1 / 16.05.2011 - Ralf Lippold CVUA Freiburg Pesticide Residue Analysis in Food of Animal Origin INTI-Workshop „Update on Pesticide Residues Ana- lytical Tools to Improve Food Control“ 16.05.2011 Buenos Aires, Argentina Ralf Lippold Chemisches und Veterinärunter- suchungsamt Freiburg (State Institute for Chemical and Veterinary Analysis of Food) 2 / 16.05.2011 - Ralf Lippold CVUA Freiburg Pesticides - Veterinary Drugs Typically, pesticides are not applied to animals Animals come in contact with pesticides e.g. by feet, water, environment ..... But, if pesticides will be applied to animals? According to the European Union legislation this will be a treatment of an animal and the pesticide will „transform“ into a veterinary drug.
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1 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Pesticide Residue Analysis in Food of Animal Origin
INTI-Workshop
„Update on Pesticide Residues Ana-lytical Tools to Improve Food Control“
16.05.2011Buenos Aires, Argentina
Ralf Lippold
Chemisches und Veterinärunter-suchungsamt Freiburg(State Institute for Chemical andVeterinary Analysis of Food)
2 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Pesticides - Veterinary Drugs
�Typically, pesticides are not applied to animals
�Animals come in contact with pesticides e.g.by feet, water, environment .....
�But, if pesticides will be applied to animals?
�According to the European Union legislation this will be a treatment of an animal and the pesticide will „transform“ into a veterinary drug.
3 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Two sets of AQC Procedures
1. Method Validation and Quality Control Procedures for Pesticide Residues Analysis in Feed and Food (Document No. SANCO /10684/2009)
2. Commission Decision 2002/657/ECImplements Council Directive 96/23/EC on measures to monitor certain substances and residues thereof in live animals and animal products
� Agreement: Currently inspectors of the Federal Veterinarian Office (FVO) will accept if pesticides in food of animal origin are validated either according the AQC procedures or CD 2002/657/EC
4 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Definition of lipophilic pesticides in EU regulations
Depending on the water solubility, the maximum levels for food of animal origin (meat, milk, eggs) are regulated either on product or fat basis as follows
• If the pesticide and/or metabolite is/are water soluble (log P ow less than 3), the MRL is expressed as mg/kg of product .
• Where the pesticide and/or metabolite is/are fat soluble (log P ow greater or equal to 3), the MRL is expressed as mg/kg of fat or product.
• Some differentiations depending on fat content
5 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Polarity range covered by trad. MRMs
-5
LogKow
-4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10
Ureas (~ 30)
1.6 - 5.9
Pyrethroids (~45)3.8 - 8.3
OPs (~95)
-0.9 - 5.7
Quats-4.5 - -2.8
OCs (~20)3.5 - 7.0
Sugars-5 - -2
Phytosterols8.5-11.5
Flavonoids/Anthocyanes0 - 6
Amino acids-5 - -1
(pH dependent)
GlyphosatepH-dependent
-3.2
Strepto-mycin
-7.5
Monoterpenes2.5-5.5
Chlorophyll17.2
Carotenoids11-18
Scope Comparisson – Classical MRMs vs. QuEChERS
Carbamates (~30)
-0.4 - 5.5
11
Fatty Acids6-8.5
Alkanoic acids (~40)
pH dependent
TGs20-24
Basic Pesticides
pH dependent
GC-Probl. ���� LC-MS
GPCLosses
GC-Problems ���� LC-MS
GC-Problems ���� LC-MS
GC-Problem ���� LC-MS
Fosetyl-AlpH-dependent
-2.7
EthephonpH-dependent
-1.9
GC-Probl.
QuEChERS-Scope
Michelangelo AnastassiadesEURL-SRM Stuttgart
6 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Pesticide and Contaminants in Food of Animal Origin
� Mostly non polar, lipophilic compounds
� Enrichment in the fat of animals� often organochlorine compounds
(application not allowed according directive 91/414/EEC)
� Residue levels going down, but some times cases of contamination (e.g. nitrofen 2002 in Germany)
� Organophosphorous compounds
� Pyrethroids� Contaminants
(e.g. organo tin compounds, moschus-xylol,indicator PCB)
7 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Pesticide and Contaminants in Food of Animal Origin
� Often volatile compounds -> GC
� Some Compounds only as metabolite identifiziert� Aldrin/Dieldrin � only Dieldrin� DDT � DDE (!)
