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European Reference Laboratory - FV
130th Annual Meeting & Exposition
130th Annual Meeting & Exposition
21st September 2016
European Reference Laboratory - FV
130th Annual Meeting & Exposition
OfLs OfLs
Main Objectives of the EURLs
European Reference Laboratory - FV
130th Annual Meeting & Exposition
EURL
EURL
EURL
EURL
NRL
NRL
NRL
NRL NRL
NRL
NRL
NRL
NRL
NRL
NRL NRL
NRL
NRL
NRL
NRL
NRL
NRL
NRL
NRL
OFL OFL OFL
OFL OFL
OFL
OFL
OFL
OFL
OFL
OFL
OFL
OFL
OFL
OFL
OFL OFL
OFL OFL
OFL
OFL
OFL OFL
OFL
OFL
OFL
OFL
OFL OFL
OFL
OFL
OFL
OFL
OFL
OFL
OFL OFL
OFL
OFL
OFL
OFL
OFL OFL
OFL OFL
OFL OFL
OFL
OFL
EURL
Network
European Reference Laboratory - FV
130th Annual Meeting & Exposition
Colaboration with – NON EU countries (18)
European Reference Laboratory - FV
130th Annual Meeting & Exposition
Organization of
Workshops
Specific trainings
and NRL visits
Technical Assistance
to the COM
Administrative
Duties
New method
Development and
Validation
Networking with NRLs
and Official Labs
Collaboration with Third
Countries’ Labs
Evaluation of new
analytical platforms
www.eurl-pesticides.eu
EURLs DUTIES/ACTIVITIES
European Reference Laboratory - FV
130th Annual Meeting & Exposition
Proficiency Tests - History
2004 2005 2006 2007 2008 2009
European Reference Laboratory - FV
130th Annual Meeting & Exposition
Proficiency Tests - History
2010 2011 2012 2013 2014
ISO/IEC 17043
2015
European Reference Laboratory - FV
130th Annual Meeting & Exposition
Acephate Difenoconazole Kresoxim-methyl Pirimicarb
Acetamiprid Diflubenzuron Lambda-Cyhalothrin Pirimphos-methyl
Acrinathrin Dimethoate Linuron Prochloraz
Aldicarb(sum) Diphenilamine Malathion Procydimone
Amitraz (DMPF&DMF) Dithiocarbamates(CS2) Mecarbam Propoxur
Azinphos-methyl Endosulfan Metalaxyl Propyzamide
Azoxystrobin EPN Metamidophos Prothiofos
Bifenthrin Fenamiphos Methidathion Pyraclostrobin
Boscalid Fenamiphos sulfone Methiocarb sum Pyridaben
Bromopropylate Fenamiphos sulfoxide Methiocarb Pyrimethanil
Bupirimate Ethion Methomyl Pyriproxifen
Buprofezin Fenhexamid Methoxyfenozide Quinoxyfen
Cadusafos Fenitrothion Methyl-Tiophanate Quintozene
Captan Fenpropathrin Mevinphos Spinosad
Carbaryl Fludioxinil Monocrotophos Spirodiclofen
Carbendazim Flufenoxuron Myclobutanil Tebuconazole
Carbofuran sum Fluopicolide Omethoate Tetraconazole
Chlorpropham Flutolanil Ortophenilphenol Thiabendazole
Chlorpyrifos Folpet Oxamyl Thiacloprid
Chlorpyrifos-methyl Fosthiazate Oxydemeton-methyl Tolclofos-methyl
Chlothalonil Hexythiazox Parathion Tolyfluanid
Cypermethrin Imazalil Penconazole Triadimenol
Cyprodinil Imidacloprid Pencycuron Triazophos
Delthamethrin Indoxacarb Pendimethalin Trifloxystrobin
Diazinon Iprodione Permethrin Triflumuron
Dichlofluanid Iprovalicarb Phosalone Vinclozolin
Dicofol Isofenphos-methyl Phosmet
Pesticides Used to Treat the Sample in last ten EUPT-FVs
183 compounds
European Reference Laboratory - FV
130th Annual Meeting & Exposition
Evolution of data dispersion along the EUPT-FV
history (Qn RSD, %)
25
European Reference Laboratory - FV
130th Annual Meeting & Exposition
Qualifying Results with
Uncertanity Measurements
U′ = 2 ∙ 𝑢
European Reference Laboratory - FV
130th Annual Meeting & Exposition
Revisions of the SANTE – AQC Document
1. Doc. 7826/VI/97 (1st EU AQC Meeting,1997, Portugal)
2. Doc. SANCO/3103/2000 (2nd EU AQC Meeting ,1999, Greece)
3. Doc. SANCO/10476/2003 (3rd EU AQC Meeting, 2003, UK)
4. Doc. SANCO/10232/2006 (4th EU AQC Meeting, 2005, Sweden)
5. Doc. SANCO/2007/3131 (2007 EURL Joint Workshop, Spain)
6. Doc. SANCO/10684/2009 (2009 EURL Joint Workshop, Denmark)
7. Doc. SANCO/12495/2011 (2011 EURL Joint Workshop, Germany)
8. Doc. SANCO/12571/2013 (2013 EURL Joint Workshop, Spain)
9. Doc. SANTE/11945/2015 (2015 EURL Joint Workshop, Germany)
www.eurl-pesticides.eu
NEXT 2017 !
