Quasimem Workshop, Amsterdam , 14-15 October 2009

Low resolution triple quadrupole MS

compared to high resolution Orbitrap MS

for the analysis of PFCs

Laboratoire d’Etude des Résidus et Contaminants dans les Aliments

Ecole Nationale Vétérinaire de Nantes - France - www.laberca.org

for the analysis of PFCs

H.Kadar, B.Veyrand, P.Marchand, B. Le Bizec

Outline

Introduction

1. Analysis of perfluorinated compounds in breast milk

2. Analysis of perfluorinated compounds in fish samples

Quasimem workshop, Amsterdam, 14-15 October 2/18

2. Analysis of perfluorinated compounds in fish samples

Conclusion

Introduction

ResiduesResidues ContaminantsContaminants

Xenobiotics Natural Hormones Halogenated Aromatics Other contaminants

CorticosteroidsPhytosanitary products

Endocrine

disruptors

General domain of activities: chemical food safety

Quasimem workshop, Amsterdam, 14-15 October 3/18

Thyreostats*

Somatotropin

ββββ-agonists*

Corticosteroids

Steroids*

Phytosanitary products

PCDD*

PCDF*

PCB*

PBB

HAP*

PFOS/PFOA

PBDE

Phytoestrogens

* National Reference Laboratory

1

9

3

2 5 6

Food industry

Environment

processes

Research activities: the chemical pollutants from their sources to human

Introduction

Quasimem workshop, Amsterdam, 14-15 October 4/18

7 84

Human

Children

Xenobiotic

administration

Feed Cattle Food product

Foetus

Metabolism, transfer studies, elimination kinetics

of animal origin

Mass spectrometric measurement

Chemometry

Quality management

• 5 PerfluoroalkylSulfonic acids

Analysed compounds

• 13 PerfluoroalkylCarboxylic acids

• 3 PerFluoroalkylPhosphonic acids

• 1 PerfluoroalkylSulfinate

F3C (CF2)n S

O

O

OH

F 3C (CF 2)n P O

O H

O HCH4

F3C (CF2)n

O

OH

O

Quasimem workshop, Amsterdam, 14-15 October 5/18

• 1 PerfluoroalkylSulfinate

• 2 PerFluoroalkylSulfonAmides

• 3 Carboxylic FluroTélomère saturated acid

• 3 Carboxylic FluroTélomère unsaturated acid

F3C (CF2)n S

O

O

N

H

H

F3C (CF2)n S

O

OH

F3C (CF2)n CH COOH

F3C (CF2)n CF CH COOH

1.Analysis of perfluorinated compounds in breast milk

Acetone precipitation

Purification steps:

SPE Oasis HLB

1 mL of breast milk Spiked with Internal standards 13 C

1.1 Sample preparation

Quasimem workshop, Amsterdam, 14-15 October 6/18

SPE Oasis HLB

SPE Envicarb

Evaporation to dryness and

reconstitution in

MeOH/Water (50/50, v/v)

Addition of

External standard

Injection in LC-MS/MS Injection in LC-HRMS

1.Analysis of perfluorinated compounds in breast milk

1.2 Analysis by LC-MS/MS vs LC-HRMS

System 1 : LC-MS/MSLow resolution

System 2 : LC-HRMSHigh resolution

Quasimem workshop, Amsterdam, 14-15 October 7/18

Identification: 2 MRM transitions for each compound

R = 30,000

Identification: The exact mass

LTQ-Orbitrap (Thermo-Finnigan®)Triple quadrupole (Agilent® 6410)

1.Analysis of perfluorinated compounds in breast milk

1.3 Comparison of sensitivity in HRMS vs MRM

PFOS498,92912 ± 5 ppm

Quantifier transition498,9 > 498,9

MRM Chromatogram (50 µL injected) HRMS Chromatogram (20 µL injected)

S/N 10

Example of PFOS in an incured breast milk (estimated conc. = 0.049 ng/ml)

Quasimem workshop, Amsterdam, 14-15 October 8/18

PFOS 13C502,94254 ± 5 ppm

Qualifier transition 498,9 > 80

Internal standard502,9 > 502,9

0,050

0,060

0,070

0,080

0,090

PF

OS

co

ncen

trati

on

(n

g/m

L o

r p

pb

)

K-S d=.10946, p> .20; Lilliefors p> .20

Theoretical Normal Distribution

0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.090

2

4

6

8

10

12

14

Num

be

r o

f O

bse

rva

tio

ns

1.4 Application to thirty breast milk samples : PFOS

1.Analysis of perfluorinated compounds in breast milk

Min 0.012Max 0.086Mean 0.039Median 0.037

Quasimem workshop, Amsterdam, 14-15 October 9/18

0,000

0,010

0,020

0,030

0,040

PF

OS

co

ncen

trati

on

(n

g/m

L o

r p

pb

)

