for urine during the process. Water was poured into polypropylene cups and transferred to polypropylene storage tubes using the same material as for urine samples. Water samples were analyzed following pentafluorobenzyl bromide derivatization by gas chromatography–tandem mass spectrophotometry (GC-MS/MS) using the method previously developed in our laboratory and described by Provencher et al. (2014) (link). Results showed that all field blanks were free of BPA and TCS contamination. All materials in contact with urine samples had been prescreened and found not to be a source of contamination.
Analytical methods. The liquid chromatography (LC)-MS/MS methods for the analysis of free and conjugated forms of BPA and TCS in urine were described previously by Provencher et al. (2014) (link). Briefly, free BPA and TCS and their isotope-labeled standards, ¹³C₁₂-BPA and ¹³C₁₂-TCS, were derivatized with dansyl chloride directly in 1 mL of urine. A liquid–liquid extraction with hexane was subsequently performed and the organic phase evaporated prior to reconstitution in a solution of acetonitrile:H₂O (50:50, vol:vol). The LC-MS/MS (UPLC Acquity and Xevo TQ-S; Waters) was operated in electrospray positive and multiple reaction monitoring mode. Chromatographic separation was achieved on an Acquity UPLC HSS T3, 1.8 μm, 50 × 2.1 mm analytical column (Waters) using a mobile phase gradient with 0.1% aqueous formic acid solution and acetonitrile.
The conjugated metabolites BPAS, BPADS, BPAG, TCSS (TCS sulfate), and TCSG (TCS glucuronide) and their isotope-labeled standards BPAS-d₆, BPADS-d₆, BPAG-d₆, TCSS-d₃, and TCSG-d₃ were extracted from 1.5 mL of urine by solid phase extraction using a weak anion exchange phase (Strata X-AW; Phenomenex). Analytes were eluted from the cartridge using a solution of 1% ammonium hydroxide (NH₄OH) in methanol. The extracts were evaporated to dryness and reconstituted in a solution of 25% methanol in water. The same LC-MS/MS instrument and analytical column were used as for the free species, but the MS/MS was operated in the electrospray-negative and multiple reaction monitoring mode. A mobile phase gradient from aqueous NH₄OH (2%) to an NH₄OH–methanol solution (0.1%) was used to obtain proper chromatographic resolution of conjugated compounds.
Laboratory quality control (QC). Several QC samples, reagents blanks, and urine blanks were incorporated into each batch of samples. In-house reference materials were prepared by spiking human urine to yield low (0.18 μg BPA/L and 0.9 μg TCS/L) and high (1.5 μg BPA/L and 7.5 μg TCS/L) concentrations. For conjugated species, human urine was spiked to obtained reference materials at three different concentration levels: low (0.2 μg/L), medium (2 μg/L for sulfate metabolites; 3 μg/L for glucuronide metabolites), and high (15 μg/L for sulfate metabolites; 60 μg/L for glucuronide metabolites). The intraday precision varied from 2.5% to 7.7%, and the interday precision ranged from 4.3% to 13% depending on the analyte. The accuracy was –3.7% for free BPA and –1.0% for free TCS. The accuracy for conjugated forms ranged from –2.1% to 13.3% depending on the analyte. Detailed quality assurance/QC procedures were described by Provencher et al. (2014) (link).
Statistical analysis. Two different approaches were used to calculate summary statistics for the biomonitoring results that were below the limits of detection (LODs). The first approach used values generated by the laboratory instrument, and observations that were reported as zero were replaced by one-half the next smallest value (other than zero) for that contaminant. In the second approach, censoring methods were used by applying survival analysis techniques to left-censored data that have been demonstrated by other authors (Helsel 2012 ; Nysen et al. 2012 (link)) to improve estimation and reduce bias. To account for nondetects, the geometric mean (GM) from a lognormal random variable with censoring was calculated using the maximum likelihood method (MLE) and compared with the empirical median from the Kaplan-Meier approach. The Greenwood estimate of variance was used for Kaplan-Meier confidence intervals. We report summary statistics for both the unadjusted and specific gravity (SG)–adjusted contaminants.
In order to compare concentrations of free and conjugated forms of BPA or TCS, we expressed the concentrations of glucuronides and sulfates as BPA (or TCS) equivalents. Total BPA or TCS was calculated by summing the free and conjugated forms, and the most conservative LOD of the components was assigned to the total to determine the percentage below the LOD.
We calculated GM urinary concentrations for each level of potential predictive variables for all analytes that had at least 50% of the data above the LOD in all groups (as justified by Helsel 2012 ). For analysis of the associations between potential predictors and the urinary metabolite, SG was included as a covariate in the regression model using analysis of covariance (ANCOVA) (Kutner et al. 2005 ). ANCOVA adjusts the mean values compared in each level of the potential predictor such that the levels are compared at the same value of the covariate (in this case, SG). The assumptions of ANCOVA are similar to those of analysis of variance (normality and constant variance of residuals) with an additional assumption, that is, the slopes of the relationship between the covariate (SG) and the urinary metabolite must be similar in each level of the potential predictor. The assumptions of normality and equal variance of residuals were tested using the Anderson Darling test and Levene’s test, respectively. The assumption of equal slopes between levels of the potential predictor and the covariate is crucial for ANCOVA to be valid. This reduces to testing the interaction between the potential predictor and SG. When the assumption of equal slopes is not validated (p < 0.05), separate treatment regression lines need to be estimated and then compared (Kutner et al. 2005 ). This implies fitting the ANCOVA model with the interaction between the potential predictor and SG and then comparing the means of the urinary metabolite in each of the groups of the potential predictor at the 25th, 50th, and 75th percentiles of the covariate SG. When the assumptions of normality and constant variance (for the ANCOVA model) were not satisfied, nonparametric methods were applied. Essentially this involved running the models on the ranks of the data. When the overall F-test for group differences of the potential predictor from ANCOVA models was significant (p < 0.05), pairwise comparisons were carried out using the Scheffé correction for multiple comparisons to determine significant group differences. This correction ensures that the overall false-positive rate from multiple comparisons is < 0.05.
Statistical analysis was performed using SAS Enterprise Guide 4.2 (SAS Institute Inc.) and R (R Development Core Team). For the censoring methods, we used functions from the R libraries NADA and SURVIVAL. Unless otherwise indicated, a 5% significance level (α = 0.05) was implemented throughout.

Modification of the lysyl group was carried out according to El-Shora [34 ]. Stock solutions of DC in acetonitrile were freshly prepared. The enzyme sample (0.5 ml) was incubated at 4^oC for 1 h with various concentrations of DC (2, 4, 6, 8, and 10 mM) in 100 mM Tris-HCl (pH 7.0). The reaction was terminated by adding 20 mM β-mercaptoethanol and 30 mM lysine.