Work carried out in the Czech Republic
CENTRAL INSTITUTE FOR SUPERVISING AND TESTING IN AGRICULTURE
Olga Nováková
CIPAC Symposium 24th June 2014, Liege, Belgium
NATIONAL REFERENCE LABORATORYDepartment of Testing Plant Protection Product
(version for CIPAC website)
CONTENT
1. Introduction 2. Steps of laboratory control of PPPs in CZ3. Example of analysis of suspicious samples4. Problems with analysis of suspicious samples5. Suggestion to discussion6. Conclusion
CIPAC Symposium 24th June 2014, Liege, BelgiumOlga Nováková 2
1997‐2013: 2 institutes• SPA (State Phytosanitary Administration)
– Postregistration Control Division• Department of Laboratory Testing Pesticide (= NRL for PPP)
• CISTA (Central Institute for Supervising and Testing in Agriculture)– Division: National Reference Laboratory (9 laboratories)
From 1.1.2014: 1 institute• SPA + CISTA = CISTA
– Division: National Reference Laboratory (9+1 laboratories)• Department of Testing Plant Protection Products = former Laboratory Testing Pesticide
Olga Nováková CIPAC Symposium 24th June 2014, Liege, Belgium 3
REORGANIZATION OF FORMER STATE PHYTOSANITARY ADMINISTRATION (SPA)
STRUCTURE OF CENTRAL INSTITUTE FOR SUPERVISING AND TESTING IN AGRICULTURE (CISTA)
Olga Nováková 4CIPAC Symposium 24th June 2014, Liege, Belgium
General Director
Authority Office
Economic and Administration Section
Section of Protection against Harmful Organisms
Section of Plant Production
Section of Agricultural Inspection
Section of Agricultural Inputs
National Reference Laboratory
Division of PPPCoordination
EvaluationPostregistration
3 Divisions
4 Divisions
7 Regional Divisions
Division of Audit and Quality Management
Division of Feed, Fertilisers and Soil
Division of Agricultural Inputs Control
STRUCTURE OF NATIONAL REFERENCE LABORATORY
Olga Nováková 5CIPAC Symposium 24th June 2014, Liege, Belgium
Director of NRL
Specialized DepartmentsRegional Departments
Brno
Opava
Plzeň
Praha
BrnoDepartment of Testing Plant Protection Products
Planá nad Lužnicí
BrnoDepartment of Proficiency Testing Programes
BrnoDepartment of Microbiology and Biochemistry
LípaDepartment of Special Plant and Feed Analyses
DEPARTMENT OF TESTING PLANT PROTECTION PRODUCTS
Olga Nováková 6CIPAC Symposium 24th June 2014, Liege, Belgium
Head of Department
Laboratoryof physicochemical
methods
Laboratoryof chromatographic
methods
Post‐registration Control Department ‐ responsibility• annual plan with cooperation of laboratory• sampling original packages ‐ PPP samples are taken directly
in the distributor stores• labeling control• control of sale, storage and use of PPP• control of technical state of application machinery
Laboratory‐ responsibility and competence• Laboratory analysis• laboratory check on physical and chemical properties of plant
protection products and other plant protection preparations and chemical compositions
• report of analysis (+results with evaluation)
Post‐registration Control Department – responsibility• reconvey the rest of samples• evaluation of results from laboratory in accord with act (law)• information of authorization holders • fine
Olga Nováková CIPAC Symposium 24th June 2014, Liege, Belgium 7
SYSTEM OF POSTREGISTRATION CONTROL –responsibility
Samplingoriginal packages
Laboratory analysis+
report
Evaluation of results in accord
with act
Planned samples • postregistration control according to annual plan
• samples within the process of PPP approvalUnplanned samples• unknown samples• suspicious samples (e.g. counterfeit)Proficiencies tests (AAPCO, AFSCA, …)
Olga Nováková CIPAC Symposium 24th June 2014, Liege, Belgium 8
LABORATORY CONTROL OF PPPs
• Registration of samples (LIMS)• Laboratory sampling
– Original sample package is higher 1L or 1kg – Original sample package is lower 1L or 1kgPartition of laboratory sample = Analytical sample
• Laboratory analysis • Evaluation of laboratory analysis
– Certificate of analysis (Agreement or disagreement with specification)
– Detailed expert reports
Olga Nováková CIPAC Symposium 24th June 2014, Liege, Belgium 9
STEPS OF LABORATORY CONTROL
AU
-0,0005
0,0000
0,0005
0,0010
0,0015
0,0020
0,0025
0,0030
0,0035
0,0040
0,0045
0,0050
0,0055
0,0060
0,0065
0,0070
0,0075
0,0080
Minutes0,00 2,00 4,00 6,00 8,00 10,00 12,00 14,00 16,00 18,00 20,00 22,00 24,00 26,00 28,00 30,00 32,00 34,00 36,00 38,00 40,00 42,00 44,00 46,00 48,00 50,00
Planned samples – postregistration control according to annual planFAO specification or existing national specification
– Identity and content of active substances – Identity and content of relevant impurities– Physical, chemical and technical properties– Storage stability tests
Xylene in EC formulations
Samples from parallel import – chromatographic and FTIR comparison with reference sample.
