analysis of hexabromocyclododecane diastereomers and...
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©2014 Waters Corporation 1
Analysis of Hexabromocyclododecane Diastereomers and Tetrabromobisphenol-A
Using a Novel Method Combining Supercritical Fluid Chromatography with Tandem Mass
Spectrometry
Douglas Stevens1, Lauren Mullin1,2, Kenneth Rosnack1, Ingrid Ericson Jogsten2, Dawei Geng2, Andy Aubin1, Bert van Bavel2
1Waters Corporation
2MTM Research Centre, Örebro University, Sweden
©2014 Waters Corporation 2
Overview
¡ Supercritical fluid chromatography (SFC) applicability to hexabromocyclododecane (HBCDD) and tetrabromobisphenol-A (TBBPA) analysis
¡ Chromatography and MS/MS method development ¡ Sample analyses results
¡ New/preliminary results: Chiral separations and additional flame retardants
¡ Conclusions
©2014 Waters Corporation 3
What is a Supercritical Fluid?
High diffusivity, low viscosity = fast, efficient
chromatography
Liquid/gas Critical point Supercritical fluid
> temp and pressure
> temp and pressure
©2014 Waters Corporation 4
UPC2 TQ-S
MS Splitter
MS Splitter
to MS
to CCM
from Make Up Pump
from CM
Make Up Pump
©2014 Waters Corporation 5
HBCDD Isomeric Separation
¡ HBCDD added to Stockholm Convention list of Persistent Organic Pollutants (POPs)
¡ Predominant HBCDD diasteromers and enantiomers
¡ Proportion dependent on sample type
©2014 Waters Corporation 6
Chromatographic Efficiency: 3 min separation of HBCDD and TBBPA
TBBPA
β-HBCDD
γ-HBCDD
α-HBCDD
©2014 Waters Corporation 7
Faster Analysis Time and Chromatographic Orthogonality
Expanded SFC Separation
LC
α- γ-
α-
β-
γ-
β- α-
β-
γ-
Rs=3.15
Rs=4.36
Rs=5.07
Rs=10.42 SFC
©2014 Waters Corporation 8
Method Development
©2014 Waters Corporation 9
MS Splitter
MS Splitter
to MS
to CCM
from Make Up Pump
from CM
Make Up Pump
UPC2 TQ-S
©2014 Waters Corporation 10
Chromatography Optimization
©2014 Waters Corporation 11
MS Splitter
MS Splitter
to MS
to CCM
from Make Up Pump
from CM
Make Up Pump
UPC2 TQ-S
©2014 Waters Corporation 12
Ionization Type Comparison
©2014 Waters Corporation 13
Make-up Flow Composition
0
5000
10000
15000
20000
25000
30000
35000
1. MeOH 2. 2-‐propanol 3. 0.1%NH4OH in MeOH
4. 0.1% NH4OH in 2-‐propanol
5. 0.2% NH4OH in 90:10 2-‐
propanol:MeOH
TIC Pe
ak Area
Make-‐up flow composition
Post Column Make-‐Up Flow Optimization
alpha HBCDD
gamma HBCDD
beta HBCDD
TBBPA
©2014 Waters Corporation 14
500 ppt HBCD isomers and TBBPA
Time0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 1.45
%
0
10004052013_10a 2: MRM of 2 Channels ES-
640.6 > 80.9 (HBCD)5.38e3
1.34
1.03
0.89
250 ppt HBCD isomers and TBBPA
Time0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 1.45
%
0
10004052013_07a 2: MRM of 2 Channels ES-
640.6 > 80.9 (HBCD)3.11e3
1.35
1.04
0.90
100 ppt HBCD isomers TBBPA
Time0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 1.45
%
0
10004052013_04a 2: MRM of 2 Channels ES-
640.6 > 80.9 (HBCD)1.15e3
1.35
1.030.89
0.81
0.97
1.151.131.28
1.47
Method Limit-of-detection (LOD) and quantification (LOQ)
0.250 pg/µl LOQ for β-HBCDD
0.500 pg/µl LOQ for α-, γ-
HBCDD
LOD Peak-to-peak S/N >3 LOQ Peak-to-peak S/N >10
γ-
α-
α-
α-
γ-
γ-
β-
β- β-
0.100 pg/µl LOD for α-,β- and γ-
HBCDD
©2014 Waters Corporation 15
ES- MS/MS conditions
¡ Capillary Voltage: 2.0 ¡ Cone Voltage: 30 ¡ Source Temperature: 150 °C ¡ Desolvation Temperature: 500 °C ¡ Desolvation Gas: 1000 L/hr ¡ Cone Gas: 150 L/hr ¡ Points per peak: 15
©2014 Waters Corporation 16
UPC2 Gradient and Conditions
¡ Column: HSS C18 SB (1.8µm 3.0 x 100 mm) ¡ Column Temperature: 40 °C ¡ Sample Temperature: 25 °C ¡ Phases: CO2 (A) and methanol (B) ¡ Injection Volume: 1 µL ¡ Make-up flow composition: 0.1% NH4OH in 2-propanol ¡ Make-up flow rate: 0.2 mL/min
Time (min.) % A % B Flow Rate (mL/min.)
