1. Is My Method Fit For Purpose ?
Rebecca Moffat
Marine Institute
27th Oct 2016

2. Determining if validation data meets acceptance criteria
For Commission Decision 2002/657/EC

3. Contents Marine Institute Documents of Reference
Validation under 2002/657/EC
Parameters Commonly Evaluated in Validation
Confirming Positive Sample Criteria
Validation Data For Dyes Analysis by LC-MS/MS
Conclusion

4. Marine Institute

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Seafood Safety – underpin consumer protection and support market
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8. Quality System Marine Institute Accredited to ISO17025 since 2002.
35 Accredited methods – 4 ext to scope in 2017.
Methods Accredited in multidisciplinary areas - Chemistry, (residues, metals, organics), biotoxins, fish diseases, microbiology, phytoplankton, molecular.
MI also holds ISO9001 Certification in the Fish Health Competent Authority Unit.

9. Residues Programme for Aquaculture
MI carries out sampling and analysis in accordance with NRCP
3 categories of Residues
Banned – Group A compounds
A3 Steroids: Methlytestosterone
Authorized – Group B compounds
B2(c) Carbamates/Pyrethroids: Cypermethrin
Unauthorized – Group B compounds
B3(e) Dyes: Malachite Green

10. Documents of Reference
Commission Decision 2002/657/EC
Eurachem: Fitness for Purpose of Analytical Methods
INAB PS15 – Guide to Method validation for Quantitative Analysis in Chemical Testing Laboratories (ISO17025)

11. Fit for Purpose: Method Validation
Defining an analytical requirement and
Confirming that the method has capabilities consistent with the application
Enables chemists to demonstrate that a method is fit for purpose

12. Parameters commonly evaluated during method validation
Performance Characteristics:
Selectivity
Working Range
Analytical Sensitivity
Trueness
Precision
Ruggedness
LOD/LOQ
CCα and CCβ
For 2002/657/EC

13. Analysis of Dyes by LCMSMS
Confirmatory method
Based on EU-RL method for Dyes
Dyes analysed include:
BG, CV, LCV, LMG, MG and VB
Awarded INAB accreditation in 2014

14. Validation under 2002/657/EC
Residues Directive Commission Decision 2002/657/EC states:
Confirmatory methods shall provide information in the chemical structure of the analyte and consequently methods based only on chromatographic analysis without the use of spectrometric detection are not suitable on their own for use as a confirmatory method

15. Suitable Confirmatory Methods for Organic Residues or Contaminants
Measuring Technique
Substance Annex 1 96/23/EC
Limitations
LC or GC with mass-spectrometric detection
Group A and Group B
Only if following either an on-line or an off-line chromatographic separation
Only if full scan techniques are used or using at least 3 (Group B) and 4 (Group A) identification points for the techniques that do not record the full mass spectra
LC or GC with IR spectrometric detection
Specific requirements for absorption in IR spectrometry have to be met
LC-full-scan DAD
Group B
Specific requirements for absorption in UV spectrometry have to be met
LC-fluorescence
Only for molecules that exhibit native fluorescence and to molecules that exhibit fluorescence after either transformation or derivatisation
2-D TLC - full scam UV/VIS
Two-dimensional HPTLC and co-chromatography are mandatory
GC-Elektron capture detection
Only if two columns of different polarity are used
LC-immunogram
Only if at least two different chromatographic systems or a second, independent detection method are used
LC-UV/VIS (single wave length)

16. Common Performance Criteria and Requirements
Internal Standards:
Where used in the method, a suitable internal standard shall be added to the test portion at the beginning of the extraction procedure.
Depending of availability, either stable isotope-labelled forms of the analyte, or compounds that are structurally related to the analyte shall be used.
Deuterated Dyes

17. Confirming Positive sample acceptance criteria: MS/MS tolerance
Criteria for confirming: MS/MS tolerance ion ratio:
Relative intensities of the detected ions, shall correspond to those of the calibration standard, at comparable concentrations, measured under the same conditions within the following tolerances:

18. Dyes Confirmation Sheet Part 1

19. Confirming Positive Sample Criteria: RRT
Criteria for confirming: Relative Retention Time:
The retention time (or relative retention time) of the analyte in the test portion shall match that of the calibration standard within a specified retention window
The ratio of the chromatographic RT of the analyte to the Internal Standard, shall correspond to that of the calibration solution at a tolerance of ±0.5% for GC and ±2.5% for LC

20. Dyes Confirmation Sheet Part 2

21. Classification of analytical methods by the performance characteristics that have to be determined
Detection limit
CCβ
Decision limit
CCα
Trueness/
Recovery
Precision
Selectivity/
Specificity
Applicability/Ruggedness/stability
Qualitative methods S + - C
Quantitative methods
S= screening methods, C= confirmatory methods, += determination in mandatory

22. Specificity Specificity: Determined by
Ability of a method to distinguish between the analyte being measured and other substances
Determined by
Analysing blank representative samples and showing no interfering peaks in mass range window
Analysing each standard individually

23. Specificity LC-MS/MS offers high degree of selectivity and specificity
Small interfering peaks were observed at RT for some analytes in chromatograms of non-fortified samples
Areas so low to be of little consequence
Quantification of each analyte not influeneced notably by levels detected in negative controls

24. Specificity Blanks - BG

25. Specificity Standards - BG

26. Working Range Linearity of matrix matched calibration curve
Upper Linear Range
Saturation Point and method range

27. Matrix matched curve
Fortify negative control salmon tissue across range of concentrations and bring through extraction procedure
Matrix can have effect on the signal produced (suppression or enhancement)
MM calibration curve used to ensure these effects are accounted for in quantification of unknowns

28. Calibration Standard Levels
~ Amount (ng analyte/g tissue)
~MRPL level 7 6
0.5
0.25 5 1 4 2 3
3.5
1.75
2.5
7.5
3.75 10

29. Example CV extracted calibration curve

30. Upper Linear Range Matrix matched Calibration Curves
Calibration standards concentrations range:
0µg/kg – 15µg/kg
Most suitable range for all analyes:
0µg/kg – 10µg/kg
Lowest standard with analyte present 0.5µg/kg
0.25*MRPL

31. Saturation point and method range
Saturation point examined in triplicate at 5 and 7.5 * MRPL for all dyes
Equivalent to 10µg/kg and 15µg/kg
Acceptable recoveries achievable for 4/6 analytes at 15µg/kg
Acceptable recoveries achievable for 6/6 analytes at 10µg/kg
Method range: 0.5µg/kg – 10µg/kg (15µg/kg)
CV, MG, LCV, LMG
BG, CV, MG, VB, LCV, LMG

32. Recovery/Trueness 2002/657/EC
The inter-laboratory CV for the repeated analysis of a reference or fortified material under reproducibility conditions, shall not exceed the level in table below:
Mass Fraction
Reproducibility CV (%)
1µg/kg
(*)
10µg/kg
100µg/kg 23
1000µg/kg 16
* For mass fraction lower than 100g/kg the Horwitz equation gives unacceptable high vales. Therefore the CV’s for concentrations lower than 100µg/kg shall be as low as possible

33. Trueness
When no CRMS are available it is acceptable that the trueness measurements are assessed through the recovery of additions of known amounts of analyte to a blank matrix.
Data corrected with the mean recovery are only acceptable when they fulfil the following:

34. Recovery/Trueness data
BG1
BG2
CV1
CV2
MG1
MG2
Mean
98.55
101.65
102.97
99.60
102.22
Std Dev
13.11
13.73
4.49
6.01
7.63
7.32
%CV
13.30
13.51
4.36
5.91
7.66
7.16
VB1
VB2
LCV1
LCV2
LMG1
LMG2
101.45
106.62
106.89
106.25
104.61
104.15
12.30
14.12
9.80
12.73
5.97
6.84
12.13
13.25
9.17
11.98
5.71
6.57