Methods: CIPAC, OECD or equivalent and the validated methods of producers, which are submitted as part of registration dossiers.
Laboratory verifies all methods according to standard operation procedure (SOP‐PP‐08‐01).
Olga Nováková CIPAC Symposium 24th June 2014, Liege, Belgium 10
LABORATORY ANALYSIS
Samples within the process of PPP approval Aim:• Verify input data (technical specification)• Verification or validation analytical methods in CZ conditions (different column…)
• Obtain knowledge of PPP• Focus on chemical composition • Time for discussion of laboratory analysis ‐analytical results with authorization holders
• Input data for postregistration control• Future ‐ Help with detection of counterfeit
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LABORATORY ANALYSIS – cont.
Unplanned control PPPs samples• Unknown samples (samples without label, confusion of active ingredient,…)
• Suspicious samples
Olga Nováková CIPAC Symposium 24th June 2014, Liege, Belgium 12
LABORATORY ANALYSIS – cont.
• Identity and content of active ingredient • Identity and content of relevant impurities• Physical, chemical and technical properties • Chemical composition of sample (co‐formulants,
impurities,…) • Comparison with reference sample (GC, LC, FTIR)
Additional tests for clarification of unregistered sample: GC/MS, FTIR, Particle size distribution (CIPAC MT187), Determination of Sulphated ash (CIPAC MT29), Pour and tap bulk density (USP2/ASTM), Density, Viscosity (CIPAC MT192), Surface tension (OECD 115), TGA method, DSC method
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LABORATORY ANALYSIS –SUSPICIOUS SAMPLES
Appearance:
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EXAMPLES OF ANALYSIS
Differences in colourbetween reference sample and suspicious sample
Differences of shapegranules between reference sample and suspicious sample
Density:
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EXAMPLES OF ANALYSIS
Density g/ml
Reference PPP 1,170
Suspicious PPP 1,126
Result:Suspicious PPP is probably diluted or hasdifferent chemical composition.
Density:
Olga Nováková CIPAC Symposium 24th June 2014, Liege, Belgium 16
EXAMPLES OF ANALYSIS
Density g/ml Dean – Stark (CIPAC MT 30.2) Amount of water
(% w/w)
Amount of active ingredient (%)
Reference PPP 1,170 44,6 30,4
Suspicious PPP 1,126 60,7 21,8
Result:Suspicious sample is diluted.
Confirmation:•determination of water by Dean‐Stark (CIPAC MT 30.2)•determination of amount active ingredients
Sulphated ash (CIPAC MT 29):
Olga Nováková CIPAC Symposium 24th June 2014, Liege, Belgium 17
EXAMPLES OF ANALYSIS
Reference PPP (different batches)
year Sulphated ash [% w/w]
2001 1,52
2003 1,30
2006 1,37
2008 1,22
2012 1,26
2013 1,05
average 1,29
Suspicious PPP
Year Sulphated ash [% w/w]
2001 ‐‐‐
2003 15,52
2006 ‐‐‐
2008 19,40
2012 10,52
2013 8,62
2013 6,83
Result: There are difference between inorganic ions in suspicious sample and reference sample ‐ the chemical composition of suspicion sample is different from reference sample.