European Reference Laboratory - FV
130th Annual Meeting & Exposition
EU Guidelines on Analytical
Quality Control and
Method Validation
Procedures for Pesticide
Residues Analysis in Food and Feed
Document Nº SANTE/11945/2015
2007 2009 2011
2013
2015
European Reference Laboratory - FV
130th Annual Meeting & Exposition
EU Guidelines on
Analytical Quality
Control and Method
Validation Procedures
for Pesticide Residues
Analysis in Food and Feed
Document Nº SANTE/11945/2015
European Reference Laboratory - FV
130th Annual Meeting & Exposition
Analytical Quality Control and Validation
Procedures for Pesticide Residues
Analysis in Food and Feed
AQC SANTE Guidance Document
Document SANTE/11945/2015
Revised by the AQC SANTE
Advisory Group - 2015
Evaluated by the Advisory Group and
then discussed and agreed by the
EU-National Reference Laboratories
European Reference Laboratory - FV
130th Annual Meeting & Exposition
Revisions of EU-SANTE – AQC Document
1. Doc. 7826/VI/97
9. Doc. SANTE/11945/2015 ----2017
• Harmonization with other international guidelines
• Updating criteria to include new technical developments
• Updating criteria to include the real labs experiences
• Improving the practical application of criteria
• Clarifying doubts and misinterpretations from labs
• Keeping the document as simple, practical and flexible as possible
• To support compliance with, and specific implementation of ISO/IEC
17025 (accreditation standard)
Reasons to justify the revisions
European Reference Laboratory - FV
130th Annual Meeting & Exposition
Structure of Document SANTE/11945/2015
A.- Introduction and legal background
B.- Sampling, transport, traceability and storage of samples
C.- Sample Analysis
D.- Identification of analytes and confirmation of results
E.- Reporting results
F.- Pesticide standards, stock and calibration solutions
G.- Analytical method validation and performance criteria
H.- Additional recommendations
Annex A and Appendices
Annex A (Commodity groups and representative matrices)
Appendix A (Method validation procedure: outline and example approaches)
Appendix B (Examples of conversion factors)
Appendix C (Examples for the estimation of measurement uncertainty)
Appendix D (Glossary)
Valid
atio
n
Qu
ality
Co
ntro
l
European Reference Laboratory - FV
130th Annual Meeting & Exposition
- High water content
- High acid content (and high water content)
- High sugar content (and low water content)
- High oil content (and very low water content)
- High oil content (and intermediate water content)
- High starch and/or protein content (low water & fat content)
- “Difficult or unique commodities”
VEGETABLES, FRUITS, and CEREALS (7 Groups)
Validation Commodity Groups for Representative Matrices
(Annex A)
Document N° SANTE/11945/2015
European Reference Laboratory - FV
130th Annual Meeting & Exposition
CG, LC-High Resolution
Accurate Mass Spectrometry
European Reference Laboratory - FV
130th Annual Meeting & Exposition
Linuron
Standard in solvent
Ion ratio: 1.8
Linuron
Real sample of coriander
Ion ratio: 2.4
FALSE NEGATIVE
LC-TQ-MS/MS
European Reference Laboratory - FV
130th Annual Meeting & Exposition
EUPT Sample of coriander analyzed by
LC-HRAMS
Linuron
Matrix interfetence
European Reference Laboratory - FV
130th Annual Meeting & Exposition
Laboratories analising by HRAMS
in the last three EUPTs-FV-SM
European Reference Laboratory - FV
53rd Annual North American Chemical Residue Workshop
HRAMs
No HRAMs Homogeneity: 0,085 mg/kg
+25%
-25%
-50%
50%
EU S-Proficiency Test
European Reference Laboratory - FV
130th Annual Meeting & Exposition
SENSITIVITY (10 µg/kg)
LINEARITY (2-3 orders)
+ REPRODUCIBILITY
(20%)
LC, GC-HRAMS performance requirements
Quant.
