rapid separation of hbcdd stereoisomers and …...©2014 waters corporation 19 min 0.600 0.800 1.000...

©2014 Waters Corporation 1

RAPID SEPARATION OF HBCDD STEREOISOMERS AND ADDITIONAL FLAME RETARDANTS

USING UPC2 MS/MS

Lauren Mullin1,2, Jennifer Burgess1, Ingrid Ericson Jogsten2, Dawei Geng2, Andy Aubin1, Bert van Bavel2

1Waters Corporation

2MTM Research Centre, Örebro University, Sweden


Overview

Supercritical fluid chromatography (SFC) applicability to hexabromocyclododecane (HBCDD) and tetrabromobisphenol-A (TBBPA) analysis

Chromatography and MS/MS Method Development Sample analyses results

New work and preliminary results: Chiral Separations and More

Flame Retardants Conclusions


What is a Supercritical Fluid?

High diffusivity, and low viscosity result in fast, efficient

chromatography

Liquid/gas Critical point Supercritical fluid

Increase temp and pressure

Increase temp and pressure


UPC2 TQ-S


HBCDD Isomeric Separation

HBCDD added to Stockholm Convention list of Persistent Organic Pollutants (POPs) Predominant HBCDD diasteromers and enantiomers

Proportion dependent on sample type


Chromatographic Efficiency: 3 minute separation of HBCDD and TBBPA

TBBPA

β−HBCDD

γ−HBCDD

α−HBCDD


Faster Analysis Time and Chromatographic Orthogonality

Expanded CC Separation

CC LC

α− γ−

α−

β−

γ−

β− α−

β−

γ−

Rs=3.15

Rs=4.36

Rs=5.07

Rs=10.42


Method Development


UPC2 TQ-S


Chromatography Optimization


UPC2 TQ-S


Ionization Type Comparison


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

Peak

Are

a

Make-up flow composition

Post Column Make-Up Flow Optimization

alpha HBCDD

gamma HBCDD

beta HBCDD

TBBPA


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


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


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


ES- MS/MS conditions

Capillary Voltage: 2.0 Cone Voltage: 30 Source Temperature: 150 degrees C Desolvation Temperature: 500 degrees C Desolvation Gas: 1000 L/hr Cone Gas: 150 L/hr Points per peak: 15


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.)

Initial 98 2 2 2 90 10 2

2.01 90 10 2.5 2.3 98 2 2.5

2.31 98 2 2


Application to Biological Matrices


Sample Analyzed

Can this method effectively separate and detect HBCDD isomers in complex matrices?

Any solvent can be injected using UPC2, so final extracted solvent for other analyses used without solvent exchange required

Extracted samples of human plasma and whale blubber were donated from previous GC analyses


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


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


05102013samples_06DL-08-007-281

9.930e+0020.89

1.361.04

min

%

0

100


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 1.8

100 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


min0.600 0.800 1.000 1.200 1.400

%

0

100


08062013samples_6110 ppb HBCD and TBBPA tetra

6.315e+0041.33

1.020.88

min

%

0

100


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


08062013samples_10DL-08-008:6b

2.294e+0040.88

min

%

0

100


08062013samples_10DL-08-008:6b

2.189e+0040.88

Whale Blubber Detection: High 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−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 2.3

10 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

late

d Co

ncen

trat

ion

ng/g

lipi

d w

eigh

t

Whale Blubber Extracted Sample

Calculated Concentrations of α-HBCDD


Ion Ratio Confirmation

Ion Ratio = Quantifier MRM area/Confirmatory MRM area All samples identified were considered to “pass” this

requirement if they were within +/- 20% of expected ion ratio Expected ion ratio was 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


Chiral Separations: Preliminary Results


Chiral HBCDD Separation

Current methods use LC with a 4.0 x 200mm 5µm PM-β-CD column with >15 minute run time

Using UPC2 with a prototype cellulose 2.1 x 150mm 2.5µm column, standards were screened

(+/-) 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


(+/-) 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


Additional Flame Retardants: Preliminary Results


Investigation of Traditionally GC-Amenable Flame Retardants

Wide variety of flame retardants are monitored in environment and biota

Polybrominated diphenyl ethers (PBDEs) traditionally use GC-MS analysis

UPC2 TQ-S APCI Overlaid SIRs

BDE 28, 33

BDE 47 BDE 99, 100

BDE 209

0.1 µg/mL

1.0 µg/mL


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]- 0.62

tris (2,3-dibromopropyl) phosphate GC and LC ESI- [M-H]- 1.11BDE 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


Conclusions

Rapid and efficient 3 minute separation of α−,β− and γ−HBCDD method shown to be proficient in the analysis of complex samples

Capability to inject wide range of solvents removes the 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


Acknowledgements

Samira Salihovic (human serum extracts) and Anna Rotander (whale blubber extracts) of MTM Research Centre, Örebro Universitet Örebro Sweden

Waters Corp., UPC2 team: Chris Hudalla for system illustration,

Baiba Cabovska for system use and guidance, Mike Jones, Ken Fountain and Mark Baynham

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.


Thank you!

rapid separation of hbcdd stereoisomers and …...©2014 waters corporation 19 min 0.600 0.800 1.000...

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