35. Precision Falls under 2 categories Within Batch Repeatability
Within Lab Reproducability

36. Within Batch Repeatability
Prepare a set f samples of identical matrices, fortified with the analyte to yield concentrations equivalent to 1, 1.5 and 2 times MRPL
Each level analysed with 7 replicates

37. Results of Repeatability 1*MRPL
BG1
BG2
CV1
CV2
MG1
MG2
Mean
97.67
99.87
103.02
101.39
102.30
104.12
Standard Dev
11.56
13.54
4.31
6.44
6.94
7.80
%CV
11.84
13.55
4.18
6.35
6.78
7.49
VB1
VB2
LCV1
LCV2
LMG1
LMG2
98.88
104.00
108.69
109.74
105.51
105.54
13.64
16.36
10.27
13.09
6.66
7.48
13.80
15.74
9.45
11.93
6.31
7.09

38. Results of Repeatability 1.5* MRPL
BG1
BG2
CV1
CV2
MG1
MG2
Mean
118.26
114.03
103.28
103.40
117.87
119.39
Standard Dev
15.84
14.28
5.33
6.70
12.17
10.65
%CV
13.39
12.52
5.16
6.48
10.33
8.92
VB1
VB2
LCV1
LCV2
LMG1
LMG2
103.42
103.69
102.31
102.01
101.53
102.26
22.08
23.63
9.14
8.84
4.93
5.75
21.35
22.79
8.93
8.67
4.86
5.63

39. Results of Repeatability 2*MRPL
BG1
BG2
CV1
CV2
MG1
MG2
Mean
118.31
113.87
103.82
104.15
113.64
116.35
Standard Dev
24.97
22.38
7.25
5.56
12.44
11.63
%CV
21.10
19.66
6.99
5.34
10.94
9.99
VB1
VB2
LCV1
LCV2
LMG1
LMG2
107.34
107.70
108.20
108.70
101.24
101.51
22.18
22.37
8.71
9.66
6.69
7.84
20.66
20.77
8.08
8.88
6.61
7.72

40. Within Lab Reproducability
Samples spiked at approx 1, 1.5 and 2*MRPL were analysed on 3 occasions by 2 analysts
Analyst A – twice using different batches of reagents and solvents
Analyst B

41. Within Lab Reproducibility 1*MRPL
BG1
BG2
CV1
CV2
MG1
MG2
Mean
100.65
104.02
102.18
99.19
100.72
100.84
Standard Deviation
10.43
11.87
3.96
4.31
5.67
6.85
%CV
10.37
11.41
3.88
4.35
5.92
6.80
VB1
VB2
LCV1
LCV2
LMG1
LMG2
99.35
104.71
105.89
107.33
102.37
102.59
8.98
9.62
9.64
12.96
3.68
4.54
9.04
9.19
9.11
12.08
3.60
4.43
Within lab reproducibility results at approximately 100%MRPL (2µg/kg)

42. Within Lab Reproducibility 1.5*MRPL
BG1
BG2
CV1
CV2
MG1
MG2
Mean
117.42
112.54
102.63
101.01
119.39
118.70
Standard Deviation
18.26
17.53
4.65
4.10
11.34
%CV
15.55
15.57
4.53
4.06
9.50
9.55
VB1
VB2
LCV1
LCV2
LMG1
LMG2
100.10
100.47
99.73
100.32
100.09
20.75
23.01
7.03
7.13
5.11
5.79
20.73
22.90
7.05
7.11
5.76
Within lab reproducibility results at approximately 150%MRPL (3µg/kg)

43. Within Lab Reproducibility 2*MRPL
BG1
BG2
CV1
CV2
MG1
MG2
Mean
121.97
119.24
104.99
104.98
122.45
124.27
Standard Deviation
24.45
23.11
7.00
5.87
6.68
7.01
%CV
20.04
19.38
6.67
5.59
5.46
5.64
VB1
VB2
LCV1
LCV2
LMG1
LMG2
113.89
115.28
107.45
108.26
101.75
102.78
25.72
26.07
9.22
10.25
8.01
8.36
22.59
22.61
8.58
6.47
7.87
8.13
Within lab reproducibility results at approximately 200%MRPL (4µg/kg)