9.7

31.3

0

9.7

31.1

5

9.7

31.1

1

9.7

31.2

8

9.7

31.1

9.7

31.1

2

9.7

31.1

3

9.7

31.1

7

9.7

31.2

2

9.7

31.4

9.7

31.6

9.7

31.2

5

9.7

31.1

0

9.7

31.1

6

9.7

31.8

9.7

31.2

1

9.7

31.2

0

9.7

31.1

4

9.7

31.1

8

9.7

31.2

3

9.7

31.2

6

9.7

31.2

9.7

31.2

7

9.7

31.2

9

9.7

31.2

4

9.7

31.3

9.7

31.1

9

9.7

31.9

9.7

31.5

9.7

31.7

Samples

0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09

PFOS Concentration (ppb)

LCHRMS

LOD

LCMSMS

LOD

0,030

0,040

0,050

0,060

PF

OA

co

ncen

trati

on

(n

g/m

L o

r p

pb

)

1.4 Application to thirty breast milk samples : PFOA

1.Analysis of perfluorinated compounds in breast milk

K-S d=.09165, p> .20; Lilliefors p> .20

Theoretical Normal Distribution

0.00 0.01 0.02 0.03 0.04 0.05 0.060

1

2

3

4

5

6

7

8

9

10

11

Num

be

r o

f O

bse

rva

tio

ns

Min 0.009Max 0.051Mean 0.030Median 0.028

Quasimem workshop, Amsterdam, 14-15 October 10/18

0,000

0,010

0,020

0,030

PF

OA

co

ncen

trati

on

(n

g/m

L o

r p

pb

)

9.7

31.9

9.7

31.2

8

9.7

31.1

0

9.7

31.1

6

9.7

31.6

9.7

31.1

1

9.7

31.2

9

9.7

31.1

7

9.7

31.8

9.7

31.2

0

9.7

31.3

9.7

31.1

8

9.7

31.2

2

9.7

31.1

3

9.7

31.2

4

9.7

31.2

5

9.7

31.1

2

9.7

31.2

7

9.7

31.3

0

9.7

31.2

3

9.7

31.1

5

9.7

31.4

9.7

31.1

9

9.7

31.2

6

9.7

31.2

1

9.7

31.1

9.7

31.2

9.7

31.7

9.7

31.1

4

9.7

31.5

Samples

0.00 0.01 0.02 0.03 0.04 0.05 0.06

PFOA Concentration (ppb)

LCMSMS

LOD

LCHRMS

LOD

PFOA-HR-10 = -.0049 + .98225 * PFOA1-MSMS

Corréla tion : r = .88971

0.030

0.035

0.040

0.045

0.050

0.055

Qu

an

tifi

cati

on

res

ult

s w

ith

LC

-HR

MS

PFOS-HR-10 = .01686 + 1.0370 * PFOS1-MSMSCorréla tion : r = .97029

0.05

0.06

0.07

0.08

0.09

Qu

an

tifi

cati

on

re

su

lts w

ith

LC

-HR

MS

PFOA PFOS

HR

MS

HR

MS

1.4 Application to thirty breast milk samples: LC-MS/MS vs HRMS

1.Analysis of perfluorinated compounds in breast milk

Quasimem workshop, Amsterdam, 14-15 October 11/18

0.010 0.015 0.020 0.025 0.030 0.035 0.040 0.045 0.050 0.055 0.060

Quantification results w ith LC-MS/MS

0.005

0.010

0.015

0.020

0.025

0.030

Qu

an

tifi

cati

on

res

ult

s w

ith

LC

-HR

MS

0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08

Quantification results w ith LC-MS/MS

0.01

0.02

0.03

0.04

0.05

Qu

an

tifi

cati

on

re

su

lts w

ith

LC

-HR

MS

a = 0.98225 a = 1.0370

LC-MS/MS

LC

-HR

MS

LC

-HR

MS

LC-MS/MS

2. Analysis of perfluorinated compounds in fish samples

LLE with Methanol

SPE dispersive

Envicarb

1 g of frozen-dryied fish Spiked with Internal standards 13 C

2.1 Sample preparation

Quasimem workshop, Amsterdam, 14-15 October 12/18

Envicarb

Evaporation to dryness and

redissolution in

MeOH/Water (50/50, v/v)

Addition of External standard

Injection in LC-MS/MS Injection in LC-HRMS

Centrifugation

2. Analysis of perfluorinated compounds in fish samples

MS/MS Chromatogram extract of fish with 25% fat (50µL injected)

HRMS Chromatogram extract of fish with 25% fat (10µL injected)

PFOA

PFOS

412,9 > 368,9412,96532; ±10 ppm

498,92912;

