determination of chloroparaffins in foodstuffs by gc ms-ms

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State Institute for Chemical and Veterinary Analysis of Food

Freiburg (Germany)

Chemisches und Veterinruntersuchungsamt (CVUA)

Freiburg

Determination of Chloroparaffins

in Foodstuffs by GC-MS/MS

Biljana Trajkovska, Karin Kypke,

Alexander Kotz, Rainer Malisch,

Ralf Lippold

Food Testing and Agriculture Solutions

Webinar

23 October 2012

Archive www.foodsafetysummit.com

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Freiburg (Germany)

Chemisches und Veterinruntersuchungsamt (CVUA)

Freiburg

Determination of Chloroparaffins

in Foodstuffs by GC-MS/MS

Biljana Trajkovska, Karin Kypke,

Alexander Kotz, Rainer Malisch,

Ralf Lippold

Food Testing and Agriculture Solutions

Webinar

23 October 2012


Freiburg (Germany)

Content

Chloroparaffins:

1. Applications, regulations, levels in the food chain

2. Analytical methods for food and human milk

Extraction and clean up

GC-MS/MS

3. Results for CPs in samples of this study


Freiburg (Germany)

Chlorinated Paraffins (CPs) (polychlorinated n-alkanes, PCAs)

general formula CnH2n+2-zClz

chain length and the number and position of the chlorines can vary

complex mixtures: > 10.000 compounds

chlorine content of the commercially available mixtures varies between 30 70 %

CPs are divided according their carbon chain length into

short chain CPs (SCCP; C10 C13)

medium chain CPs (MCCP; C14 C17)

long chain CPs (LCCP; > C17)

Molecular Structure


Freiburg (Germany)

Applications of Chlorinated Paraffins (CPs)

metal industry (lubricant, cutting oil)

flame retardants in rubber, in paints, for coatings, sealants

textile industry as flame retardants, for repelling water and as anti-fouling agent

leather industry for fat liquoring

plasticiser in PVC, especially MCCP (alternative to phthalates)

Use


Freiburg (Germany)

SCCP

classified as persistent

high potential for bioaccumulation

classified as toxic to aquatic organisms

carcinogenic to rats and mice

Toxicology

categorized by IARC in group 2 B

as possible carcinogenic to humans


Freiburg (Germany)

Directive 2002/45/EC

SCCP were banned for certain applications

- in metal and leather industry

EU Regulations

Production, the marketing and the use of SCCP is prohibited,

whether on their own, in preparations or as constituents of articles

COMMISSION REGULATION (EU) No 519/2012

and Annex I of the Regulation (EC) No 850/2004


Freiburg (Germany)

Stockholm Convention on Persistent Organic Pollutants (POPs)

Adopted and signed at Diplomatic Conference in Stockholm, May 2001;

Convention entered into force on 17 May 2004;

Aim:

Protection of human health and the environment

Reduction or elimination of releases of certain POPs


Freiburg (Germany)

Stockholm Convention on Persistent Organic Pollutants (POPs)

Comprises: Pesticides, industrial chemicals, by-products

Initial 12 POPs

New POPs (number: 10)

Candidate POPs (number: 5;

among them: Short-Chained Chlorinated Paraffins)

http://chm.pops.int


Freiburg (Germany)

Parties to Stockholm Convention

October 2012: 178 Parties (with152 Signatories)

http://chm.pops.int


Freiburg (Germany)

CP levels in fish from Germany and Switzerland

Reth (2006): Total CP concentrations

in various fish species (muscle

tissue) captured in Germany and

Switzerland. Determination by

HRGC-EI-MS/MS:

Range 0.5 27 % lipid

Range 10 250 ng CP/g ww

Range 500 8000 ng CP/g lw


Freiburg (Germany)

CP levels in human milk from Germany

Reth M, Kypke K, Schchtele J and Oehme M, Organohalogen Compounds (2005) 67:1671-1673


Freiburg (Germany)

CP levels

in human milk from UK

Gareth OT, Farrar D, Braekevelt E, Stern G,

Kalantzi OI, Martin FL, Jones KC in

Environment International 32 (2006) 34 40


Freiburg (Germany)

