Determination of Chloroparaffins in Foodstuffs by GC MS-MS

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Chemisches und Veterinruntersuchungsamt (CVUA)

Freiburg
Food Testing and Agriculture Solutions
Webinar
23 October 2012
Determination of Chloroparaffins
in Foodstuffs by GC-MS/MS
Biljana Trajkovska, Karin Kypke,

Alexander Kotz, Rainer Malisch,
Ralf Lippold
State Institute for Chemical and Veterinary Analysis of Food

Freiburg (Germany)
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Chemisches und Veterinruntersuchungsamt (CVUA)

Freiburg
Food Testing and Agriculture Solutions
Webinar
23 October 2012
Determination of Chloroparaffins
in Foodstuffs by GC-MS/MS
Biljana Trajkovska, Karin Kypke,

Alexander Kotz, Rainer Malisch,
Ralf Lippold

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)
Molecular Structure
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)

Freiburg (Germany)
Use
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)

Freiburg (Germany)
Toxicology
SCCP
classified as persistent
high potential for bioaccumulation
classified as toxic to aquatic organisms
carcinogenic to rats and mice
categorized by IARC in group 2 B

as possible carcinogenic to humans

Freiburg (Germany)
EU Regulations
Directive 2002/45/EC
SCCP were banned for certain applications
- in metal and leather industry
COMMISSION REGULATION (EU) No 519/2012

and Annex I of the Regulation (EC) No 850/2004
Production, the marketing and the use of SCCP is prohibited,
whether on their own, in preparations or as constituents of articles

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

http://chm.pops.int
October 2012: 178 Parties (with152 Signatories)

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)
Literature
35 references found with

chlorinated paraffins in titles of
presentations given at the
annual dioxin conferences

Freiburg (Germany)
Scientific basis for development of analytical procedure
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

Freiburg (Germany)
Analytical procedure
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

Freiburg (Germany)
Analytical procedure
Internal standards:
1.[13C10]-trans-Chlordane
2. -Hexachlorocyclo-hexane

Freiburg (Germany)
Fat Extraction
samples with a high fat content

for example: bacon, kidney fat
2 5 g sample
heated in a microwave oven
melted fat

Freiburg (Germany)
Fat Extraction
raw milk / human milk

50 g milk
centrifuged at 4C
(3000 rpm, 10 min)
homogenize the cream with

water free sodium sulphate
fat extraction with n-hexane /
dichloromethane 1+1 (v+v)
solvent is removed by using a turbo vap 500

Freiburg (Germany)
Fat Extraction
meat / cheese / eggs

10 g sample
homogenize with water free
sodium sulphate
mixture is packed into
a glass column
lipid is extracted with n-hexane /
dichloromethane (1:1)
solvent is removed by using a Turbo Vap 500

Freiburg (Germany)
Na2SO4
Removal of Fat by Oxidation
conditioning:
silica gel/
sulfuric acid (44 %)
n-hexane / dichloromethane (1:1)
elution:
Na2SO4
glas wool
evaporation with Turbo Vap

Freiburg (Germany)
plant oil
conditioning:
elution:
silica gel/

Na2SO4
glass wool
Clogging of column can be caused by compounds difficult to oxidize (e.g. waxes)

Freiburg (Germany)
plant oil
silica gel/
sodium hydroxide
conditioning:
silica gel/
Na2SO4
glass wool
elution:

Freiburg (Germany)
C
conditioning:
n-hexane
Na2SO4
florisil
deactiviated with
1.5 % water
Na2SO4
glass wool
Florisil column
pre-wash:
n-hexane
dichloromethane
elution:
dichloromethane
evaporate with turbo vap
dissolve residue in
cyclohexane

Freiburg (Germany)
Florisil column
Separation of organochloro pesticides and PCBs from chlorinated paraffins

mix standard solution with
PCB, DDT, nonachlor
(c = 0.4 ng /l)
Figure 1: mix standard solution

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

Freiburg (Germany)
Content
Chloroparaffins:
GC-MS/MS

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

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

Freiburg (Germany)
GC-MS/MS (EI mode)

GC-ECNI-MS

Freiburg (Germany)
Analysis of CP
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

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)
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]-transChlordane
383 [M-Cl]+
276 [M-4Cl]+
-20
-Hexachlorocyclohexane
181 [M-3Cl]+
145 [M-4Cl]+
-15
-Hexachlorocyclohexane
181 [M-3Cl]+
109 [M-5Cl]+
-31
Analyte