European Reference Laboratory - FV
130th Annual Meeting & Exposition
MASS ACCURACY (5 ppm)
RESOLUTION (¿?)
𝟐𝟎𝟎. 𝟎𝟎𝟏 − 𝟐𝟎𝟎. 𝟎𝟎𝟎
𝟐𝟎𝟎. 𝟎𝟎𝟎=
𝟎. 𝟎𝟎𝟏
𝟐𝟎𝟎. 𝟎𝟎𝟎= 𝟓 ∙ 𝟏𝟎−𝟔
Qual.
European Reference Laboratory - FV
130th Annual Meeting & Exposition
Ethirimol 0.1 mg/kg in tomato, MS2 spectrum
ppm or mDa?
European Reference Laboratory - FV
130th Annual Meeting & Exposition
0
20
40
60
80
0.1 0.01 0.005 0.001 0.0005
495 49 25 5 2 (Da)
(ppm)
Thiabendazole
C10H7N3S (m/z 202.0433)
C0-25H0-50N0-5O0-5Cl0-5Br0-5S0-5
Nu
mb
er
of
po
ssib
le m
ole
cula
r fo
rmu
las
5 ppm
~ 2 000 ~ 20 000 ~ 40 000 ~ 200 000 ~ 400 000 (FWHM) Resolution
Resolution ~ 70 000 FWHM
9 molecular formulas
5 molecular formulas
- Cl, Br
~ 70 000 ~25 000
Absolute mass error relative to Thiabendazole
Resolution ~ 25 000 FWHM
25 molecular formulas
12 molecular formulas
- Cl, Br
RESOLUTION (¿?)
European Reference Laboratory - FV
130th Annual Meeting & Exposition
MS detector/ characteristics
Typical
systems (examples)
Acquisition
Requirements for identification
minimum number of ions other
Unit mass resolution
quadrupole,
ion trap, TOF full scan, limited m/z range, SIM 3 ions
S/N ≥ 3e)
Analyte peaks in
the extracted ion
chromatograms
must fully overlap.
Ion ratio within
±30% (relative)
of average
of calibration
standards from same sequence
MS/MS
triple
quadrupole,
ion trap,
Q-trap,
Q-TOF, Q-Orbitrap
selected or multiple reaction
monitoring (SRM, MRM), mass
resolution for precursor-ion
isolation equal to or better than unit mass resolution
2 product ions
Accurate mass
measurement
High resolution
MS: (Q-)TOF
(Q-)Orbitrap
FT-ICR-MS
sector MS
full scan, limited m/z range, SIM,
fragmentation with or without
precursor-ion selection, or combinations thereof
2 ions with
mass accuracy
≤ 5 ppma,b, c)
combined single stage MS and
MS/MS with mass resolution for
precursor-ion isolation equal to or better than unit mass resolution
2 ions:
1 molecular ion,
(de)protonated molecule
or adduct ion with mass acc. ≤ 5 ppma,c
plus
1 MS/MS product iond)
a) preferably including the molecular ion, (de)protonated molecule or adduct ion
b) including at least one fragment ion
c) < 1 mDa for m/z < 200
d) no specific requirement for mass accuracy
e) in case noise is absent, a signal should be present in at least 5 subsequent scans
Conditions for Identification
By using Target List (with retention time)
•Table 4 in Document Nº SANTE/2015/11945 . Identification criteria for different MS techniques
European Reference Laboratory - FV
130th Annual Meeting & Exposition
MS mode MS1 MS/MS MS1 and MS/MS
EIC: signal requirement
(absolute)
A criterion to be set by one of the following methods:
(1) a S/N threshold ≥ 3;
(2) an intensity ratio relative to the comparison standard
equal or above a preset threshold
EIC: retention time,
relative to comparison
standard
A criterion to be set by one of the following methods:
(1) ≤ 0.2 min, or
(2) within ±2.5%, not to exceed 0.5 min, or
(3) within an established error range, not to exceed 0.5 min
MS: number of structurally
significant ions Minimum 2 Minimum 2
Minimum 2
combined
MS: mass accuracy ≤ 5 ppm ≤ 10 ppm MS1: ≤ 5 ppm;
MS/MS: ≤ 10 ppm
Table 1. Summarized Requirements for Confirmation of Identity
FDA Acceptance Criteria for Confirmation
of Identity of Chemical Residues using Exact Mass Data
European Reference Laboratory - FV
130th Annual Meeting & Exposition
64%
30%
5% 1%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0-0.2 mDa 0.21-0.5 mDa 0.51-1 mDa >1 mDa
Errors in full scan MS (n = 7444)
45% 38%
16%
1% 0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0-0.2 mDa 0.21-0.5 mDa 0.51-1 mDa >1 mDa
Errors in ddMS2 (n = 30623)
Is it necessary 10 ppm
in MS2?