Surface tension (OECD 115):
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EXAMPLES OF ANALYSIS
Result: Suspicious PPP probably does not contain surface active agent.
PPP Surface tension of 0,01% w/w solution (mN/m)
Surface tension of 0,5% w/w solution
(mN/m)
Surface tension of 1,0 % w/w solution
(mN/m)
Reference PPP 57,0 32,9 32,7
Suspicious PPP 59,4 69,9 71,8
Particle size distribution (CIPAC MT 187) :
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EXAMPLES OF ANALYSIS
Reference PPP Suspicious PPP
Particle size distribution (CIPAC MT 187) :
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EXAMPLES OF ANALYSIS
Result: Particle size distribution of particles which disperse in water in suspicious PPP are different. It means that it can be different technology of production.
Reference PPP Suspicious PPPDiameter at 10% 0,68 μm 0,80 μm
Diameter at 50% 2,45 μm 3,77 μm
Diameter at 90% 6,07 μm 9,68 μm
Mean diameter 2,97 μm 4,56 μm
HPLC screening by HPLC/UV:
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EXAMPLES OF ANALYSIS
Result: Suspicious PPP 1 has probably identical chemical composition with reference PPP, Suspicious PPP 2 has not identical chemical composition with reference PPP.
AU
-0,0005
0,0000
0,0005
0,0010
0,0015
0,0020
0,0025
0,0030
0,0035
0,0040
0,0045
0,0050
0,0055
0,0060
0,0065
0,0070
0,0075
0,0080
Minutes0,00 2,00 4,00 6,00 8,00 10,00 12,00 14,00 16,00 18,00 20,00 22,00 24,00 26,00 28,00 30,00 32,00 34,00 36,00 38,00 40,00 42,00 44,00 46,00 48,00 50,00
Reference PPP (black), Suspicious PPP 1 (blue) Suspicious PPP 2 (green)
GC/FID – identification of co‐formulant:
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EXAMPLES OF ANALYSIS
Reference PPP (blue) Suspicious PPP (red)
min2.5 5 7.5 10 12.5 15 17.5 20
Norm.
10
15
20
25
30
35
FID1 B, (130708\ZK000005.D)
11.
644
FID1 B, (130708\ZK000008.D)
11.
658
GC/FID – identification of co‐formulant:
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EXAMPLES OF ANALYSIS
Result: Suspicious PPP does not contain surface‐active agent which is present in reference PPP.
Reference PPP (blue) Suspicious PPP (red)Surface‐activ agent (green)
min10 11 12 13 14 15 16 17
pA
10
15
20
25
30
35
FID1 B, (130708\ZK000005.D) FID1 B, (130708\ZK000008.D) FID1 B, (130708\ZK000004.D)
11.
644
Overlay chromatograms of reference PPP and suspicious PPP and Surface‐active agent, extract 10‐18min
GC/MSD identification of co‐formulants:
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EXAMPLES OF ANALYSIS
Reference PPP (green), Suspicious PPP (black)
GC/MSD chromatographic profile of PPP (split 100:1, extract 2‐4min)
2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.800
100000
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Time-->
Abundance
TIC: 11101210.DTIC: 11101211.D (*)
N,N‐dimethylformamide
Ethylene glycol
MSD spectrum of ethylene glycol
20 40 60 80 100 120 140 160 180 200 220 240 260 2800
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m/z-->
Abundance
Scan 27 (2.206 min): 11102502.D31
43
6284 207133 28198
MSD spectrum of N,N‐dimethylformamid
20 30 40 50 60 70 80 90 1001101201301401501601701801902002100
200000
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m/z-->
Abundance
Scan 127 (2.671 min): 11113005.D73
44
30 58
20721 91 191
GC/MSD identification of co‐formulants:
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EXAMPLES OF ANALYSIS
Reference PPP (green), Suspicious PPP (black)
GC/MSD chromatographic profile of PPP (split 10:1, extract 4‐8min)MSD spectrum of naphthalene
4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.500
50000
100000
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Time-->
Abundance
TIC: 11101205.DTIC: 11101206.D (*)
naphthalene20 40 60 80 100 120 140 160 180 200 220 240 260 280
0
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m/z-->
Abundance
Scan 770 (5.660 min): 11102604.D128
1026451 75 873928 113 207 281
GC/MSD identification of co‐formulants:
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EXAMPLES OF ANALYSIS