� Pig fat and meat from the market � tested to be “free” of pesticide residues (NPD ZB 50)
IS
21 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Detection using MSD (Pig-fat)
� Pig fat and meat from the market � tested to be “free” of pesticide residues (MSD SIM)
22 / 16.05.2011 - Ralf Lippold CVUA Freiburg
� Pig fat and meat from the market� tested to be free of pesticide residues
� Certified spiking solution� custom made by LGC Promochem GmbH, Germany� checked against EURL-standards
� Spiking of 1 kg pig fat with 20 ml solution� 2 h stirring (40°C)
EUPT AO 05: GC-MSD
IS
ISIS
IS
23 / 16.05.2011 - Ralf Lippold CVUA Freiburg
EUPT AO 05: GC-MS/MS
� Pig fat and meat from the market� tested to be free of pesticide residues
� Certified spiking solution� custom made by LGC Promochem GmbH, Germany� checked against ERL-standards
� Spiking of 1 kg pig fat with 20 ml solution� 2 h stirring (40°C)
24 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Extraction of Fat
Cleanup efficient and well tested for all three groups of pesticides
�Some clean-up procedures like aluminium oxide column or sulphuric acid don’t give good recovery rates for all groups of pesticides�Especially several organophosphorus and pyrethroidcompounds are not eluted quantitatively
The CVUA Freiburg favorites gel chromatography
25 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Extraction of Fat
� Cleanup efficient and well tested
� How to get the fat-phase containing the non-polar pesticides?
26 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Extracting fat in food of animal origin at CVUA Freiburg
Examples:
• Centrifugation to separate cream for raw milk
• Liquid / liquid distribution for homogenized milk
• Addition of Na2SO4 and extraction with organic solvents (meat, eggs)
27 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Extraction of milk 1
� Extract milk with diethylether
� Evaporate to „moderate dryness“
28 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Extraction of milk 2 (Sandmeier-Methode)
� Centrifuge Milk using a high performance centrigue
� Phase containing the fat will be on top of the milk
� Mix fat containing phase with dry sodium sulphate and extract the fat using petrol ether
� Filter the extract and evaporate to „moderate dryness“
29 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Results (main findings)
In total, more than 80 (fat-soluble) analytes are checked. For evaluation and comparing the results, the relevant and representative compounds were chosen.
30 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Method of Soxhlet
� Homogenisation of 10 g of sample with 40 g Celite(or a mixture of sand and sodium sulphate)
� Extract with diethylether under re-flushing conditions (4 to 8 hours)
� Evaporate to „moderate dryness“
� Good for matrices like meat, fish, eggs, cheese
� Not applicable for liquid matrices (like milk)
31 / 16.05.2011 - Ralf Lippold CVUA Freiburg
EN 1528-2: Modular System for Pesticide Methods
European Stan-dard
Description German Standard Modul Description Validated me-thods of CVUA Freiburg
Samples / Compounds
EN 1528-2: 1996-10 (confirmed 2001)
Extraction of fat, pestici-des and PCBs and de-termination of fat content
§ 64 LFGB: L 00.00-34 (confirmed 1999)
E 8 Extraction of fat with hexan/acetone
PV 31 P01601 meat, fish
Extraction of fat, pestici-des and PCBs and de-termination of fat content
� Mean values for these analytes are shown in the charts� No more than slight differences between organic and
conventional foods regarding the levels of organochlorineresidues and contaminants.
� Fact: persistent organochlorine and organobromine compounds are accumulated via the food chain in the fat tissue of animals.
� For dairy products, mean levels for every analysed compound were not higher than 0.005 mg/kg fat
40 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Regulation 396/2005Footnotes in Annex I
41 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Footnote 5 - Meat and Preparations of Meat
�Where the pesticide and/or metabolites (included in the residue definition) is/are water soluble (log Pow less than 3) the MRL is expressed as mg/kg of meat (including fat), preparations of meat, offal and animal fats. Where the pesticide and/or metabolite (included in the residue definition) is/are fat soluble (log Pow greater than or equal to 3) the MRL is expressed as mg/kg of fat contained in the meat, preparations of meat, offal and animal fats. In the case of foodstuffs with a fat content of 10 % or less by weight, the residue is related to the total weight of the boned foodstuff. In such cases, the maximum level is one-tenth of the value related to fat content, but must be no less than 0.01 mg/kg. The latter does not apply when the MRL is set at the LOD.