European Reference Laboratory - FV
130th Annual Meeting & Exposition
Full scan MS 20-70k
GC-EI-HRAMS
Real Samples: Fluopyram
Orbitrap GC-EI-MS System
50 µg/kg Std in Tomato Real Sample: Pear
C = 26 µg/kg
Ratio1: 50%
Ratio1: 30%
Ratio1: 35%
Ratio1: 52%
Ratio1: 29%
Ratio1: 34%
The ratios are within ± 4% with
respect to the standard and
mass accuracy below 0.2 mDa
Ion 145.0260: 0.14 mDa
Ion 195.0057: -0.08 mDa
Ion 223.0244: 0.02 mDa
Ion 173.0209: 0.01 mDa
European Reference Laboratory - FV
130th Annual Meeting & Exposition
20-70k 20-70 k
TARGET ANALYSIS (DATA DEPENDENT ACQUISITION)
Full scan MS MS2
Time Time
20-70k
NON TARGET MS2 (DATA INDEPENDEN ACQUISITION)
20-70 k
LC-ESI-HRAMS Is it necessary ion ratio within 30%?
Is it achievable when MS and MS2
Is selected?
European Reference Laboratory - FV
130th Annual Meeting & Exposition
80%
85%
90%
95%
100%garlic
onion
spinach
green bean
lettuce
appleorange
cucumber
parsley
leek
tomato
0.1 mg/kg 0.01 mg/kg
80%82%84%86%88%90%92%94%96%98%
100%garlic
onion
spinach
green bean
lettuce
appleorange
cucumber
parsley
leek
tomato
0.1 mg/kg 0.01 mg/kg
0.1 mg/kg without ion ratio 0.01 mg/kg without ion ratio
a) b)
Percentage of identified compounds with:
a) dd MS2 [one fragment ion with mass error <5 ppm]
b) AIF [one fragment ion with mass error <5 ppm and variation of ion ration < 30%]
Ion ratio + 30% Ion ratio+ 30%
European Reference Laboratory - FV
130th Annual Meeting & Exposition
Fragment ion Abundance in solvent Abundance in leek
114.9615 39% 43%
142.9386 100% 100%
170.9699 82% 74%
199.0013 29% 26%
Ethion
Full scan
384.9949 ± 5 ppm
ddMS2
384.9949 ± 0.5 Da
Solvent Leek
10,66 10,66
114.9615
142.9386
113.0423
170.9699
199.0013
142.9384
170.9696
199,0008
Dd MS2
(two fragment ions with mass error < 5 ppm)
European Reference Laboratory - FV
130th Annual Meeting & Exposition
C:\Xcalibur\...\Puerro_10ppb_AIF 09/13/16 14:04:11
RT: 9.06 - 10.21
9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8 9.9 10.0 10.1 10.2
Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
9.50
9.51
9.49
9.529.48
9.539.47 9.549.46
9.569.45 9.589.43
NL:
0
m/z=
152.5253-
152.5268 MS
Puerro_10ppb_
AIF
NL:
1.46E6
Base Peak
m/z=
153.1012-
153.1028 MS
Puerro_10ppb_
AIF
Puerro_10ppb_AIF #1808 RT: 9.52 AV: 1 NL: 6.58E7
T: FTMS + p ESI Full ms2 560.0000@hcd30.00 [66.7000-1000.0000]
100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200
m/z
0
1
2
3
4
5
6
7
8
Re
lative
Ab
un
da
nce
107.0860
194.9968
147.0298142.9985
177.1123
136.9549119.0858
149.0234
121.1015163.0247
185.0067123.0807
186.9713169.0795 199.1254138.9706 179.0019153.0346
188.9683159.1170
165.0404 173.1326
128.0623
193.9889184.0733
196.0002
Diazinon (305.1083) MS2 spectrum Retention time 9.48 ± 0.1 min Precursor selection Quadrupole window width 1 Da
Diazinon MS2 spectrum No precursor selection Quadrupole window width 934 Da (66 – 1000 Da)
Puerro_10ppb_EC_opt_012min 09/13/16 00:34:23
RT: 9.05 - 10.19
9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8 9.9 10.0 10.1
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
9.48 9.50
9.47
9.51
9.52
9.529.45
9.53
9.53
