AFSSA-Fougères LERMVD

French National and E.U. Community

Reference Laboratory for Veterinary Drug
Antimicrobial Residue Control in Food

WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS

TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION
AND RELIABLE QUANTIFICATION
OF RESIDUES MONITORED IN BIOLOGICAL MATRICES ?
WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

What kind of residue

in biological matrices ?

Veterinary Drug
Residue
At AFSSA-Fougères

2 ERIC VERDON SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007

WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

2 main regulations
FR Décret 2006/7 - E.U. Directive 96/23/EC

• National Reference Laboratory

– All Veterinary Drug Residues except for group A1 to A5
(stilbenes, anti-thyroid agents, steroids, resorcyclic acid lactones, β-agonists)

• Community Reference Laboratory

– B1 : Antimicrobials including sulphonamides and quinolones
– A6 : Chloramphenicol, Nitrofurans, Dapsone
– B3e : Dyes (Malachite green)

• All foods from animal origin

3 ERIC VERDON SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007

WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

What kind of methods

in biological matrices ?

Monitoring : Screen & Confirm

4 ERIC VERDON SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007

WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

Main Objectives for Analytical Methods in Residue Testing

• Screening
– Large sieving and low cost analysis
– Detection with a low rate of false negative
– Microbiological, Immunological, Physico-chemical methods

• Confirmation
– Unequivocal Identification
– Mass Spectrometry compulsory for non authorized substances (several signals)
– Other physico-chemical detections accepted for authorized substances

• Quantification (confirmation)
– Accuracy of measurement
– Comparison with MRL (Maximum Residue Limit - Regul. 2377/90/EC)

5 ERIC VERDON SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007

WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

What performance for

vet drug residue methods
in biological matrices ?

6 ERIC VERDON SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007

WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

Performance characteristics of analytical methods

• Validation of methods = fit for the purpose

= In-house validation according to criteria of Decision 657/2002/EC

• Screening methods (Microbiological, Immunological, Physico-chemical)

• False negative rate < 5 %

• Confirmatory methods (Physico-chemical)

• False positive rate < 1 % (non authorized subst.) and < 5 % (MRL authorized subst.)
• UV & FLD criteria for identification (RT, UV spectra, Std comparison)
• MS : Identification points from Ion earning (3: authorized subst.; 4: non authorized subst.)

• Quantitative methods
• Specified Accuracy objectives (Trueness-bias and Precision-variable error)

7 ERIC VERDON SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007

WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

What detection limits for

residue methods
in biological matrices ?

8 ERIC VERDON SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007

WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

ANALYTICAL SIGNAL and ANALYTICAL LIMITS

XIC of +MRM (10 pairs): 189.0/102.0 amu from Sample 3 (003-sup3(foie)) of 031127.wiff (Turbo... Max. 1.2e4 cps.

6.66
1.20e4

1.10e4
Area:6.059e+4

1.00e4

9000.00

8000.00

7000.00

6000.00

5000.00

4000.00

3000.00

2000.00 4.80 6.28

3.61
3.87
3.17
1000.00 8.26

0.00
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
Time, min

Signal
X I C o f + M R M ( 1 0 p a i r s ) : 1 8 9 . 0 / 1 0 2 . 0 a m u f r o m S a m p l e 1 0 ( 0 1 0 - b l c f o i e 3 ) o f 0 3 1 1 2 7 . w i f f ( T u r b . . . M a x . 1 3 1 0 . 0 c p s .

6 . 2 9
1 3 0 0

1 2 0 0

4 . 7 8
1 1 0 0

1 0 0 0

9 0 0 5 . 9 0

8 0 0
6 . 4 2
7 0 0 4 . 9 3 8 . 5 5
3 . 5 8 4 . 4 0 8 . 3 1

7 . 2 8 7 . 8 9 8 . 6 2
6 0 0

3 . 1 2
5 0 0

4 0 0

3 0 0

2 0 0

1 0 0

0
0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5 5 . 0 5 . 5 6 . 0 6 . 5 7 . 0 7 . 5 8 . 0 8 . 5 9 . 0 9 . 5
T i m e , m i n

Noise or Signal ?

9 ERIC VERDON SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007

 ANALYTICAL LIMITS

A QUESTION of SIGNAL and NOISE

WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

SIGNAL and NOISE

• Case 1 • Case 2
Analytical Variations recorded Signal close to Noise Signal >> Noise
from the signal in biological samples

- linked to noise and Significant Not significant

to matrix interferences
X I C

1
3

0
o

0
f

0
+ M R M ( 1 0 p a i r s ) : 1 8 9 . 0 / 1 0 2 . 0 a m u f r o m S a m p l e 1 0 ( 0 1 0 - b l c f o i e

4
3

. 7
)

8
o f 0 3 1 1 2 7 . w i f f ( T

6
u r b

. 2 9
. . . M a x . 1 3 1 0 . 0 c p s .