Content

Chloroparaffins:




GC-MS/MS



Freiburg (Germany)

Literature

35 references found with

chlorinated paraffins in titles of presentations given at the

annual dioxin conferences


Freiburg (Germany)

Michael Oehme and his former workgroup at University of Basel (Switzerland):

Reth M. and Oehme M. Limitations of low resolution mass spectrometry in the electron capture negative ionization mode for the analysis of short and medium chain chlorinated paraffins Anal. Bioanal. Chem. 2004, 378, 1741-1747

Reth M., Zencak Z. and Oehme M. First study of congener group patterns and concentrations of short- and medium-chain chlorinated paraffins in fish from the North and Baltic Sea Chemosphere 2005, 58, 847-854

Z. Zencak, M. Reth and M. Oehme Determination of Total Polychlorinated n-Alkane Concentration in Biota by Electron Ionization-MS/MS Anal. Chem. 2004 76, 1957-1962

Z. Zencak, A. Borgen, M. Reth and M. Oehme Evaluation of four Mass Spectrometric Methods for the Analysis of Polychlorinated n-Alkanes J. Chromatogr. A 2005, 1067, 295-301

Scientific basis for development of analytical procedure


Freiburg (Germany)

Extraction and clean up:

1. fat extraction

different procedures depending on matrix and fat content

2. removal of fat

column chromatography with silica gel impregnated with sulphuric acid

3. clean up adsorption chromatography on Florisil

4. Determination by GC/MS

Analytical procedure


Freiburg (Germany)

Internal standards:

1.[13C10]-trans-Chlordane

2. -Hexachlorocyclo-hexane

Analytical procedure


Freiburg (Germany)

samples with a high fat content for example: bacon, kidney fat

2 5 g sample

A Fat Extraction

melted fat

heated in a microwave oven


Freiburg (Germany)

raw milk / human milk

50 g milk

fat extraction with n-hexane /

dichloromethane 1+1 (v+v)

solvent is removed by using a turbo vap 500

centrifuged at 4C (3000 rpm, 10 min)

homogenize the cream with

water free sodium sulphate

A Fat Extraction


Freiburg (Germany)

meat / cheese / eggs

A Fat Extraction

10 g sample

lipid is extracted with n-hexane /

dichloromethane (1:1)

solvent is removed by using a Turbo Vap 500

homogenize with water free

sodium sulphate

mixture is packed into

a glass column


Freiburg (Germany)

B Removal of Fat by Oxidation

glas wool

silica gel/

sulfuric acid (44 %)

Na2SO4

Na2SO4

conditioning: n-hexane / dichloromethane (1:1)

elution: n-hexane / dichloromethane (1:1)

evaporation with Turbo Vap


Freiburg (Germany)

plant oil

glass wool

silica gel/


Na2SO4

Clogging of column can be caused by compounds difficult to oxidize (e.g. waxes)






Freiburg (Germany)

plant oil

glass wool

silica gel/


Na2SO4

silica gel/

sodium hydroxide






Freiburg (Germany)

C Florisil column

conditioning:

n-hexane

pre-wash:

n-hexane

dichloromethane

elution:

dichloromethane

evaporate with turbo vap

dissolve residue in

cyclohexane

glass wool

florisil

deactiviated with

1.5 % water

Na2SO4

Na2SO4


Freiburg (Germany)

Figure 1: mix standard solution

mix standard solution with

PCB, DDT, nonachlor

(c = 0.4 ng /l)

after clean up on Florisil

Figure 2: mix standard solution, after clean up on Florisil

C Florisil column

Separation of organochloro pesticides and PCBs from chlorinated paraffins


Freiburg (Germany)

Content

Chloroparaffins:




GC-MS/MS



Freiburg (Germany)

GC/low resolution EI-MS:

strong fragmentation of CPs under EI conditions (resulting mass spectra without

compound-specific information, e.g. chain length, number of chlorine atoms).

Fragments present in all CPs are below m/z 110.

Thus increased risk of interferences, high background from remaining sample.