2.2 Specificity comparison: HRMS vs MRM

Quasimem workshop, Amsterdam, 14-15 October 13/18

PFOS

PFUnDA

PFTeDA

498,9 > 80

562,9 > 518,9

712,9 > 668,9

498,92912; ± 10 ppm

562,95578; ± 10 ppm

712,94616; ± 10 ppm

2. Analysis of perfluorinated compounds in fish samples

2.3 Application to fish samples

- Observation : significant difference between quantitative results obtained inLC-MS/MS and LC-HRMS for PFOS

-Hypothesis : interference on the PFOS diagnostic signal in LC-MS/MS

Quasimem workshop, Amsterdam, 14-15 October 14/18

-Hypothesis : interference on the PFOS diagnostic signal in LC-MS/MS

- Experiment : extraction of the PFOS diagnostic signal with various mass precision

RT: 5.05 - 5.94

30

40

50

60

70

80

90

100

Rela

tive A

bundance

RT: 5.47

AA: 1552672

NL: 5.12E5

m/z=

498.87923-498.97901

F: FTMS - c ESI Full

ms [200.00-920.00]

MS Genesis

210809020

RT: 5.02 - 5.94

30

40

50

60

70

80

90

100

Rela

tive A

bundance

RT: 5.47

AA: 9253651

NL: 3.03E6

m/z=

498.87923-498.97901

F: FTMS - c ESI Full

ms [200.00-920.00]

MS Genesis

210809015

Lavaret fishGardon fish

498.92912 ±100 ppm

RT: 5.05 - 5.97

30

40

50

60

70

80

90

100

Rela

tive A

bundance

RT: 5.46

AA: 8196252

NL: 2.50E6

m/z=

498.87923-498.97901

F: FTMS - c ESI Full

ms [200.00-920.00]

MS Genesis

210809010

PFOS standard

498.92912 ±100 ppm

498.92912 ±100 ppm

2. Analysis of perfluorinated compounds in fish samples

2.4 Interference issue

Quasimem workshop, Amsterdam, 14-15 October 15/18

5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9

Time (min)

0

10

20

30

40

50

60

70

80

90

100

0

10

20

5.46

5.49

5.44

5.66

5.305.28 5.51

5.08 5.39 5.61 5.685.16 5.70

5.73

NL: 2.24E4

m/z=

498.23855-498.33821

F: FTMS - c ESI Full

ms [200.00-920.00]

MS 210809020

5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9

Time (min)

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

RT: 5.47

MA: 3968180

5.05 5.905.24 5.595.09 5.28 5.865.67 5.71

NL: 1.10E6

m/z=

498.23855-498.33821

F: FTMS - c ESI Full

ms [200.00-920.00]

MS 210809015

498.28838 ±100 ppm

5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9

Time (min)

0

10

20

30

40

50

60

70

80

90

100

0

10

20

5.09

5.63

5.52

NL: 3.97E3

m/z=

498.23855-498.33821

F: FTMS - c ESI Full

ms [200.00-920.00]

MS 210809010

498.28838 ±100 ppm

498.28838 ±100 ppm

2. Analysis of perfluorinated compounds in fish samples

Benskin et al. report presence of this compound in biological matrix which can complicate PFOS quantification

S

O

O

O-FF

F F

F F

F F

F F

F FF F

F

F

F

2.4 Interference issue

Quasimem workshop, Amsterdam, 14-15 October 16/18

• Benskin et al., 2007, Simultaneous Characterization of Perfluoroalkyl Carboxylate, Sulfonate, and Sulfonamide Isomers by Liquid Chromatography-Tandem Mass Spectrometry. Anal. Chem. 2007, 79, 6455-6464.

TDCAMW = 498.29

PFOSMW = 498.93

- Same transition M- > SO3-

- Only transition M- > FSO3- can discriminate PFOS from TDCA

sensitivity ?

Conclusion

Sample preparation procedures dedicated to the monitoring of an extended range of PFC compounds have been developped for milk and fish samples

LC-MS/MS and LC-HRMS evaluated as measurement techniques

HRMS advantages :• Sensitivity increased with high resolution compared to low resolution• Better specificity for complex biological matrice• Provides good discrimination between compounds and interferents

Quasimem workshop, Amsterdam, 14-15 October 17/18

• Provides good discrimination between compounds and interferents• Efficient confirmatory methodHRMS limitations :• High cost compared to low resolution• Difficulty to be applied in routine laboratories

Other applications are running on other matrices (water, bakery products, human serum…)

First exposure and body burden data are generated through various projects

Quasimem Workshop, Amsterdam , 14-15 October 2009

Thank you for your attention

Laboratoire d’Etude des Résidus et Contaminants dans les Aliments

Ecole Nationale Vétérinaire de Nantes - France - www.laberca.org

H.Kadar, B.Veyrand, P Marchand, B Le Bizec