1. Analysis of combustion byproducts on firefighter protection equipment and in firefighter urine using novel high resolution GC/Q-TOF and bioassay
Christiane Hoppe-Jones, Shawn Beitel, Leanne Flahr, Sofia Nieto, Nathan Eno, Craig Marvin, and Shane Snyder
National Environmental Monitoring Conference
August 11th, 2017

2. Specific Aims
Evaluate exposure to carcinogens throughout the work shift, fire scene and station life
Measure biomarkers of carcinogenic effect in relation to workplace exposures
3) Reduce fire service carcinogen exposure and effects (personal protective equipment and post- fire policies and procedures)

3. Biomarker Testing
Tier 1. Quantification of select biomarkers of exposure in urine
Hydroxylated PAH, methoxyphenols, and volatile brominated compounds
Tier 2. In vitro bioassays
Measures biological response of the sum of compounds in the urine
Tier 3. GC/Q-TOF Non-targeted analysis of urine for the identification of new biomarkers of exposure

4. Study of Exposure – Analysis of Urine and Dermal Wipes

5. Quantification of hydroxylated PAHs
3 mL urine
Addition of Surrogate Standards
Enzyme digestion (18h)
Solid Phase extraction
Derivatization
GC-MS/MS and Data Analysis

6. Concentrations of urinary 1-Naphthol before and after fires for different occupational groups

7. Concentrations of urinary 2-Naphthol before and after fires for different occupational groups

8. Sample preparation and GC-qTOF analysis
Samples taken from helmet and neck prior to and post fire
4 in2 area wiped with cotton cloth and IPA
Extraction of cloth with 10mL DCM twice
Evaporation of extract to 1mL
Column
DB-5MS, 30 m, 0.25 mm, 0.25 µm
Injection volume
1 µL
Injection mode
Splitless
Split/Splitless inlet temperature
280 °C
Oven temperature program
50 °C for 3 min
10 °C/min to 300 °C, 7 min hold
Carrier gas
Helium at 1.5 mL/min, constant flow
Transfer line temperature
300 °C
Ionization mode
Standard EI at 70 eV; low energy EI at 15 eV and 12 eV
Source temperature, 70eV/15 eV or less
240°C/200°C
Quadrupole temperature
150°C
Mass range
50 to 1200 m/z
Spectral acquisition rate
5 Hz

9. Data analysis
Data processed using SureMass in Unknowns Analysis B.08.00
Compound identification using NIST14 EI library, confirmation by retention index (RI)
Molecular ions of unknown brominated compounds were identified with the help of low electron energy
Molecular ions were confirmed by evaluating the complete cluster for m/z, relative isotope abundance and isotope ratios using Molecular Formula Generator (MFG)

10. Identification of PAHs on the
helmet post-fire
Compound/formula
m/z
Avg mass error (ppm)
Formula
Regulated PAHs
Other PAHs
O-containing PAHs
Naphthalene [C10H8]
1.01
C10H12
0.91
C13H8O
0.90
Acenaphthylene [C12H8]
0.82
C11H14
1.07
C16H10O
1.47
Acenaphthene [C12H10]
0.35
C11H10
0.55
C17H10O
1.99
Fluorene [C13H10]
1.15
C12H16
1.22
C17H10O2
1.27
Phenanthrene [C14H10]
1.25
C12H12
C18H10O
2.03
Anthracene [C14H10]
0.96
C13H12
1.14
C18H10O2
Fluoranthene [C16H10]
1.06
C14H14
0.59
C20H12O
1.68
Pyrene [C16H10]
1.51
C15H12
0.53
Benz[a]anthracene [C18H12]
C15H10
2.57
Chrysene [C18H12]
C18H22
Benzo[b]fluoranthene [C20H12]
1.81
C18H18
1.04
Benzo[k]fluoranthene
[C20H12]
2.23
C17H12
0.87
Benzo[a]pyrene [C20H12]
1.79
C18H10
0.92
Indeno[1,2,3-cd]pyrene
[C22H12]
1.65
C20H14
1.05
N-containing PAHs
Dibenz[a,h]anthracene
[C22H14]
1.55
C19H14
0.58
C13H9N
1.24
Benzo[ghi]perylene [C22H12]
1.73
C19H12
2.17
C12H9N
C22H14
C15H9N
1.52
C24H18
1.67
C17H11N
0.63
C22H12
2.25
C19H11N
1.08
C13H8O
C16H10O
C18H10O
C10H8
C16H10
C20H12
C22H14
C11H10
C12H12
C13H9N
C15H9N

11. Detection of Volatile Brominated Organic Compounds
ICP-MS technique is uniquely sensitive to detect halogens (I>Br>>Cl) when coupled with LC or GC.
Brominated flame retardants and other halogenated metabolites will be targeted.

12. Detection of Volatile Brominated Organic Compounds (GC-ICP-MS)
Br-I-benzene
Br-F-benzene
Further Identification using Mass Spectrometry and Correlation with Exposure needed

13. Detection of Flame Retardants on dermal wipes
Organic flame retardants were found on the skin post fire.

14. Detection brominated flame retardants

15. Detection brominated flame retardants
70 eV
15 eV
12 eV
MFG Score
95.40
99.34
99.47

16. Other brominated compounds
70 eV
15 eV M+
Contaminant ions
70 eV
15 eV
12 eV M+
Contaminant ions Br OH Br OH

17. Other brominated compounds
Formula
m/z
Mass error,
ppm*
Resolving
power
MFG
score
Verified
by standard
Neck –
Pre-fire,
% abundance
Post-fire,
Helmet –
C6H4Br2O
1.7
27336
98.34
n.d.
15.26
C14H21BrO
0.92
26911
90.92
31.17
C7H8Br2N2
0.22
27495
88.15
5.72
C7H6Br2N2O
0.99
26072
90.08
13.84
C10H9BrN2
1.27
27136
97.41
1.53
C16H9Br
1.69
26187
98.71
4.39
C15H14Br2O2
0.25
29880
95.36
0.32
C12H7Br3O
0.45
31609
90.67
Tri-BDE
1.1
0.29
0.23
29157
96.13
1.41
0.4
C12H6Br4O
1.36
30425
91.87
Tetra-BDE
0.9
2.62
31364
95.40
58.84
13.09
1.15
27322
93.01
0.69
0.3
C12H5Br5O
2.59
30367
95.05
Penta-BDE
8.99
2.74
2.03
30813
94.57
25.29
9.26
1.24
29448
91.29
0.35
C12H4Br6O
0.7
30817
91.38
Hexa-BDE
0.83
0.51
31299
94.73
0.85
0.43
* mass error is calculated as weighted average mass error for the entire isotopic cluster Br OH

18. In Vitro Bioassays
Testing de-conjugated urine extracts as an indicator of exposure to biologically active compounds that:
Interact with the arylhydrocarbon receptor (AhR)
Responsive to PAHs, dioxins, furans, etc.
PAH CALUX®

19. Aryl hydrocarbon receptor assay
Greater concentrations of agonists and greater binding affinity lead to:
Luminescence

20. AhR Bioassay Results
Sum of PAH-OH
does not explain

21. Conclusions
Hydroxylated PAHs in post-fire urines were elevated for all occupational groups present at fires
Brominated flame retardants were detected on the skin of firefighters post-fire and could be potential biomarkers of exposure
Sensitive GC-qTOF analysis can help identifying possible biomarkers and the low energy EI source facilitated the identification
Further investigation into bioassay results and correlation with hydroxylated PAH results needed. Br

22. Questions?