Methanol, acetonitrile, ammonium hydroxide (Fisher Scientific, Pittsburgh, PA, USA), and formic acid, acetic acid, (Sigma-Aldrich, St. Louis, MO, USA) were all HPLC-grade. Deionized water was purified using a NANOpure Infinity ultrapure water system (Barnstead/Thermolyne, IA, USA). Diphenyl phosphate (DPhP), DPhP-d10, di-m-cresyl phosphate (DmCP), di-o-cresyl phosphate (DoCP), DoCP-d14, di-p-cresyl phosphate (DpCP), DpCP-d14, bis(1-chloro-2-propyl) phosphate (BCPP), BCPP-d12, bis(2-chloroethyl) phosphate (BCEtP), and BCEtP-d8 were purchased from Toronto Research Chemicals, TRC (Toronto, Canada). Bis(1,3-dichloro-2-propyl) phosphate (BDCPP), BDCPP-d10, TBBA, and TBBA-¹³C6 were purchased from Wellington Laboratories (Guelph, Canada). DBuP, DBuP-d18, DBzP, DBzP-d14, β-glucuronidase Type H-1 from Helix pomatia, and 4-Methylumbelliferyl β-D-glucuronidase hydrate (UMB) were purchased from Sigma-Aldrich. DMP, DMP-d6, DMTP, DMTP-d6, DMDTP, DMDTP-d6, DEP, DEP-d10, DETP, DETP-d10, DEDTP, DEDTP-d10, UMB-13C4 were purchased from Cam-bridge Isotope Laboratories (Andover, MA, USA). 2-((isopropyl) phenyl) phenyl phosphate (iPPPP) and 4-((tert-butyl) phenyl) phenyl phosphate (tBPPP) were purchased from Duke University Small Molecule Synthesis Facility (Durham, NC, USA). All chemicals and standard materials were used without further purification. The analytes chemical structures and abbreviations are shown in Fig. 1.

Individual stock solutions of standards and labeled internal standards were prepared by dissolving or diluting in appropriate solvent (according to manufacturers’ solubility recommendations). Using these individual stock solutions, three intermediate stock solutions with all target analytes were prepared in 1:1 (v/v) methanol/water giving a concentration of individual compounds of 1000 ng mL⁻¹, 500 ng mL⁻¹, or 50 ng mL⁻¹. Ten calibration standard solutions containing all target analytes were prepared by diluting appropriate amounts from intermediate stock solutions in 1:4 (v/v) methanol/water. A 200 μL spike from these calibration standards onto 200 μL of urine would cover a final concentration range of 0.05 ng mL⁻¹ to 100 ng mL⁻¹ for DMP, DMTP, DMDTP, and DEP, and 0.05 ng mL⁻¹ to 40 ng mL⁻¹ for the rest of the analytes. To monitor the extent of the enzymatic reaction, 4-methylumbelliferyl β-D-glucuronide hydrate and ¹³C4–4-methylumbelliferone stock solutions prepared in methanol were used as deconjugation standards. By mixing appropriate amounts from isotope-labeled standards and deconjugation standards in 1:4 (v/v) methanol/water, the spiking solution of isotope-labeled standards and deconjugation standards mixture was prepared, so that a 100 μL spike onto 200 μL of urine would result in 10 ng mL⁻¹ concentration of the individual labeled compounds, 1500 ng mL⁻¹ of 4-methylumbelliferyl β-D-glucuronide hydrate, and 75 ng mL⁻¹ of ¹³C4–4-methylumbelliferone. All stock solutions and standards were stored in amber polypropylene vials at or below −10°C.

To prepare quality control (QC) pools and for method validation, urine samples were collected anonymously in Atlanta, GA in 2015 from a diverse group of male and female adult volunteers with no documented occupational exposure to target flame retardants or OP pesticides. CDC’s Human Subjects Institutional Review Board reviewed and approved the study protocol. A waiver of informed consent was requested under 45 CFR 46.116(d). No personal or demographic data were collected.

These individual samples were screened for endogenous amounts of target analytes. Individual samples with overall lowest concentrations of endogenous target analytes were combined to form a blank pool. The blank urine was stored at or below −20 °C in glass vials. QC materials were prepared by spiking portions of blank urine with native target compounds. The low-concentration QC (QCL) was about 4 ng mL⁻¹; the high concentration QC (QCH) was about 15 ng mL⁻¹. The spiked QC materials were refrigerated, mixed for over 24 h, aliquoted into polypropylene vials (1 mL portions), and stored at or below −20 °C until use.