Ini$al 98 2 2 2 90 10 2
2.01 90 10 2.5 2.3 98 2 2.5 2.31 98 2 2
©2014 Waters Corporation 17
Application to Biological Matrices
©2014 Waters Corporation 18
Sample Analyzed
¡ Can method separate and detect HBCDD isomers in complex matrices?
¡ Wide solvent compatibility with SFC so final extract used without solvent exchange
¡ Extracts of human plasma and whale blubber were donated from prior GC analyses
©2014 Waters Corporation 19
min0.600 0.800 1.000 1.200 1.400
%
0
100
F2:MRM of 2 channels,ES-640.6 > 78.9
05102013samples_35500 ppt HBCD and TBBPA tetra
3.250e+0031.35
1.03
0.89
min
%
0
100
F2:MRM of 2 channels,ES-640.6 > 80.9
05102013samples_35500 ppt HBCD and TBBPA tetra
3.171e+0031.35
1.030.89
min0.600 0.800 1.000 1.200 1.400
%
0
100
F2:MRM of 2 channels,ES-640.6 > 78.9
05102013samples_06DL-08-007-281
9.930e+0020.89
1.361.04
min
%
0
100
F2:MRM of 2 channels,ES-640.6 > 80.9
05102013samples_06DL-08-007-281
9.670e+0020.89
0.55 0.61 0.731.260.94 1.03
1.171.36 1.40
Human Plasma Detection: Low Level
Calibration Curve Results
Quantifier MRM
Confirmatory MRM
Quantifier MRM
Confirmatory MRM
S A M P L E
S T A N D A R D
%RSDs (n=5)Concentration (pg/µl) α -‐HBCDD β-‐HBCDD γ-‐HBCDD
0.25 <LOQ 7.9 <LOQ0.5 5.6 11.6 16.71 12.6 4.0 13.15* 6.8 2.3 5.310 3.0 2.2 3.725 5.5 2.0 1.650 2.3 2.2 1.8100 3.7 1.3 1.0Fit Linear Linear Linear
Weighting 1/x 1/x 1/xR2 0.998 0.999 0.999*n=4
Calculated Concentrations ng/g lipid weightSample α -‐HBCDD β -‐HBCDD γ -‐HBCDDHS1 4.21 <LOD <LOD
©2014 Waters Corporation 20
min0.600 0.800 1.000 1.200 1.400
%
0
100
F2:MRM of 2 channels,ES-640.6 > 78.9
08062013samples_6110 ppb HBCD and TBBPA tetra
6.315e+0041.33
1.020.88
min
%
0
100
F2:MRM of 2 channels,ES-640.6 > 80.9
08062013samples_6110 ppb HBCD and TBBPA tetra
6.624e+0041.33
1.020.88
min0.600 0.800 1.000 1.200 1.400
%
0
100
F2:MRM of 2 channels,ES-640.6 > 78.9
08062013samples_10DL-08-008:6b
2.294e+0040.88
min
%
0
100
F2:MRM of 2 channels,ES-640.6 > 80.9
08062013samples_10DL-08-008:6b
2.189e+0040.88
Whale Blubber Detection: High Level
%RSDs (n=5)Concentration (pg/µl) α−HBCD β−HBCD γ−HBCD
0.25 <LOQ 11.7 <LOQ0.5 9.8 6.4 15.11 16.8 9.4 5.75 4.3 1.0 2.310 5.4 1.0 4.425 3.4 2.2 3.450 1.6 0.6 1.3100 3.4 2.6 3.5Fit Linear Linear Linear
Weighting 1/x 1/x 1/xR2 0.998 0.999 0.999
1.15 0.80
13.25
2.255.17
23.86
0.00
5.00
10.00
15.00
20.00
25.00
30.00
WB 1 WB 2 WB 3 WB 4 WB 5 WB 6
Calcu
lated Co
ncen
tration ng/g lipid weight
Whale Blubber Extracted Sample
Calculated Concentrations of α-‐HBCDD Quantifier MRM
Confirmatory MRM
Quantifier MRM
Confirmatory MRM
S A M P L E
S T A N D A R D
Calibration Curve Results
©2014 Waters Corporation 21
Ion Ratio Confirmation
¡ Ion Ratio = Quantifier MRM area/Confirmatory MRM area ¡ Pass = +/- 20% of expected ion ratio ¡ Expected ratio determined by averaging all points’ ion ratio in
calibration series
Ion RatiosCalculated Concentrations pg/µl α -‐HBCDD β -‐HBCDD γ -‐HBCDD