9.559.43
9.429.57 9.57
9.41 9.63 9.68
NL:
3.92E6
Base Peak m/z=
305.1068-
305.1098 MS
Puerro_10ppb_
EC_opt_012min
Puerro_10ppb_EC_opt_012min #2085 RT: 9.53 AV: 1 NL: 3.35E5
T: FTMS + p ESI d Full ms2 305.1082@hcd30.00 [50.0000-330.0000]
100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200
m/z
0
1
2
3
4
5
6
7
8
Re
lative
Ab
un
da
nce
105.0700
131.0856107.0858112.0344 119.0855
145.1008121.1012
109.0649 135.0801
157.1009143.0854123.0804
149.0958
129.0696 161.0956
124.9821
101.0426163.1118 171.1171138.0107
114.9614 153.1020
169.0791
114.9617
153.1024
169.0795
Puerro_10ppb_EC_opt_012min 09/13/16 00:34:23
RT: 9.05 - 10.19
9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8 9.9 10.0 10.1
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
9.48 9.50
9.47
9.51
9.52
9.529.45
9.53
9.53
9.559.43
9.429.57 9.57
9.41 9.63 9.68
NL:
3.92E6
Base Peak m/z=
305.1068-
305.1098 MS
Puerro_10ppb_
EC_opt_012min
Puerro_10ppb_EC_opt_012min #2085 RT: 9.53 AV: 1 NL: 1.96E5
T: FTMS + p ESI d Full ms2 305.1082@hcd30.00 [50.0000-330.0000]
153.00 153.02 153.04 153.06 153.08 153.10 153.12 153.14 153.16 153.18 153.20
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
153.1020
C:\Xcalibur\...\Puerro_10ppb_AIF 09/13/16 14:04:11
RT: 9.06 - 10.21
9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8 9.9 10.0 10.1 10.2
Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
9.50
9.519.4910.14
9.52 10.13 10.1510.029.489.53 10.0710.009.93 10.169.469.22 9.26 9.299.07 9.929.439.17 9.659.309.13 9.769.33 9.949.899.56 9.989.70 9.779.37 9.659.58 9.79 9.829.71
9.66
9.50
9.51
9.49
9.529.48
9.539.47 9.549.46
9.569.45 9.589.43
NL:
3.52E6
m/z=
168.2896-
169.9762 MS
Puerro_10ppb_
AIF
NL:
1.46E6
Base Peak
m/z=
153.1012-
153.1028 MS
Puerro_10ppb_
AIF
Puerro_10ppb_AIF #1808 RT: 9.52 AV: 1 NL: 1.03E6
T: FTMS + p ESI Full ms2 560.0000@hcd30.00 [66.7000-1000.0000]
153.00 153.02 153.04 153.06 153.08 153.10 153.12 153.14 153.16 153.18 153.20
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
153.0346
153.0548
153.1024
153.0733
153.0912
153.1276
153.0701
Not necessary
80
168.90 168.92 168.94 168.96 168.98 169.00 169.02 169.04 169.06 169.08 169.10 169.12 169.14 169.16 169.18 169.20
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
169.0791
Puerro_10ppb_EC_opt_012min 09/13/16 00:34:23
RT: 0.00 - 12.11
0 1 2 3 4 5 6 7 8 9 10 11 12
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
NL: 1.43E6
TIC F: FTMS + p ESI d
Full ms2
305.1082@hcd30.00
[50.0000-330.0000] MS
Puerro_10ppb_EC_opt_0
12min
Puerro_10ppb_EC_opt_012min #2085 RT: 9.53 AV: 1 NL: 2.10E5
F: FTMS + p ESI d Full ms2 305.1082@hcd30.00 [50.0000-330.0000]
114.70 114.75 114.80 114.85 114.90 114.95 115.00 115.05 115.10 115.15 115.20
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
114.9614
115.0379
114.9614
Not necessary
Not necessary
European Reference Laboratory - FV
130th Annual Meeting & Exposition
INFORMATION + INTERACTION + HARMONIZATION
European Reference Laboratory - FV
130th Annual Meeting & Exposition
INFORMATION + INTERACTION + HARMONIZATION
PT
AQC
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