9 0 0
5 . 9 0

8 0 0
6 . 4 2
7 0 0 4 . 9 3 8 . 5 5
3 . 5 8 4 . 4 0 8 . 3 1

6 0 0 7 . 2 8 7 . 8 9 8 . 6 2

3 . 1 2
5 0 0

4 0 0

3 0 0

2 0 0

1 0 0

0
0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5 5 . 0 5 . 5 6 . 0 6 . 5 7 . 0 7 . 5 8 . 0 8 . 5 9 . 0 9 . 5
T i m e , m i n

- linked to signal and XIC of +MRM (10 pairs): 189.0/102.0 amu from Sample 3 (003-sup3(foie)) of 031127.wiff (Turbo... Max. 1.2e4 cps.

Not significant Significant

to calibration
6.66
1.20e4

1.10e4
Area:6.059e+4

1.00e4

9000.00

8000.00

7000.00

6000.00

5000.00

4000.00

3000.00

2000.00 4.80 6.28

3.61
3.87
3.17
1000.00 8.26

0.00
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
Time, min

ANALYTICAL
Consider the noise at concentration Consider the signal variations at
LIMITS of interest close to analytical zero the concentration of interest far from
level analytical zero level

11 ERIC VERDON SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007

WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

SIGNAL and NOISE

• Case 1 → Signal close to Noise

– 3 < S/N < 6-10 (LOD and LOQ)

• Analytical methods for non authorized substances (Group A)

• Analytical methods for authorized substances (Group B) with signal at the

regulated limits nearing the noise on the analytical instrument

• Case 2 → Signal >> Noise

– S/N > 6-10 and often S/N >>> 6-10 (>>> LOQ)

• Analytical methods for authorized substances (Group B) with signal at the

regulated limits pretty far from the noise on the analytical instrument

12 ERIC VERDON SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007

WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

HOW WE CALCULATE ACCORDING TO 657/2002/EC

METHOD 1 METHOD 2
NOISE approach SIGNAL CALIBRATION approach
20 samples from blank matrices : Sets of matrix calibrants (around MRL or MRPL):
. Samples from independant batches, . Calibrations from independant batches,
. Representative of the field of application of the method. . Representative of the field of application of the method.

• Non authorized Substances MRPL / Zero Level • Authorized Substances MRL

Calculate variability from the analytical Noise Calculate variability of analytical Signal
over the 20 blank samples at the MRL by analysis of the regression
and estimate the critical concentration (Standard ISO11843)
achievable above the Zero Level and estimate the critical concentration
(below the MRPL) achievable above the MRL

• Authorized Substances MRL • Non authorized Substances MRPL/Zero Level

Calculate variability of the analytical Signal Calculate variability of analytical Signal
over the 20 blank samples at MRPL level by analysis of the regression
fortified at the MRL (Standard ISO11843)
and estimate the critical concentration and estimate the critical concentration
achievable above the MRL achievable above the Zero Level
(below the MRPL)
13 ERIC VERDON SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007
WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

CCα/CCβ calculation and decision making

• CC alpha
= limit of decision for confirmatory analysis

• CC beta
= capacity of detection for screening analysis

14 ERIC VERDON SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007

WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

CCα/CCβ calculation and decision making

Screening Methods Confirmatory Methods

Non authorized substances Non authorized substances
(S/N < 6-10) (S/N < 6-10)
CCβscreening : Statistically calculated from “critical” signal for the most sensitive CCα confirmation : Statistically calculated from “critical” signal for the less sensitive (identification)
(detection, screening) and the most variable

Compliant Suspect Compliant Non Compliant

Result of Confirmation Result of

Screening measurement measurement
CCβ screening CCαconfirmation
0.0 MRPL 0.0 MRPL
or or
UNCERTAINTY OF MEASUREMENT
WAL WAL
In practice CCβ minimal : ½ MRPL

Authorized MRL Substances Authorized MRL Substances

(S/N >> 6-10) (S/N >> 6-10)
CCβscreening : Statistically calculated from “critical” signal for the most sensitive and below the MRL CCα confirmation : Statistically calculated from “critical” signal for the less sensitive (identification)
(detection, screening) and the most variable