Analytical results are calculated in mg/kg meat.
Reference:
mg/kg whole product
42 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Footnote 5 - Meat and Preparations of Meat
�Where the pesticide and/or metabolites (included in the residue definition) is/are water soluble (log Pow less than 3) the MRL is expressed as mg/kg of meat (including fat), preparations of meat, offal and animal fats. Where the pesticide and/or metabolite (included in the residue definition) is/are fat soluble (log Pow greater than or equal to 3) the MRL is expressed as mg/kg of fat contained in the meat, preparations of meat, offal and animal fats. In the case of foodstuffs with a fat content of 10 % or less by weight, the residue is related to the total weight of the boned foodstuff. In such cases, the maximum level is one-tenth of the value related to fat content, but must be no less than 0.01 mg/kg. The latter does not apply when the MRL is set at the LOD.
Analytical results are calculated in mg/kg fat.
Reference:
mg/kg fat
43 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Footnote 5 - Meat and Preparations of Meat
�Where the pesticide and/or metabolites (included in the residue definition) is/are water soluble (log Pow less than 3) the MRL is expressed as mg/kg of meat (including fat), preparations of meat, offal and animal fats. Where the pesticide and/or metabolite (included in the residue definition) is/are fat soluble (log Pow greater than or equal to 3) the MRL is expressed as mg/kg of fat contained in the meat, preparations of meat, offal and animal fats. In the case of foodstuffs with a fat content of 10 % or less by weight, the residue is related to the total weight of the boned foodstuff. In such cases, the maximum level is one-tenth of the value related to fat content, but must be no less than 0.01 mg/kg. The latter does not apply when the MRL is set at the LOD.
Analytical results are calculated in mg/kg meat.
Reference:
mg/kg whole productMRL is one-tenth
44 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Problem: Determination of FatFat Content of the Test Material
Min 4.20Max 36.00Median 17.32Average 16.08Robust mean (A) 16.47Standardabw. 4.49Robust standard deviation Q [µg/kg] // coefficient of variation3.36Robust standard deviation (algorithm A of 13528) [µg/kg] // coefficient of variation3.38<10% 9>25% 1
45 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Problem in EUPT AO 05: Determination of Fat
� Problem:
Labs analysing pesticides using test material and determine fatin second method -> results of pesticides depend on the resultof the fat determination!
� Follow up => questionnaire
46 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Evaluation: Fat-Determination in EUPT AO 05
EUPT AO 05: Fat Content
5
7
9
11
13
15
17
19
21
23
25
Column extraction ASE Cold extraction Soxhlet Weibull-Stoldt
Fat
con
tent
[%]
Median (all data)17.32 µg/kg
47 / 16.05.2011 - Ralf Lippold CVUA Freiburg
0
5
10
15
20
25
TW
, FD
Ultr
a-T
urra
x
AS
E, H
ydro
mat
rix
AS
E, D
A
TW
, Na2
SO
4
TW
, FD
Sox
ther
m, n
o pr
e-tr
.
