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Modified QuEChERS Extraction and Shoot-and-Dilute GC: Fast Sample Preparation and
Analysis of Halogenated Flame Retardants in Fish
Michelle Misselwitz, Jack Cochran, Julie Kowalski; Restek Corporation, 110 Benner Circle; Bellefonte, PA, USA
Introduction
Ø Halogenated flame retardants (HFRs), including polybrominated diphenyl ethers (PBDEs), have been added to numerous household and office products including polyurethane foams, electronics and plastics, and can
eventually migrate into household dust and the fatty tissue of animals.
Ø The health concerns of PBDEs are similar to that of polychlorinated biphenyls (PCBs) and at the most recent Stockholm Convention on Persistent Organic Pollutants, two technical mixtures (pentaBDE and octaBDE)
were added to the priority pollutant list. The production of the last remaining technical mixture (decaBDE) has been recently phased out.
Ø Other halogenated flame retardants are now being used to replace the PBDEs and there is still debate whether these replacements will be more environmentally and health friendly than their PBDE counterparts.
Ø Monitoring HFRs in food and environmental matrices can be difficult due to the complexity of the sample, structural isomers that must be separated chromatographically, and thermally-labile compounds that can
break down during gas chromatography (GC).
Ø Screening fish and other fatty foods for the presence of HFRs is important from a human health perspective and some of the newer high-production flame retardants do not have any available food occurrence data.
Ø In order to develop a screening method for halogenated flame retardants in fish, we paired a modified QuEChERS extraction and a quick extract pass-through with a PSA (primary secondary amine) cleanup cartridge.
Ø While splitless injections have been primarily used for trace-level analysis by GC, relying on the sensitivity of the electron capture detector (GC Micro-ECD) or tandem mass spectrometry (GC-MS/MS) for multiply
halogenated compounds allows the possibility to perform a split injection.
Ø This “Shoot-and-Dilute” technique is analogous to Dilute-and-Shoot for LC-MS/MS analysis in that it can reduce the impact of matrix-changed compound response, and increase instrumental ruggedness. Shoot-andDilute GC (split injection) is also advantageous over typical splitless injection because it decreases the residence time of thermally labile compounds in the hot GC inlet.
Ø GC inlet and column maintenance is especially important for BDE analysis, because nonvolatile material persists in sample extracts and deposits onto the inlet liner and front of the column. This can cause poor
transfer of BDEs ,which compromises quantification, sensitivity and also leads to poor peak shapes.
Materials and Methods
Sample Cleanup – PSA Cartridge Pass Through
Modified QuEChERS Extraction
1. Homogenize with LN2
5. Vortex 30 min using Ø Rinse PSA Cartridge (6 mL, 500 mg) with
10
mL
acetone
Glas-Col Shaker
2. Weigh 5 g sample
Ø Dry cartridge under vacuum for 2 min
+ 5 mL water
6. Add Extraction Salts Ø Add magnesium sulfate (MgSO4) to the
3. Add 100 µL IS, and
top of the cartridge (~ 0.5 g or 1 cm)
4 g MgSO4, 1 g NaCl
hydrate 30 min
Ø Add 1 mL extract then pull vacuum
7. Shake 1 min
quickly to elute sample through
4. Add 10 mL
completely (~ 10 sec)
Hexane:Acetone* (1:1)
8. Centrifuge 5 min
Ø
Transfer
to
limited
vial
insert
for
analysis
4,4’-dibromooctafluorobiphenyl, pentachloronitrobenzene, PCB 122 and decachlorobiphenyl were used as internal standards.
Analysis Conditions
Column: Rtx-1614, 15 m x 0.25 mm x 0.10 µm
Oven: 75°C (1.5 min) to 330°C (3.57 min)
at 18.3°C/min
Injection: 1µL split (10:1)
Inlet Temp: 300°C
Inlet Liner: 4mm Sky Split Precision® with wool
Carrier Gas: He, constant flow, 1.4 mL/min
GC-Micro ECD Temp: 350°C
Data collection: 10 Hz
N2 makeup + column flow constant: 50 mL/min
Results and Discussion
Ø Sample preparation is often the bottleneck in the analytical laboratory. Soxhlet or pressurized liquid extractions (PLE) are commonly used for the analysis of flame retardants and can take many hours and hundreds of
milliliters of solvents. With the modified QuEChERS extraction and the quick PSA cSPE pass-through the solvent usage is cut to 50 mL and preparation time is reduced to a few hours per sample.
Ø Experiments to determine the best extraction solvent resulted in using hexane:acetone (1:1) as the extraction solvent because it yielded more consistent recoveries in the 70 – 130% range compared to acetonitrile
(Table I). This also correlates with a previous project where hexane:acetone was used to extract flame retardants and PCBs from human breast milk.