Compliant Suspect Compliant Non Compliant

Screening Result of Confirmation Result of

CCβ screening measurement measurement
0.0 MRL CCαconfirmation
0.0
MRL
In practice CCβ minimal ½ MRL UNCERTAINTY OF MEASUREMENT

15 ERIC VERDON SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007

WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

Exemple : Malachite green residue analysis by LC-MSMS

(Confirmation)

• Malachite green: non-authorized substance in food

• Marker Residues in fish products :

malachite green (MG) + its leucobase (LMG)
(easily metabolized) (persistant)

H3C CH3 H 3C CH 3
N + N

CH
C

CH 3
CH3
N
N

CH 3
CH3
Malachite green
Leucomalachite green

16 ERIC VERDON SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007

WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

Exemple : Malachite green residue analysis by LC-MSMS (Confirmation)

• Minimum Required Performance Limit

for monitoring labs
• Decision 2004/25/EC : EU-MRPL set at 2 µg/kg

• LC-MSMS = 4 MRM Signals

– MRM tr1 for MG + MRM tr1 for LMG
– MRM tr2 for MG + MRM tr2 for LMG

17 ERIC VERDON SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007

WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

Exemple : Malachite green residue analysis by LC-MSMS (Confirmation)

MG LMG

MRM tr 1

MRM tr 2

18 ERIC VERDON SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007

WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

Exemple : Malachite green residue analysis by LC-MSMS (Confirmation)

LMG

MRM tr 2

19 ERIC VERDON SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007

WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

Exemple : Malachite green residue analysis by LC-MSMS (Confirmation)

20 ERIC VERDON SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007

WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

Response (Area ratio)

3.0
y = 0.9596x - 0.0256
Exemple : Malachite green residue analysis byR LC-MSMS
= 0.9984 (Confirmation) 2.5 2
2.0
1.5
1.0
0.5
0.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Conc (µg/kg)

21 ERIC VERDON SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007

WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

Exemple : Malachite green residue analysis by LC-MSMS

(Confirmation)
0,8

0,4

yc
0,0
0,0 Xc: 0.12 0,5 1,0

0,8

0,4

0,0
0,0 0,5 1,0

22 ERIC VERDON SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007

WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

Exemple : Malachite green residue analysis by LC-MSMS (Confirmation)

Screening Methods Confirmatory Methods

Non authorized substances Non authorized substances
(S/N < 6-10) (S/N < 6-10)
CCβscreening : Statistically calculated from “critical” signal for the most sensitive CCα confirmation : Statistically calculated from “critical” signal for the less sensitive (identification)
(detection, screening) and the most variable

Compliant Suspect Compliant Non Compliant

Result of Confirmation Result of

Screening measurement measurement
CCβ screening CCαconfirmation
MRPL MRPL
or or
WAL WAL

Leuco-Malachite green LMG1 Leuco-Malachite green LMG2

CC alpha = 0.09 µg/kg CC alpha = 0.12 µg/kg

CC beta = 0.12 µg/kg CC beta = 0.15 µg/kg

23 ERIC VERDON SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007

WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

Exemple : Nitrofuran metabolites analysis by LC-MSMS (Confirmation)

Confirmatory monitoring of 4 analytes for 12 series over several months :

nitrofuran metabolites in chicken muscle tissues

Control chart displaying CCa variations (MRM tr2)

Extension matrice muscle de volaille - Relevé des CCa et CCb AHD MRM tr2
AOZ MRM tr2
1.50 SEM MRM tr2
AMOZ MRM tr2
Concentrations µg/kg

1.00

LPMR

0.50
1/2 LPMR

0.00
0 1 2 3 4 5 6 7 8 9 10 11 12 13

Séries

24 ERIC VERDON SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007

WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

CONCLUSION

Analytical limits :

• Calculation during routine confirmatory analysis = variable parameter

• Setting borders between compliant and non compliant results at specified levels
(MRL, MRPL)

• Checking performance especially for confirmatory purpose

• Calculated as a critical concentration (CCalpha)

statistical evaluation of the uncertainty of measurement

25 ERIC VERDON SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007

WHAT CRITICAL LIMIT FOR ANALYTICAL METHODS TO GIVE CONFIDENCE IN UNEQUIVOCAL IDENTIFICATION AND RELIABLE QUANTIFICATION OF RESIDUES MONITORED IN BIOLOGICAL MATRICES?

Thank you
For Your Attention
AFSSA-Fougères LERMVD
French National and E.U. Community
Reference Laboratory for Veterinary Drug
26 ERIC VERDON
Antimicrobial Residue Control in Food
SASKVAL07 – SASKATOON – SASKATCHEWAN – CANADA – 12 JUNE 2007