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:
injection:
injection volume:
280 C
splitless
2.5 l
transferline:
ion source temperature:
electron energy:
carriergas:
280 C
200 C
70 eV
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

SCCP 55.5 % Cl; 102 -> 67
MCCP 57 % Cl; 102 -> 67
SCCP 55.5 % Cl; 102 -> 65
MCCP 57 % Cl; 102 -> 65
SCCP 55.5 % Cl; 91 -> 53
MCCP 57 % Cl; 91 -> 53

Freiburg (Germany)
Chromatograms of SCCP and MCCP

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

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)
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
Freiburg (Germany)
GC-MS/MS (EI mode)

GC-ECNI-MS
published by Reth (2006)

Freiburg (Germany)
Determination of CPs using GC-ECNI-MS
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
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
Chemisches und Veterinruntersuchungsamt
Freiburg
Comparability of CP levels in fish determined by ECNIMS and EI-MS/MS

Freiburg (Germany)

Freiburg (Germany)

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
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
blank values shall be below 0.1 ng/ l

(corresponding to 5 ng/g lipid with use of the
established method)

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
mean [ng/ l]
1.88
94
10.1
standard deviation [ng/ l]
0.19
0.49
rel. standard deviation [%]
20
10
101

Freiburg (Germany)
Validation of the Matrix Raw Milk

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
LOD = xblind + 3 * sblind
LOQ = xblind + 10 * sblind

xblind = 0.12 ng/ l
LOD = 0.26 ng/ l
sblind = 0.05 ng/ l
LOQ = 0.60 ng/ l

LOD = 0.5 ng/ g milk
LOQ = 1.2 ng/ g milk

Freiburg (Germany)
Content
Chloroparaffins:
GC-MS/MS

Freiburg (Germany)
Samples
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
Matthias Koranzki / pixelio.de
mirco1 / pixelio.de

Freiburg (Germany)
Peter Hebgen / pixelio.de
Raw Milk
pooled samples of
severals farms
CP
CP
[ng / g fat]
[ng / g wet weight]
raw milk 1
Tour I
< LOD
< LOD
raw milk 2
Tour 13
< LOD
< LOD
raw milk 3
Tour 7
< LOD
< LOD
raw milk 4
Tour 6
< LOD
< LOD
raw milk 5
Tour 8
< LOD
< LOD
raw milk 6
Tour 1
< LOD
< LOD
raw milk 7
Tour 12
< 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
Brie
lipid
content
[%]
CP
CP
[ng / g fat]
[ng / g wet
weight]
24,1
41
5.0
country of
origin
The Netherland
animal
organic
goat
Feta cheese
24,4
122
24
Greece
organic
sheep and
goat
Feta cheese
26,2
94
19
Germany
organic
sheep
Feta cheese
28,4
38
7.5
Germany
organic
sheep
Cheese
26,5
< LOQ [18]
< LOQ [3.6]
France
conventional
cow
Mozzarella
21,0
55
11
Italy
conventional
buffalo
Cheese
32,0
< LOQ [28]
< LOQ [5.5]
Italy
organic
sheep
Feta cheese
22,7
47
9.3
Greece
organic
sheep
Goat cheese
27,1
42
8.4
Germany
organic
goat
10
Goat cheese
27,8
< LOQ [18]
< LOQ[3.6]
Germany
organic
goat

Freiburg (Germany)
Cheese
Brie
lipid
content
[%]
CP
CP
[ng / g fat]
[ng / g wet
weight]
24,1
41
5.0
country of
origin
The Netherland
animal
organic
goat
Feta cheese
24,4
122
24
Greece
organic
sheep and
goat
Feta cheese
26,2
94
19
Germany
organic
sheep
Feta cheese
28,4
38
7.5
Germany
organic
sheep
Cheese
26,5
< LOQ [18]
< LOQ [3.6]
France
conventional
cow
Mozzarella
21,0
55
11
Italy
conventional
buffalo
Cheese
32,0
< LOQ [28]
< LOQ [5.5]
Italy
organic
sheep
Feta cheese
22,7
47
9.3
Greece
organic
sheep
Goat cheese
27,1
42
8.4
Germany
organic
goat
10
Goat cheese
27,8
< LOQ [18]
< LOQ[3.6]
Germany
organic
goat

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
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.