Sox
ther
m, N
a2S
O4
AS
E, D
A
AS
E, H
ydro
mat
rix
TW
, Na2
SO
4
TW
, FD
Wei
bull-
Sto
ldt
Lipi
d co
nten
t [%
]
Extraction of fat from homogeneized meat samples
Cyclohexan / Toluene
Pet
r ole
um e
t he r
Cyc
l oh e
xane
n -H
exa n
e
Pet
r olu
emet
her Dry mass: 34 %
TW: Twisselmannextraction
DA: Drying agentFD: Freeze-dried
Raw extract
Ethanol /Toluene(tert. BME)C
ycl o
h exa
ne2 -
Pro
p an o
l
48 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Evaluation: Fat-Determination in EUPT AO 05
Results� Simple stirring or mixing of solvents with test material
(“cold extraction”) leads to unequal resultsinfuenced by� Duration of stirring/mixing/shaking� Ratio between solvent and matrix� Repeated extraction steps
(as using column extraction, ASE or soxhlet)
49 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Polarity range covered by trad. MRMs
-5
LogKow
-4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10
Ureas (~ 30)
1.6 - 5.9
Pyrethroids (~45)3.8 - 8.3
OPs (~95)
-0.9 - 5.7
Quats-4.5 - -2.8
OCs (~20)3.5 - 7.0
Sugars-5 - -2
Phytosterols8.5-11.5
Flavonoids/Anthocyanes0 - 6
Amino acids-5 - -1
(pH dependent)
GlyphosatepH-dependent
-3.2
Strepto-mycin
-7.5
Monoterpenes2.5-5.5
Chlorophyll17.2
Carotenoids11-18
Scope Comparisson – Classical MRMs vs. QuEChERS
Carbamates (~30)
-0.4 - 5.5
11
Fatty Acids6-8.5
Alkanoic acids (~40)
pH dependent
TGs20-24
Basic Pesticides
pH dependent
GC-Probl. ���� LC-MS
GPCLosses
GC-Problems ���� LC-MS
GC-Problems ���� LC-MS
GC-Problem ���� LC-MS
Fosetyl-Al pH-dependent
-2.7
Ethephon pH-dependent
-1.9
GC-Probl.
QuEChERS-Scope
50 / 16.05.2011 - Ralf Lippold CVUA Freiburg
51 / 16.05.2011 - Ralf Lippold CVUA Freiburg
52 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Polar Pesticides in Milk - Quechers
� Shake 10 g milk with 10 ml acetonitrile� Add magesium salfate (4 g), sodium chloride (1g) and citrate (0,5
g Dinatriumhydrogencitrat + 1 ± 0,05 g Trinatriumcitrat)� Shake and centrifugate� Add magnesium sulfate (900 mg) and PSA (150 mg Bondesil-
PSA 40 µm, Varian) to 6 ml of the acetonitrile phase� Shake and centrifugate� LC-MS/MS
Hint: Quechers is suitable for polar pesticides in food of animal origin
53 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Pyrrolizidine Alcaloids in Milk - Quechers
� Group of about 200 alcaloids� Occurance in plant of species senecio (groundsel) � Problem: derivates are toxic (poisoning of cattle feeding silage)� Findings in milk and poisoning of humans possible� Analysis possible using Quechers-method for milk and LC-
MS/MS-Detection
54 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Pyrrolizidine Alcaloids in Milk - Quechers
55 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Polar Pesticides in Honey
� Modern Pesticides are more/less polar
� No enrichment in fat of animals� Compounds rapidly metabolized and/or excreted
Is it necessary to analyse for all pesticides in fo od of animal origin?
� Example Honey (Cleanup QuEChERS-method)
- Database research for findings (e.g. pesticides-online, publications)- Development/Validation of a method with the “relevant” pesticides (~30)
- Time to time addition of more pesticides (validation in smaller packages of 20 to 30 pesticides)
10 µg/kg in honey matrix45 analyts, 3 ISTD =>98 transisitions
Liquid chromatography with MS/MS-Detektion (Agilent LC 1200 with Agilent 6410 QQQ)
LC column: Zorbax Eclipse Plus C18 (Agilent) 150 mm x 2,1 mm i.d x 3.5 µm
Eluent A: 5 mMol/L ammonium formiate
Eluent B: acetonitrile
Flow: 0.3 ml/min
Gradient: time (min) % B
0 5 1 5 30 100 32.5 100 33 5
Temp.: 25 °C
Injection: 5.0 µl
MS Mode ESI Positive
58 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Honey Examples of FindingsBienenhonig
Liquid chromatography with MS/MS-Detektion (Agilent LC 1200 with Agilent 6410 QQQ)
LC column: Zorbax Eclipse Plus C18 (Agilent) 150 mm x 2,1 mm i.d x 3.5 µm
Eluent A: 5 mMol/L ammonium formiate
Eluent B: acetonitrile
Flow: 0.3 ml/min
Gradient: time (min) % B
0 5 1 5 30 100 32.5 100 33 5
Temp.: 25 °C
Injection: 5.0 µl
MS Mode ESI Positive
ISTD
ISTD
ISTD
Azoxystrobin
Dimoxystrobin
Thiacloprid
59 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Polarity range covered by trad. MRMs
-5
LogKow
-4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10
Ureas (~ 30)
1.6 - 5.9
Pyrethroids (~45)3.8 - 8.3
OPs (~95)
-0.9 - 5.7
Quats-4.5 - -2.8
OCs (~20)3.5 - 7.0
Sugars-5 - -2
Phytosterols8.5-11.5
Flavonoids/Anthocyanes0 - 6
Amino acids-5 - -1
(pH dependent)
GlyphosatepH-dependent
-3.2
Strepto-mycin
-7.5
Monoterpenes2.5-5.5
Chlorophyll17.2
Carotenoids11-18
Scope Comparisson – Classical MRMs vs. QuEChERS
Carbamates (~30)
-0.4 - 5.5
11
Fatty Acids6-8.5
Alkanoic acids (~40)
pH dependent
TGs20-24
Basic Pesticides
pH dependent
GC-Probl. ���� LC-MS
GPCLosses
GC-Problems ���� LC-MS
GC-Problems ���� LC-MS
GC-Problem ���� LC-MS
Fosetyl-AlpH-dependent
-2.7
EthephonpH-dependent
-1.9
GC-Probl.