Reference PPP (green), Suspicious PPP (black)
GC/MSD chromatographic profile of PPP (split 10:1,extract 8‐11,6 min)
MSD spectrum of N‐octyl‐2‐pyrrolidone
8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.500
50000
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500000
550000
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1000000
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Time-->
Abundance
TIC: 11101205.DTIC: 11101206.D (*)
N‐octyl‐2‐pyrrolidone
20 40 60 80 100 120 140 160 180 200 220 240 260 2800
20000
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60000
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100000
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180000
200000
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m/z-->
Abundance
Scan 1341 (8.315 min): 11113009.D98
19770 126
112
41
86 14028 55 154168180 281209
GC/MSD identification of co‐formulants:
Olga Nováková CIPAC Symposium 24th June 2014, Liege, Belgium 27
EXAMPLES OF ANALYSIS
Reference PPP (green), Suspicious PPP (black)
GC/MSD chromatographic profile of EW formulation PPP ‐ active ingredient (extract 11,6‐12,6 min)
11.60 11.70 11.80 11.90 12.00 12.10 12.20 12.30 12.40 12.50 12.60
500000
1000000
1500000
2000000
2500000
3000000
3500000
Time-->
Abundance
TIC: 11101210.DTIC: 11101211.D (*)
active ingredient
GC/MSD identification of co‐formulants:
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EXAMPLES OF ANALYSIS
Result: The chemical composition of suspicious PPP are different from reference sample.
Summary:
Identified components Reference PPP Suspicious PPP Content % w/w
N,N‐dimethylformamide No Yes 16,78
N‐octyl‐2‐pyrrolidone No Yes 0,306
Ethylene glycol No Yes 1,525
Naphthalene No Yes 0,018
UV‐VIS spectroscopy:
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EXAMPLES OF ANALYSIS
Reference PPP Suspicious PPPVIS spectrum:
Sample λ max v DI water [ nm]
Reference sample 628 nm
Suspicious sample 620 and 657 nm
Result: The suspicious PPP does not contain the same dye as reference sample.
FTIR spectroscopy :
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EXAMPLES OF ANALYSIS
Reference PPP (blue), Suspicious PPP (red)
FTIR spectroscopy :
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EXAMPLES OF ANALYSIS
FTIR spectrum of Insoluble residues of PPP in CH2Cl2
Reference PPP (blue), Suspicious PPP (red)
FTIR spectroscopy :
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EXAMPLES OF ANALYSIS
Result: The suspicious PPP contains kaolin = the chemical composition is different from reference sample.
FTIR spectrum of Insoluble residues of suspicious PPP in CH2Cl2 and kaolin CAS No. 1332‐58‐7
Suspicious PPP (blue), kaolin CAS No. 1332‐58‐7(red)
Differential Scanning Calorimetry (DSC):
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EXAMPLES OF ANALYSIS
Reference PPP Suspicious PPP
Result: The chemical composition of suspicious sample is different from reference sample.
Thermogravimetric analysis (TGA):
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EXAMPLES OF ANALYSIS
Reference PPP Suspicious PPP
Result: Different chemical composition of volatile compounds present in PPP or/and other compounds in PPP.
• Active ingredients and relevant impurities in suspicious PPP mostly agree with specification.
• We usually find differences in chromatography profile and/or in FTIR spectra between reference and suspicious samples:
– we use the other technics and methods for identification.
• Necessity of cooperation between laboratories of national authority and producers (in interpretation of raw data, support of analytical method, standards…)
• Necessity of clear legislation.
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CONCLUSION
THANK YOU FOR THE ATTENTION!
CENTRAL INSTITUTE FOR SUPERVISING AND TESTING IN AGRICULTURE
NATIONAL REFERENCE LABORATORYDepartment of Testing Plant Protection Product
Name: Olga Novákováe-mail: [email protected]: Department of Testing Plant Protection ProductAddress: Zemědělská 1a, 613 00 Brno, Czech Republicweb: www.ukzuz.cz