44. Ruggedness Examined using the Youden approach
Choose a subset of 8 combinations that have a balance between capital and small letters
8 determinations are made which will use a combination of the chosen factors A-G

45. Chosen Factors Method Parameter Changed Normal Change
Time sample left after spiking
A= 15 mins
a= 20 mins
Time vortex after addition hydroxylamine
B= 30 sec
b= 1 mins
Time left in dark
C= 10 mins
c= 15 mins
Time shake after MgSO4
D= 30 sec
d= 1 min
Time on mechanical shaker
E= 10 mins
e= 15 mins
Turbo Vap Temperature
F= 50˚C
f= 45˚C
Time on mechanical shaker at end
G= 5mins
g= 6 mins

46. Experimental Design for Ruggedness Study
Factor Value F
Combination of determination number 1 2 3 4 5 6 7 8
A/a A a
B/b B b
C/c C c
D/d D d
E/e E e
F/f F f
G/g G g
Observed Result S T U V W X YS Z

47. Ruggedness Results BG1 CV1 MG1 VB1 LCV1 LMG1 100% MRPL %Recovery S
82.7
93.9
84.9
97.8
103
110 T
78.5
102
86.4
117
100
98.5 U
91.1 94
87.3
109
91.3 V
90.3
93.8
85.1
115
107
105 W
72.4
93.6
94.5
97.7
101 X
87.2
89.6
94.3
106
96.8 Y
81.3
93.7
92.2
116
91.0
99.9 Z
91.9
85.7
88.0

48. Decision Limit (CCα) Decision Limit Substances with no permitted limit
The limit at and above which it can be concluded with error on probability, α, that a sample is non-compliant
Substances with no permitted limit
CCα established by analysing 20 blanks and calculate the signal to noise at the time window analyte is expected
3 times S/N can be used as CCα
Weakest qualifier used to determine S/N

49. S/N Ratio Data for Transition 2
Identification
BG2
CV2
MG2
VB2
LCV2
LMG2
Approx. signal range (min)
3.9 – 4.3
3.6 – 4.0
3.3 – 3.6
5.4 – 5.9
5.3 – 5.9
Approx. noise range (mins
2.5 – 3.0
3.0 – 3.5
6.0 – 6.5
Average S/N
7.33
99.00
11.92
7.54
4.76
4.72
3* Average S/N
21.98
297.00
35.76
22.62
14.29
14.17
S/N Ext 6
126
2520
350
408
47.3
311
Ext 6 is the lowest standards in the calibration curve and its S/N ration is greater than 3* S/N of blanks
CCα for confirmatory analysis if 0.5µg/kg for each analyte

50. Detection Capability CCβ
The lowest concentration of an analyte that may be detected, identified and/or quantified in a sample with an error of probability β
Substances with no permitted limit
CCβ established by analysing 20 blanks fortified at the decision limit.
Decision limit times the std dev of within laboratory reproducibility of the measured content equals detection capability (β = 5%)

51. Recovery data for blank matrix spiked at 0.5µg/kg
BG1
BG2
CV1
CV2
MG1
MG2
Average
91.16
112.64
108.24
108.39
111.92
104.15
Std Dev
20.21
22.65
15.30
15.46
28.44
14.82
1.64 * (SD/AV)*0.5
0.18
0.17
0.12
0.21
CCβ
0.68
0.67
0.62
0.71
VB1
VB2
LCV1
LCV2
LMG1
LMG2
103.56
103.39
111.68
109.90
111.89
114.64
14.88
16.74
11.74
14.96
0.09
0.11
0.59
0.61

52. Conclusion Method for analysis of Dyes by LCMSMS was validated.
Validation data shows the performance of analytical method is Fit For Purpose and acceptable within acceptance criteria as stated in Commission 2002/657/EC