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Page 6 of 17 Rawn et al. J Environ Expo Assess 2024;3:16 https://dx.doi.org/10.20517/jeea.2024.04

Once PBDE analyses were completed, sample extracts were concentrated just to dryness using a gentle
stream of nitrogen and diluted with the performance standard, deuterated d HBCD (α-, β- and γ-), in
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100 µL methanol: water (80:20) to allow for LC-MS/MS analysis.

Analysis
PBDE analysis
A Waters Autospec Ultima high resolution mass spectrometer (Waters Corporation, Mississauga, ON)
operating at 10,000 resolution, coupled to an Agilent 6890 gas chromatograph (Agilent Technologies,
Mississauga, ON), was used for all PBDE analyses (15, 17, 28, 37, 47, 66, 71, 75, 77, 85, 99, 100, 119, 126, 138,
153, 154, 160, 181, 183, 190, 205, 209). Separation of 23 congeners was achieved using a DB-5MS column
(15 m × 0.25 mm i.d. × 0.1 µm film thickness; J&W; Agilent Technologies, Mississauga, ON). One µL
aliquots were injected using a cool on-column injector, set to track the oven temperature. The starting oven
-1
temperature was 80 °C and held for 1 min, after which the temperature was increased at a rate of 32 °C·min
-1
until it reached 225 °C. Then, the oven was set to increase at 3 °C·min to 230 °C where it was held for
1 min and the final increase was set at 40 °C·min to a maximum temperature of 295 °C where it remained
-1
for 8 min. Helium was the carrier gas set at variable pressure (28-173 kPa), and the mass spectrometer was
operating in EI positive ion mode at 50 eV with the trap current set to 650 μA and the source temperature to
250 °C.

HBCD analysis
HBCD analyses were performed using a Waters Acquity ultra-high pressure liquid chromatograph I-Class
coupled to a Waters Xevo TQ-XS triple quadrupole mass spectrometer with electrospray ionization
operating in the negative ion detection mode. A 2.1 mm × 150 mm Kinetex C18, 2.6 µm column
(Phenomenex, USA) was used to separate the three HBCD isomers considered. Mobile phase A (water) and
acetonitrile: methanol (2:1) (mobile phase B) were used to separate α-, β- and γ-HBCD using gradient
elution, starting with 60% mobile phase B for 1 min, transitioning to 80% mobile phase B by 4 min, holding
until 11 min and increasing to 85% mobile phase B by 14 min and remaining there until 16 min, before
returning to the starting conditions by 16.1 min, where it was allowed to stabilize until 22 min. The flow rate
was 0.175 mL·min and the column temperature was set to 25 °C to support complete resolution of the d ,
-1
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13 C- and native HBCD isomers. The capillary and cone voltage were -2.5 kV and 20 V, respectively. The
source temperature was set to 150 °C, while the desolvation temperature was 400 °C. The desolvation gas
-1
-1
flow was 1,000 L·h , with the cone gas flow set to 170 L·h . Argon was used as the collision gas at a flow rate
-1
of 0.15 mL·min . The transitions monitored for native, C, and d HBCD isomers were: 639 → 79, 641 →
13
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79, 641 → 81, and 643 → 81 (native); 651 → 79, 653 → 79, 653 → 81, and 655 → 81 ( C surrogates); and
13
656 → 79, 658 → 79, 658 → 81, and 660 → 81 (d - performance standards), respectively. Dwell times were
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set to 24 msec.
Quality assurance/quality control
Two method blanks prepared using reagents alone, following the same protocol as for the unknown
samples, were included with each set of samples to allow for the removal of laboratory background
concentrations. Additionally, either one or two standard reference materials of human milk containing
known concentrations of PBDEs [standard reference material (SRM) 1953 (unfortified), SRM 1954
(fortified)] were included with each set of samples prepared for analysis. For those sets where a single SRM
was included, an internal sample of human milk that has been used in the laboratory as an internal quality
control (QC) over time was also included. PBDE concentrations and patterns determined in the SRMs were
as anticipated, with concentrations generally within two standard deviations of the expected values. The
internal QC sample PBDE concentrations were generally within one standard deviation of the mean values.