Ø Using a split injection when analyzing high fat matrices, like fish, that have not been through an extensive cleanup procedure, increases system uptime over a splitless injection by depositing less nonvolatile material
onto the column (Figure 1). Chub mackerel has a high fat content (14%) and had 38 mg/mL of nonvolatile fat remaining after the PSA cleanup.
Ø More frequent inlet and column maintenance would need to be performed using a splitless injection compared to a split (10:1) injection. With a split injection, the system remained operational for a total of 15
mackerel injections compared to less than 3 mackerel injections using splitless injection.
Ø Recovery experiments were also evaluated using a Thermo TSQ 8000 GC-MS/MS. Analysis conditions were translated from the GC-ECD to vacuum outlet using the Restek EZGCTM Method Translator. Two transitions
were monitored for each analyte and target ion ratios within 30% confirmed a match (Table II).
Ø Potential incurred flame retardants include TCEP and TDCPP. These chlorinated Tris flame retardants have been linked to reproductive effects and potential neurotoxicity. Other possibly incurred flame retardants, TBB
and TBPH, are the main components of Firemaster 550®, a newer high production flame retardant used in replacement of pentaBDE (Table III).
Figure 1:
Ruggedness study
comparing splitless
injections (top) and
split injections
(bottom) of a 10 pg
on-column HFR
standard.
A chub mackerel
extract was
repeatedly injected
and alternated with
a standard in split
and splitless
injection mode to
monitor analyte
response and
system uptime. The
response for
Dechlorane Plus
isomers (red circle)
in the splitless
injection were
dramatically
reduced after only a
few (3) mackerel
injections.
Splitless
Table I: Comparison of the average (n=3) percent
recovery of spiked chub mackerel using different
extraction solvents.
0 µg fat
400
500
600
700
800
900
114 µg fat
400
500
600
700
800
900
380 µg fat
Tetrabromo-o-chlorotoluene
Pentabromotoluene
TDCPP
BDE 47
BDE 100
BDE 99
TBB
BB 153
BDE 153
TBPH
syn-Dechlorane Plus
anti-Dechlorane Plus
Hexane:Acetone
74
60
108
108
120
125
118
97
104
102
106
104
Acetonitrile
51
49
253
109
104
93
80
129
156
113
160
161
Table III: Possible incurred flame retardants found in
albacore tuna. Text in red represents values where the
target confirmation ion ratio was > 30%.
TCEP
TDCPP
TBB
TBPH
Dechlorane Plus
(total)
Prep Blank
Avg
ng/g
0.75
0.56
0.05
0.06
Tuna No
Spike 1
ng/g
1.67
1.90
0.39
1.77
Tuna No
Spike 2
ng/g
1.98
1.67
0.17
0.57
Average
Incurred(?)
ng/g
1.82
1.78
0.28
1.17
0.02
0.08
0.06
0.07
Table II: Percent recoveries of tuna fortified at 10 ng/g and 1 ng/g analyzed by GC-MS/MS with a split injection (10:1).
Text in red represents values where the target confirmation ion ratio was > 30%.
400
500
600
700
800
900
Split (10:1)
0 µg fat
400
500
600
700
800
900
114 µg fat
400
500
600
700
800
900
380 µg fat
400
500
600
700
800
900
tris(2-chloroethyl)phosphate (TCEP)
Pentabromobenzene
Tetrabromo-o-chlorotoluene
Pentabromotoluene
tris(1,3-dichloroisopropyl)phosphate (TDCPP)
BDE 47
BDE 100
BDE 99
2-Ethylhexyl-2,3,4,5-tetrabromobenzoate (TBB)
BB 153
BDE 153
Bis(2-ethylhexyl)tetrabromophthalate (TBPH)
syn-Dechlorane Plus
anti-Dechlorane Plus
Percent Recovery, 10 ng/g spike Tuna
(500 fg on-column)
S1
S2
S3
Avg %RSD
73
61
55
63
12
83
98
104
95
9
95
93
121
103
12
100
104
103
102
1
94
79
90
88
7
89
97
102
96
6
91
98
99
96
4
98
96
118
104
10
107
104
103
105
2
100
85
103
96
8
85
94
97
92
6
155
161
195
171
10
109
110
117
112
3
106
112
125
115
7
Percent Recovery, 1 ng/g spike Tuna
(50 fg on-column)
S1
S2
S3
Avg %RSD
295
359
287
314
10
69
94
116
93
20
130
95
112
112
13
114
105
104
108
4
362
313
281
319
10
106
135
115
119
10
119
103
115
113
6
132
109
101
114
11
165
171
123
153
14
110
100
130
113
11
118
93
125
112
12
341
147
392
293
36
132
114
127
124
6
117
119
119
118
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