Human Serum Samples α -‐HBCDD β -‐HBCDD γ -‐HBCDD [1.123] [1.097] [1.11]HS 1 0.575 ND ND 1.342 ND ND
Whale Blubber Samples [1.112] [1.163] [1.127]WB 1 12.88 ND ND 1.108 ND NDWB 2 8.33 ND ND 1.109 ND NDWB 3 47.7 ND ND 1.117 ND NDWB 4 19.8 ND ND 1.16 ND NDWB 5 45.5 ND ND 1.088 ND NDWB 6 85.9 ND ND 1.003 ND ND
©2014 Waters Corporation 22
Chiral Separations Preliminary Results
©2014 Waters Corporation 23
Chiral HBCDD Separation
¡ Prior LC method 4.0 x 200mm 5µm PM-β-CD column with >15 min run
¡ UPC2 prototype cellulose 2.1 x 150mm 2.5µm column on standards
¡ (+/-) α- and γ- separated using same co-solvent, (+/-) β- with different co-solvent
¡ Further method optimization
underway
Chiral Separation EtOH co-solvent
α-HBCDD standard
α-HBCDD detected in matrix
©2014 Waters Corporation 24
(+/-) α-HBCDD in Whale Blubber
0
50
100
150
200
250
300
350
WB5 WB1 WB2 WB4
Area Cou
nts
Whale Blubber Samples
α-‐HBCDD Enantiomers in Whale Blubber
alpha-‐HBCDD E1
alpha-‐HBCDD E2
Total alpha-‐HBCDD
©2014 Waters Corporation 25
Additional Flame Retardants Preliminary Results
©2014 Waters Corporation 26
Investigation of Traditionally GC-Amenable Flame Retardants
¡ Wide variety of FRs monitored in environment and biota ¡ Polybrominated diphenyl ethers (PBDEs) traditionally require
GC-MS UPC2 TQ-S APCI Overlaid SIRs
BDE 28, 33
BDE 47 BDE 99, 100
BDE 209
0.1 µg/mL
1.0 µg/mL
©2014 Waters Corporation 27
Diverse Flame Retardants on Single Platform: Preliminary Detection
Flame RetardantCurrent Chromatographic
TechniqueOptimum Ionization Technique (UPC2)
Predominantly Observed Ion
Retention Time (min.)
triphenylphosphate LC ESI+ [M+H]+ 1.01α−,β−,γ−HBCDD LC ESI-‐ [M-‐H]-‐ 0.88,1.01,1.33
TBBPA LC ESI-‐ [M-‐H]-‐ 2.11tris (1,3-‐dichloro-‐2-‐propyl) phosphate GC and LC ESI-‐ [M-‐H]-‐ 0.62tris (2,3-‐dibromopropyl) phosphate GC and LC ESI-‐ [M-‐H]-‐ 1.11
BDE 28 GC APCI-‐ [M-‐H]-‐ 0.88BDE 33 GC APCI-‐ [M-‐H]-‐ 0.67BDE 47 GC APCI-‐ [M-‐Br+O]-‐ 1.12BDE 99 GC APCI-‐ [M-‐Br+O]-‐ 1.4BDE 100 GC APCI-‐ [M-‐Br+O]-‐ 1.43BDE 209 GC APCI-‐ [M-‐C6Br5]-‐ 4.19
©2014 Waters Corporation 28
Conclusions
¡ Rapid and efficient 3 min separation of α-,β- and γ-HBCDD demonstrated effective for complex samples
¡ Ability to inject wide range of solvents eliminates need for
solvent exchange required for RP-LC ¡ Chiral separation method development underway for HBCDD
enantiomers
¡ UPC2 shows potential for single chromatographic platform for GC and LC amenable flame retardants
©2014 Waters Corporation 29
Acknowledgements
¡ Samira Salihovic (human serum extracts) and Anna Rotander (whale blubber extracts), MTM Research Centre, Örebro Universitet, Örebro Sweden
¡ Waters Corp - Baiba Cabovska, Mike Jones, Ken Fountain and
Mark Baynham, Chris Hudalla
¡ Reference for APCI Ionization of PBDEs: S. Zhou, E. Reiner, C. Marvin, P. Helm, N. Riddell, F. Dorman,
M. Misselwitz, L. Shen, P. Crozier, K. MacPherson and I. Brindle, Analytical and Bioanalytical Chemistry, 2010, 396, 1311-1320.