Freiburg (Germany)
Human Milk

Freiburg (Germany)
Human Milk
23 500 ng/ g fat

Freiburg (Germany)
Human Milk
Conc. [ng/ g fat]
Asia
Pacific Island
Europe

Freiburg (Germany)
Human Milk
3026 ng/ g fat
8492 ng/ g fat
Asia
Pacific Island
Europe

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]
wild boar, fat
12
< LOQ [18,1]
951
< LOD
< LOD
< LOD
wild boar, fat
12
< LOQ [17,3]
8.4
2.9
23.0
< LOD
wild boar, fat
< LOQ [29,8]
14.2
78.1
54.1
< LOD
wild boar, fat
12
< LOQ [26,2]
12.9
12.4
5.9
< LOD
200
200
100
20
maximum amount by law
alpha-HCH
[ng/ g fat]
beta-HCH
[ng/ g fat]
lindane [ng/
g fat]
Chloroparaffins
LOD: 13 ng/ g fat
LOQ: 30 ng/ g fat

Freiburg (Germany)
Meat
CP
[ng/ g fat]
chicken
country of origin
< LOQ [26]
Germany
turkey
< LOD
Germany
goose
34
Germany
beef
< LOQ [33]
Germany
beef
60
Germany
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]
olive oil
< LOD
olive oil
75
olive oil
< LOQ [20]
olive oil
98
olive oil
38
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
Patricia Fischer,
Sebastian Schelb,
Katrin Thoma,
Nadine Singler
Thank you for your attention

Freiburg (Germany)

Determination of Chloroparaffins in Foodstuffs by GC MS-MS

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Interested in conducting your own webinar? Email webinars@bnpmedia.

Chemisches und Veterinruntersuchungsamt (CVUA)

Biljana Trajkovska, Karin Kypke,

State Institute for Chemical and Veterinary Analysis of Food

Chemisches und Veterinruntersuchungsamt (CVUA)

Biljana Trajkovska, Karin Kypke,

State Institute for Chemical and Veterinary Analysis of Food

State Institute for Chemical and Veterinary Analysis of Food

Chlorinated Paraffins (CPs)

general formula CnH2n+2-zClz

(SCCP; C10 C13)

medium chain CPs

(MCCP; C14 C17)

long chain CPs

(LCCP; > C17)

State Institute for Chemical and Veterinary Analysis of Food

Applications of Chlorinated Paraffins (CPs)

metal industry (lubricant, cutting oil)

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

leather industry for fat liquoring

plasticiser in PVC, especially MCCP (alternative to phthalates)

State Institute for Chemical and Veterinary Analysis of Food

categorized by IARC in group 2 B

State Institute for Chemical and Veterinary Analysis of Food

COMMISSION REGULATION (EU) No 519/2012

State Institute for Chemical and Veterinary Analysis of Food

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

Convention entered into force on 17 May 2004;

Protection of human health and the environment

Reduction or elimination of releases of certain POPs

State Institute for Chemical and Veterinary Analysis of Food

Comprises: Pesticides, industrial chemicals, by-products

Parties to Stockholm Convention

October 2012: 178 Parties (with152 Signatories)

CP levels in fish from Germany and Switzerland

Reth (2006): Total CP concentrations

Range 0.5 27 % lipid

State Institute for Chemical and Veterinary Analysis of Food

CP levels in human milk from Germany

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

State Institute for Chemical and Veterinary Analysis of Food

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

State Institute for Chemical and Veterinary Analysis of Food

State Institute for Chemical and Veterinary Analysis of Food

35 references found with

State Institute for Chemical and Veterinary Analysis of Food

35 references found with

State Institute for Chemical and Veterinary Analysis of Food

Scientific basis for development of analytical procedure

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

Z. Zencak, M. Reth and M. Oehme Determination of Total Polychlorinated n-Alkane

State Institute for Chemical and Veterinary Analysis of Food

Extraction and clean up:

State Institute for Chemical and Veterinary Analysis of Food

State Institute for Chemical and Veterinary Analysis of Food

samples with a high fat content

heated in a microwave oven

State Institute for Chemical and Veterinary Analysis of Food

raw milk / human milk

homogenize the cream with

solvent is removed by using a turbo vap 500

State Institute for Chemical and Veterinary Analysis of Food

meat / cheese / eggs

solvent is removed by using a Turbo Vap 500