Florisil

Wristband and hand wipe samples were extracted and analyzed using previously published methods for each matrix for the aforementioned organophosphate flame retardants: TDCIPP, TCIPP, TPHP, and mono-ITP.^{12 (link),28 (link)} Each wristband, including the field blanks, was cut using solvent-rinsed scissors into two equal pieces to ensure that the wristbands would be submerged under the solvent within a Soxhlet apparatus used for extraction. Prior to beginning the extraction, the bands were spiked with d₁₅-TDCIPP (162 ng) and ¹³C-TPHP (100 ng) as internal standards. The wristbands were Soxhlet extracted with 1:1 hexane:acetone (v/v) for 12 hours, and the extracts were concentrated using an automated nitrogen evaporation system (Turbo Vap II, Zymark Inc.). Extracts were filtered with a 25mm syringe filter with a 0.2 micrometer PTFE membrane to remove larger particles. This concentrated extract were later cleaned using a Florisil solid-phase extraction cartridge (Supelclean ENVI-Florisil, 6 mL, 500-mg bed weight; Supelco), eluting the F1 fraction with 10 mL hexane (brominated compounds) and the F2 fraction with 10 mL ethyl acetate (PFRs), which was adapted from the method developed by Van den Eede, et. al (2012).^{32 (link)} Using a nitrogen evaporator system, each fraction was concentrated to about 1 mL then transferred to an autosampler vial for gas chromatography-mass spectrometry (GC/MS) analysis (Agilent Technologies, Models 6890N and 5975, respectively). The F1 concentrated fraction was stored at −20°C for future analysis. To measure recovery of the organophosphate internal standards in the wristbands, d₉-tris(2-chloroethyl) phosphate (d₉-TCEP; 227 ng) and d₁₅-TPHP (429 ng) were spiked into each sample to measure recovery of d₁₅-TDCIPP and ¹³C-TPHP, respectively. Recoveries of d₁₅-TDCIPP and ¹³C-TPHP averaged 33 ± 5% and 99 ± 6%, respectively, in all samples. Prior to the Florisil cleanup, recovery of the d₁₅-TDCIPP was 94 ± 5%, which suggests some of the compound were lost with additional processing. It is possible that short oligomers were extracted from the wristbands during the Soxhlet extraction which may have sorbed both labeled and unlabeled TDCIPP which were retained on the Florisil SPE column. However, due to the use of isotope-labeled standards, we still have confidence in the accuracy of our results following the cleanup step. Four lab blanks were analyzed alongside the wristbands and field blanks for quality assurance and quality controls.
The whole hand wipe samples were each spiked with d₁₅-TDCIPP (180 ng) and ¹³C-TPHP (50 ng) as internal standards and extracted three times via sonication with 1:1 hexane: acetone (v/v). The combined extract of roughly 45 mL was concentrated to 1 mL using a nitrogen evaporator system, then transferred to an autosampler vial for GC/MS analysis. Recoveries of the internal standards in the hand wipes were measured by spiking all samples with d₁₅-TPHP (429 ng). Recoveries of d₁₅-TDCIPP and ¹³C-TPHP averaged 119 ± 14% and 97 ± 12%, respectively.
For both wristbands and hand wipes, mono-ITP was quantitated using a commercial mixture of FM550 and assuming the percent of mono-ITP in FM550 by mass is 32%.⁸ All isomers at 368 m/z were integrated over a retention time of 15.90 minutes to comprise the mass of mono-ITP in each sample. PFR concentrations in wristbands and hand wipes were blank corrected based on the average concentrations measured in the field blanks (Supplemental Table S1). Higher background levels were measured in the wristbands relative to the hand wipes for some chemicals. The Soxhlet extraction process may have contributed to the higher wristband field blank levels in wristbands; however, the wristband field blanks were still significantly lower than levels measured in the wristband samples. Method detection limits (MDLs) were calculated as three times the standard deviation of the levels in the field blanks. MDLs for the PFRs ranged from 5.7 ng for TPHP to 30.3 ng for TCIPP on wristbands and 0.19 ng for TPHP to 11.21 ng for TDCIPP on hand wipes (Table 2).

All wristbands were prepared and processed according to our previously published methods^{5 (link), 7 (link)}. Briefly, silicone wristbands (24hourwristbands.com, Houston, TX) were solvent cleaned in three different organic solvents prior to distribution to study participants. Prior to cleaning dog tags were prepared by cutting a wristband to one-fourth the size and affixing them to a metal key ring to allow for easy attachment to a dog collar. At the end of the study period, participants removed their wristband and the collar tag from their dog, placed them in separate clean pieces of aluminum foil. The silicone band and tags were sealed in an air tight bag and stored in a domestic freezer until transport or shipment back to the laboratory. Silicone wrist bands and dog tags used as field blanks were treated the same way except they remained stored in foil at room temperature in the laboratory. All wristbands and tags were stored in −20°C until extraction, which was no longer than 4 months for any sample.
Prior to processing, each dog tag was cut in half and each wristband was cut into similarly sized pieces (ranging from 0.6–0.9g) for extraction. Individual pieces were weighed and then placed in a glass tube, spiked with a suite of isotopically labeled compounds (Table S1.) and extracted in 10 mL of a 50:50 (v:v) mixture of hexane:dichloromethane in a 15 minutes sonication extraction. This step was repeated three times for a total extraction volume of 30 mL. Prior to column chromatography extracts were concentrated to ~1.0 mL with nitrogen gas using a Savant SPD121P SpeedVac™ Concentrator (Thermo Scientific). Extracts were cleaned using 8.0 g deactivated Florasil® (Acros Organics™ Florisil™, 100–200 mesh, FisherScientific). Three fractions were collected, F1, F2, and F3 using elution solvents hexane, ethyl acetate, and methanol, respectively. F1 and F2 were combined and concentrated to 1.0 mL using an automated nitrogen evaporation system (TurboVap II, Zymark Inc.). Samples were concentrated to near dryness and reconstituted in 1.0 mL hexane. A suite of isotopically labelled (Table S2) recovery standards were spiked into each sample prior to mass spectrometry analysis. The average percent recovery across all surrogate standards was 68 ± 22%. The average recovery for each individual labelled compounds can be found in Table S1. Samples were analyzed for a suite of target compounds using a Q Exactive GC hybrid quadrupole-Orbitrap GC-MS/MS system (Thermo Scientific) operated in full scan in full scan Electron Ionization (EI) mode. Samples were analyzed for brominated flame retardants using a single quadrupole GC-MS (Agilent 6890N and 5975, respectively) operated in negative chemical ionization (NCI) mode. Field blanks (n=8, four wristbands and four dog tags) and lab blanks (n=7) were processed and analyzed with each batch of wristbands for quality control and quality assurance. Method detection limits were based on the field blanks if analytes were detected in field blanks, using three times the standard deviation of the field blank responses, or equal to 10 times a signal to noise value if the analyte was not detected in the field blanks.