We also used 145 urine samples collected in 2010–2011 from firefighters after performing structural firefighting while wearing full protective clothing and self-contained breathing apparatus (SCBA) (Fent et al., 2014; Pleil et al., 2014). All participants gave consent to have their residual urine stored without identifiers for future research purposes, and the study protocol was approved by the U.S. National Institute for Occupational Safety and Health’s IRB. The analysis of these de-identified specimens was determined not to constitute engagement in human subjects research.

The extraction procedure was adapted from our previously published method (Jayatilaka et al., 2017), but adjusted for 200 μL urine. In brief, after adding 100 μL of labeled/deconjugation standard spiking mixture to each well, 200 μL of calibration standard solutions were spiked onto the wells assigned for each calibration level, 200 μL of deionized water was added to the solvent blank well, and 200 μL of QCs or study urines were added to the designated wells. Then, 400 μL of enzyme solution was dispensed to each well (a minimum of 1000 units of β-glucuronidase, 33 units of sulfatase per sample in 0.2 M sodium acetate buffer) and incubated overnight (typically 17 h) at 37 °C. Samples were cleaned up and concentrated by SPE as previously described (Jayatilaka et al., 2017), but eluted with 800 μL of freshly prepared 2% (v/v) NH₄OH in methanol and reconstituted with 100 μL of 95:5 (v/v) water: acetonitrile/methanol mixture after the drying step.

High-performance liquid chromatography (HPLC) was performed on an Agilent 1290 (Agilent Technologies, Santa Clara, CA, USA) system equipped with a binary pump, an autosampler with a cooling thermostat module, and a temperature controlled column compartment. Mobile phase A was 0.1% (v/v) acetic acid in deionized water and mobile phase B was a mixture of 1:1 acetonitrile: methanol. The gradient started at 5% of mobile phase B for 0.5 min, increased to 50% B in 4.0 min, ramped to 100% in 9.5 min and held for 1.5 min, returned to 5% B in 1 min and held for 4.0 min to equilibrate the column for the next sample. All analytes eluted within 10 min. The injection volume was 10 μL and the flow rate was constant at 0.7 mL/min. Chromatographic separation was performed on a Hypersil GOLD aQ column (150 × 4.6 mm, 3 μm; Thermo scientific, San Jose, CA, USA) preceded by inline filters (2 μm and 0.5 μm, Upchurch Scientific, Oak Harbor, WA, USA). During the sample analysis, the autosampler was kept at 4 °C and the column at 45 °C.

Mass spectrometry was performed on an AB Sciex 5500 Qtrap mass spectrometer (Applied Biosystems, Foster City, CA, USA) equipped with a TurboIonSpray^® source. The parameters were set equipped with a TurboIonSpray^® source. The parameters were set as follows: curtain gas 20, collision gas medium option, IonSpray voltage −4500 V, temperature 450 °C, and ion source gases 45. The mass spectrometer was operated in scheduled multiple reaction monitoring (sMRM) mode using negative polarity. Table 1 shows the transitions and collision energies used for each analyte.

The operation system and the mass spectrometry data acquisition were controlled by Analyst software version 1.6.3 (Applied Biosystems). This software controls all components of the analytical system. The data were processed and integrated with Multiquant software version 3.0.3 (Applied Biosystems).

Each analytical run prepared in a 96-well plate included a solvent blank, ten calibration standards, two aliquots each from QC low-and high-concentration (QCL, QCH) samples, and the study samples. The concentrations of the QCs, averaged to obtain one measurement of high- and one of low-concentration QC for each run, were evaluated by use of standard statistical probability rules (Caudill et al., 2008). For quantification, we used 10 standard solutions to construct daily calibration curves, weighted by the reciprocal of the standard amount (1/x), of the response factor (calculated as the peak area of each analyte ion divided by the peak area of its internal standard) versus the standard amount. Calibration curves were linear up to 3e4 orders of magnitude, depending on the analyte. Samples with concentrations exceeding the highest calibration standard were diluted, re-extracted, and reanalyzed so that the measured values were within the calibration range. Appropriate dilution factors were used to get the final concentrations. Because standards and study samples went through the same extraction procedure, reagent contributions were automatically corrected by the calibration curve intercept.

The purpose of this work was to add six OP pesticide metabolites and three additional FR metabolites to an existing method which was able to quantify nine FR metabolites in urine (Jayatilaka et al., 2017). Therefore, all analytes were optimized to existing mass spectrometry parameters using negative electrospray ionization (ESI). ESI has been the method of choice for quantifying FRs and DAPs before (Moller et al., 2004; Odetokun et al., 2010; Reemtsma et al., 2011; Van den Eede et al., 2013b; Butt et al., 2014; Been et al., 2017).