QuEChERS-Scope
60 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Streptomycin in Honey and Fire Blight
� Fire blight is spreading over wide areas in Germany, Switzerland, Austria, Northern Italy and the upper Rhone valley (France).
� Fire blight is a disease induced by bacteria (Erwiniaamylovora) causing lot of problems in pome plantations with losses of trees and therefore large crop failures.
61 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Background Information: Fire blight
� Germany issued yearly a certificate of exemption for use of streptomycin containing pesticide formulations because the lack of alternatives.
� Monitoring programmes for streptomycin during spraying in pome plantations and for residues of streptomycin in honey were introduced.
62 / 16.05.2011 - Ralf Lippold CVUA Freiburg
Streptomycin - Analytics
LC Säule: ZIC-HILIC PEEK (Merck Sequant)
100 x 2.1 mm i.d. x 3.5 µm
Eluent A: 0.02 M Natriumformiat + 0.1% Ameisensäure
Eluent B: Acetonitril + 0.1% Ameisensäure
Flow: 0.30 ml/min
Gradient: Time (min) %B
0 90
0,5 90
1,5 10
6,5 10
8 90
Temp.: 25 °C
Injektion: 10 µl
LC mit MS-MS-Detektion
MS Mode ESI PosLC mit MS-MS-Detektion
M+HCone (V)
product ion 1 / coll (eV)
product ion 2 / coll (eV)
Streptomycin 582.4 135 263.2 / 32 246.2 / 40
Dihydrostreptomycin 584.4 135 263.2 / 31
Honey [1] Extraction of samples
Weighted sample weigh 20 g of honey into a 200 ml centrifuge tube (poly propylene)
Internal standard add 50 µl of internal standard solution (dihydrostreptmycin, ρ = 10 ng/µl); wait 5 minutes before extraction
add 87 g water
shake 20 minutes
Extraction
filter through a fluted filter
conditioning of SPE-Cartridge (Sep-Pak Vac 6 cc (500 mg) Accell Plus CM) with 10 ml washing solu-tion (980 ml water + 20 ml acetic acid) and 10 ml water
load 50 ml (53.5 g) of the filtrate onto the cartridge
wash with 10 ml water
SPE-Cleanup step
elute with 7.5 ml elution solution (780 ml water + 20 ml acetic acid + 200 ml acetonitrile)
concentrate eluate to about 4 ml Final extract
add water to 5.0 g
Standards in solution spike aliquots of streptomycin and 50 µl of internal standard solution into 10 g of washing solution
Final quantification for quantification of streptomycin in honey the stan-dard addition procedure is recommended
63 / 16.05.2011 - Ralf Lippold CVUA Freiburg
ISTD Dihydrostreptomycin
Streptomycin I
Streptomycin II
Dot. 10+15 µg/kg Streptomycin I
ISTD Dihydrostreptomycin
Streptomycin II
honey 15 µg/kg
Streptomycin - Analytics
Streptomycin Monitoring Results
about 8,4 t of honey (0,24 % of the yearly honey production in our state
since 2009MRL = 10 µg/kgapplication limited to 2 sprayings � few residues found