GC-NICI-MS: (with CH4/CH2Cl2)

Advantage: nearly exclusive formation of [M+Cl]- and suppression of formation

of other fragment ions; enhances selectivity and sensitivity; allows reliable

quantification independent from degree of chlorination

Disadvantage: not suitable for routine analysis - cleaning of instrument

necessary after 48 hours

GC/HRMS

Advantage: avoids interferences from other contaminants

Disadvantage: HRMS expensive and not available in many laboratories

Options for determination of CP with GC-MS (1)


Freiburg (Germany)

GC-MS/MS (EI mode)

GC-ECNI-MS



Freiburg (Germany)

Analysis of CP

CPs are complex technical mixtures with thousands of isomers

Chromatograms with broad humps instead of single peaks

Determination of all homologue groups very time-consuming

Quantification of CP extremely difficult

response factor depends on the chlorine content

Analysis of CP


Freiburg (Germany)

GC-MS/MS (EI mode)

GC-ECNI-MS



Freiburg (Germany)

Determination of total concentration with GC-MS/MS (EI-mode)

EI Scan; SCCP 55.5 % Cl

m/z 110

EI Scan; MCCP 42 % Cl

m/z 110


Freiburg (Germany)

Determination of total concentration with GC-MS/MS (EI-mode)

Analyte Precursor Ion

(m/z)

Product Ion

(m/z)

Collision Energy

(V)

CPs 91 [C7H7]+ 53 [C4H5]

+ -16

CPs 102 [C5H7Cl]+ 65 [C5H5]

+ -10

CPs 102 [C5H7Cl]+ 67 [C5H7]

+ -18

[13C10]-trans-

Chlordane 383 [M-Cl]+ 276 [M-4Cl]+ -20

-Hexachlorocyclo-hexane

181 [M-3Cl]+ 145 [M-4Cl]+ -15

-Hexachlorocyclo-hexane

181 [M-3Cl]+ 109 [M-5Cl]+ -31


Freiburg (Germany)

Parameters of GC-EI-MS/MS

instrument: Agilent Technologies 7000 GC/MS Triple Quad,

7693 Autosampler, 7890A GC System

column: HP-5MS

fused silica capillary column (15 m length x 0.25 mm i.d.),

coated with 0.25 m of 5%-phenyl-methylpolysiloxane

injector temperature: 280 C

injection: splitless

injection volume: 2.5 l

transferline: 280 C

ion source temperature: 200 C

electron energy: 70 eV

carriergas: helium, constant flow of 2 ml/ min

temperature programme: 1 min isothermal at 100 C, increased at 50 C/ min to 300 C,

isothermal for 5 min


Freiburg (Germany)

Chromatograms of SCCP and MCCP

MCCP 57 % Cl; 102 -> 67

MCCP 57 % Cl; 102 -> 65

MCCP 57 % Cl; 91 -> 53

SCCP 55.5 % Cl; 102 -> 67

SCCP 55.5 % Cl; 102 -> 65

SCCP 55.5 % Cl; 91 -> 53


Freiburg (Germany)

MCCP 57 % Cl; TIC SCCP 55.5 % Cl; TIC

SCCP 55.5 % Cl + MCCP 57 % Cl; TIC

Chromatograms of SCCP and MCCP


Freiburg (Germany)

GC-EI-MS/MS

SCCP 55.5 % [102 -> 67]

SCCP 55.5 % [102 -> 65]

SCCP 55.5 % [91 -> 53]

low LODs resp. LOQs for all three transitions

Relevant range: for analysis of CPs in food between 0.5 20 ng/ l

GC-MS/MS is suitable for the

determination of CP in food


Freiburg (Germany)

GC-MS/MS (EI-mode):

Allows determination of total CP concentration

Advantage

low variation of response factor (according to Zencak, quantification does

not depend on chain length and degree of chlorination.)

fast determination of total concentrations of SCCP and MCCP (possible

further characterization by ECNI-LRMS)

Disadvantage:

strong fragmentation of CPs under EI conditions gives mass spectra without

compound-specific information (e.g. chain length, number of chlorine

atoms): fragments present in all CP are below m/z 110 (thus increased risk

of interferences, high background from remaining sample)