Several reversed phase HPLC columns with different dimensions and particle sizes were tested (data not shown). With most of the columns, including ZORBAX Eclipse XDB-C8 column used in our previous work (Jayatilaka et al., 2017), DMP eluted near the injection front and co-eluted with matrix interferences. Hypersil GOLD aQ (4.6 × 150 mm, 3 mm) was selected because it retained DMP a little longer than the other columns without co-eluting with matrix interferences, and had better chromatography for all the analytes considered. None of the columns evaluated separated DoCP, DmCP, and DpCP isomer peaks completely, and the isomers were quantified together (i.e., total DCP concentration). . Fig. 2 shows typical ion chromatograms for all target analytes in urine.

Strata-X-AW (60 mg per well, 96-well plate, Phenomenex, Torrance, CA, USA), used to quantify FR metabolites in urine before (Jayatilaka et al., 2017) was selected as the SPE column. Portions from break-through and wash steps were tested to evaluate any losses during sample loading and washing steps. Samples were eluted with 2% of NH4OH in methanol. Care was taken to control the sample pass through flow rate to as low as possible, a critical step to achieve good recoveries for DMP and DMDTP. Because Strata-X-AW extracts eluted with 2% NH4OH in methanol provided satisfying results for all new target analytes, performance for other sorbents was not evaluated.

Previous studies have shown that DAPs are directly excreted into urine, while hydroxylated OPFR metabolites need glucuronide conjugation before excretion (Ballesteros-Gómez al., 2015; Eede et al., 2015; Su et al., 2016). These conjugates must be hydrolyzed to measure total (conjugated plus free) concentration of the target analytes. Therefore, enzymatic treatment has been used for quantification of urinary flame retardant metabolites (Van den Eede et al., 2013a; Van den Eede et al., 2015; Su et al., 2016; He et al., 2018). Our previous work showed that the enzyme treatment only had a significant effect on DBuP and DPhP, and showed insignificant differences with the type of enzyme used (Jayatilaka et al., 2017). We tested the same enzyme, β-glucuronidase/sulfatase (Helix Pomatia, type H-1), on all new target analytes. As expected, enzyme treatment did not significantly affect the concentrations of the six DAPs. Also, no significant differences were observed for iPPPP and tBPPP with the enzyme considered. Tested samples had no detectable DCP concentrations to evaluate.

We assessed the suitability of the method by analyzing 158 urine samples collected anonymously from a convenience group of adults without known occupational exposure to these chemicals. We also tested the method by analyzing 145 urine samples collected from firefighters after performing structural firefighting while wearing full protective clothing and SCBA respirators. All of these samples were tested once with our previous method (Jayatilaka et al., 2017). With the current method, we were able to quantify concentrations of additional compounds, including DAPs. None of the samples tested had detectable DCP or DBzP (Table 4).

FR metabolite and DAPs concentrations in the general population group are within the ranges reported in the U.S. general population from NHANES (US-CDC, 2009; Ospina et al., 2018), and with those reported from previous research involving non- occupationally exposed populations (Cooper et al., 2011; McKelvey et al., 2013; Meeker et al., 2013; Butt et al., 2014; Dodson et al., 2014; Jayatilaka et al., 2017). For example, BDCPP and DPhP were also the FR biomarkers most frequently detected and at the highest concentrations in studies of 33 men from couples seeking fertility treatment in Massachusetts, USA (Meeker et al., 2013), 21 North Carolinian mothers and their toddlers (Butt et al., 2014), and nine other North Carolinian adults (Cooper et al., 2011), and 16 Californian adults (Dodson et al., 2014).

Compared to the general population samples, both DAPs and FR metabolite concentrations were higher in the firefighters group, and varied between two times (BCPP: 0.11 vs 0.24 ng mL⁻¹) and 37 times (DMTP: 0.39 vs 15 ng mL⁻¹) the concentrations of the non-occupationally exposed group. A recent study reported that U.S. fire stations were contaminated with higher levels of flame retardants than residences and other occupational settings (Shen et al., 2018), which can result in higher concentrations of flame retardant metabolites in firefighters. Polyurethane spray foam workers have also higher levels of urinary flame retardant metabolites than general population groups (Estill et al., 2019). Although dietary exposure to organophosphate insecticides or their degradation products (e.g., DAPs) influences the measured concentrations of DAPs (Lu et al., 2008; Curl et al., 2015), the reason(s) for the differences between the two populations examined are unknown. We did not have access to diet or other lifestyle related information for these groups of samples. It is possible that DEP and DMP also resulted from metabolism of trimethylphosphate (TMP) and triethylphosphate (TEP). TMP has been used as a gasoline additive for controlling surface ignition and spark-plug fouling; it is also used as a methylating agent, as a catalyst in the preparation of resins and polymers and as a flame retardant in paints and polymers (Connor, 1979). TEP is mostly used as flame retardant and plasticizer. TMP and TEP are metabolized in rats and mice to DMP and DEP, respectively and excreted in urine (Jones, 1970).