Separate quantification of SCCP and MCCP not possible

GC-MS/MS (EI-mode)


Freiburg (Germany)

GC-MS/MS (EI mode)

GC-ECNI-MS


published by Reth (2006)

Chemisches und Veterinruntersuchungsamt Freiburg

Advantage

no strong fragmentation of CPs under ECNI conditions

leads to formation of [M-Cl]- as major fragment

allows the evaluation of all congener and homologue groups of SCCP and MCCP

allows a differentation between SCCP and MCCP

Determination of CPs using GC-ECNI-MS

Disadvantage

GC-ECNI-MS does not detect lower chlorinated CPs

high number of runs per sample is necessary

the quantification depends on the degree of chlorination of the used standard

GC-ECNI-MS is time consuming


Freiburg (Germany)

Comparability of CP levels in fish determined by ECNI-

MS and EI-MS/MS


Freiburg (Germany)

Method Blanks

possible sources causing contamination in laboratory:

glassware CP bind to active surface

technical equipment (in contact with samples or extracts)

chemicals

air (air condition)


Freiburg (Germany)

Method Blanks

measures to reduce the risk of contamination during clean up

Soaking of glassware in detergent solution

Heating of glassware up to 450 C for 2 hours

Covering of glassware in alumina foil for storage and during analysis

Rinsing of glassware with solvent immediately before use


Freiburg (Germany)

Method Blanks

blank values shall be below 0.1 ng/ l (corresponding to 5 ng/g lipid with use of the

established method)

Measures to reduce the risk of contamination during clean up

no plastic materials

teflon cleaned with solvent for use

heating of chemicals at 600 C overnight

use only of high purity solvents

extraction of glass wool with solvent


Freiburg (Germany)

Validation of the Matrix Raw Milk

Level 1

[2 ng/g]

Recovery

[%] Level 2

[10 ng/g]

Recovery

[%]

Sample 1 1.82 91 9.50 95

Sample 2 1.66 83 10.0 100

Sample 3 1.46 73 9.30 93

Sample 4 2.42 121 9.74 97

Sample 5 2.06 103 11.7 117

number of samples 5 5

mean [ng/ l] 1.88 94 10.1 101

standard deviation [ng/ l] 0.19 0.49

rel. standard deviation [%] 20 10


Freiburg (Germany)

xblind = 0.12 ng/ l

sblind = 0.05 ng/ l

LOD = 0.26 ng/ l

LOQ = 0.60 ng/ l

LOD = xblind + 3 * sblind

LOQ = xblind + 10 * sblind

Calculation of LOD and LOQ depending on blank value:

Collection of results of individual blank values over a period of 3 month

Calculation of mean and standard deviation from these collected data

Validation of the Matrix Raw Milk

LOD = 0.5 ng/ g milk

LOQ = 1.2 ng/ g milk


Freiburg (Germany)

Content

Chloroparaffins:




GC-MS/MS



Freiburg (Germany)

Samples

mirco1 / pixelio.de

Peter Hebgen / pixelio.de Matthias Koranzki / pixelio.de

Samples analysed:

10 samples of raw milk

10 samples of cheese

25 samples of human milk

4 samples of wild boar

6 samples of meat

6 samples of plant oil


Freiburg (Germany)

Raw Milk

pooled samples of

severals farms CP

[ng / g fat]

CP [ng / g wet weight]

raw milk 1 Tour I < LOD < LOD

raw milk 2 Tour 13 < LOD < LOD






raw milk 8 Tour Ellrich < LOD < LOD

raw milk 9 Tour Frankenhardt < LOD < LOD

raw milk 10 Tour Eckartshausen < LOQ [26] < LOQ [1.0]

Chloroparaffins LOD: 13 ng/ g fat LOQ: 30 ng/ g fat


Freiburg (Germany)

Raw Milk

SCCP 55.5 % chlorine content MCCP 57 % chlorine content

raw milk sample 10


Freiburg (Germany)

Cheese

lipid

content

[%]

CP [ng / g fat]

CP [ng / g wet

weight]

country of

origin

animal

1 Brie 24,1 41 5.0 The Netherland organic goat

2 Feta cheese 24,4 122 24 Greece organic sheep and

goat

3 Feta cheese 26,2 94 19 Germany organic sheep

4 Feta cheese 28,4 38 7.5 Germany organic sheep

5 Cheese 26,5 < LOQ [18] < LOQ [3.6] France conventional cow

6 Mozzarella 21,0 55 11 Italy conventional buffalo

7 Cheese 32,0 < LOQ [28] < LOQ [5.5] Italy organic sheep

8 Feta cheese 22,7 47 9.3 Greece organic sheep

9 Goat cheese 27,1 42 8.4 Germany organic goat

10 Goat cheese 27,8 < LOQ [18] < LOQ[3.6] Germany organic goat

Chloroparaffins LOD: 13 ng/ g fat LOQ: 30 ng/ g fat


Freiburg (Germany)

Cheese


Feta cheese, greece

122 ng/ g CP

Feta cheese, germany

94 ng/ g CP


Freiburg (Germany)

Human Milk


Freiburg (Germany)

Human milk is particulary well suited for biomonitoring of POPs

easy way to get high fat content

Study was organised by WHO and UNEP with CVUA Freiburg as

Reference Laboratory.

The study is performed as contribution to the Stockholm Convention.

The aim is to get comparable data for the background concentration of

POPs and for evaluation of time trends.

Each country participating in the study sent one representative sample

prepared by pooling (mixing) of at least 50 individual human milk

samples.

Human Milk


Freiburg (Germany)

Human Milk


Freiburg (Germany)

Human Milk

23 500 ng/ g fat


Freiburg (Germany)

Human Milk

Asia

Europe Pacific Island

Conc. [ng/ g fat]


Freiburg (Germany)

Human Milk

Asia

Europe Pacific Island

3026 ng/ g fat 8492 ng/ g fat


Freiburg (Germany)

Human Milk

Conc. [ng/ g fat]


Freiburg (Germany)

Human Milk


Freiburg (Germany)

Wild Boar

Sample

age

[month]

CP

[ng/ g fat]

HCB

[ng/ g

fat]

alpha-HCH

[ng/ g fat]

beta-HCH

[ng/ g fat]

lindane [ng/

g fat]

1 wild boar, fat 12 < LOQ [18,1] 951 < LOD < LOD < LOD

2 wild boar, fat 12 < LOQ [17,3] 8.4 2.9 23.0 < LOD

3 wild boar, fat - < LOQ [29,8] 14.2 78.1 54.1 < LOD

4 wild boar, fat 12 < LOQ [26,2] 12.9 12.4 5.9 < LOD

maximum amount by law - 200 200 100 20

Chloroparaffins

LOD: 13 ng/ g fat

LOQ: 30 ng/ g fat


Freiburg (Germany)

Meat

CP [ng/ g fat] country of origin

1 chicken < LOQ [26] Germany

2 turkey < LOD Germany

3 goose 34 Germany

4 beef < LOQ [33] Germany

5 beef 60 Germany

6 beef < LOQ [28] Germany

Chloroparaffins

LOD: 13 ng/ g fat

LOQ: 30 ng/ g fat


Freiburg (Germany)

Meat


beef

60 ng/ g fat CP

goose

34 ng/ g fat CP


Freiburg (Germany)

Olive Oil

CP [ng/ g fat]

1 olive oil < LOD

2 olive oil 75

3 olive oil < LOQ [20]

4 olive oil 98

5 olive oil 38

6 olive oil 37

LOD: 13 ng/ g fat

LOQ: 30 ng/ g fat


Freiburg (Germany)

Outlook

Actual activity:

Determination of CPs with GC-ECNI-MS as second type of

detection

for comparison of results of both ionization

techniques (confirmation)

for determination of CP patterns


Freiburg (Germany)

Final

Thank you for your attention

Patricia Fischer,

Sebastian Schelb,

Katrin Thoma,

Nadine Singler

determination of chloroparaffins in foodstuffs by gc ms-ms

Documents

food chain

humans state institute

study state institute

favorites state institute

certain pops state institute

ralf lippold food testing

zclz chain length

carbon chain length