FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 1 Validation of confirmatory methods for MRPL and MRL substances in matrices of

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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT

Validation of confirmatory methods forMRPL and MRL substances

in matrices of veterinary origin according to 2002/657/EC:

Case Study

VALIDATION OF METHODS

Dr. Apr. S. Impens and Ir. S. Carbonnelle

Department of Pharmaco-Bromatology

Drugs and Hormones

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• Allowed substances: maximum residue limits (MRL)

• Forbidden components: Minimum Required Performance Levels (MRPL)

Dxm (EMEA) MRLb,p,e 2 µg/kg

Btm (EMEA) MRLb,p 2 µg/kg

Prolon (EMEA)

MRLb 10 µg/kg

Mprolon (EMEA)

MRLb 10 µg/kg

Flm (B) MRPL 2 µg/kg

TrmAt (B) MRPL 2 µg/kg

Example: corticosteroids in liver

b = bovinep = porcinee = equidae

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• Analysis portion: 20 5 g

• Hydrolysis: H. Pomatia extract (2 h – 60 5 °C)

• L/L-extraction: methanol – diethylether

• Purification: Solid Phase Extraction (C18-column)

• Separation – Detection: LC-MSn (ThermoFisher Deca XP ion trap)

Corticosteroids in liver

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2002/657/EC: Performance Characteristics

Detection Capability

CCß

Decision Limit CC

Trueness/ Recovery

Precision Selectivity/ Specificity

Applicability/ Ruggedness/

Stability

S + - - - + + Qualitative methods C + + - - + +

S + - - + + + Quantitative methods C + + + + + +

• Allowed substances: maximum residue limits (MRL)

QUANTITATIVE PARAMETERS

• Forbidden components: Minimum Required Performance Levels (MRPL)

QUALITATIVE PARAMETERS

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Criteria for specificity

2002/657/EC:

- Chemically related components (metabolites, isomers,…)

- Representative number of blank samples

Practical work:

- 20 blank samples

- 20 blank samples fortified with possibly interfering compounds

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Criteria for trueness

2002/657/EC:

- 6 replicates of Certified Reference Materials (CRM) if available

- Calculation of mean, standard deviation and coefficient of variation- Trueness (%)= mean recovery-corrected concentration detected x 100 certified value- Remark: CRM not available recovery

Practical work:

-6 identical blank samples fortified at MRL/MRPL

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Criteria for recovery, repeatability and reproducibility

2002/657/EC:- Recovery: 18 samples fortified at :

- 6 x 0.5 MRL or 1 MRPL- 6 x 1 MRL or 1.5 MRPL-6 x 1.5 MRL or 2 MRPL

- Repeatability: 3 series of recovery analyses

-Within-laboratory reproducibility: 3 series of recovery analyses (different operators, different environmental conditions, …)

-For between-laboratory reproducibility: Proficiency tests

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Criteria for applicability and ruggedness

2002/657/EC:

-Determination of factors influencing measurements results

(Method of Youden)

Practical work:

-Development of the method: minimalisation of the

factors influencing the results

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Criteria for stability

2002/657/EC:- Analyte in solution at each concentration level

Practical work:- Preparation of working solutions- Conditions of conservation: tabel- Analysis at time 0, after 1 week, 2 weeks, … until instability

- 20 °C + 4 °C + 20 °C

Dark 10 aliquots 10 aliquots 10 aliquots

Light 10 aliquots

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Criteria for stability

2002/657/EC:

- Analyte in matrix at detection capability level

Practical work:

- Analysis of incurred material - preparation of fortified blank samples

- Conservation at min. – 20 °C

- Analysis at time 0, after 1 week, 2 weeks, … until instability

(max. 20 weeks)

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Criteria for calibration curve

2002/657/EC:

- At least 5 levels (including 0)

- Description of working range

- Determination of the mathematical formula, goodness of fit and

acceptability ranges

Practical work:

-5 levels of concentrations: 0 – 0,5 – 1 – 1,5 – 2 MRL/MRPL

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Criteria for Decision Limit (CCα)

2002/657/EC:

- MRL Substances (permitted limit – α = 5%):

1. Calibration curve: analysis of blank samples and blanks fortified

around the MRL

CCα = MRL+ 1,64 σRW

OROR

2. 20 blank samples fortified at MRL

CC α = MRL+1.64 σRW

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Criteria for Decision Limit (CCα)2002/657/EC:

- Banned Substances (no permitted limit – α = 1%):

1. Calibration curve: analysis of blank samples and blanks fortified

at and above the MRPL

CCα = Yintercept+ 2.33 σRW

OROR

2. 20 blank samples

CCα = 3 x S/NRT

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Criteria for Detection Capability (CCβ)

2002/657/EC:

- MRL Substances (permitted limit):

1. Calibration curve: analysis of blank samples and blanks

fortified around the MRL

CCβ = CCα + 1,64 σRW

OROR

2. 20 blank samples fortified at CCα

CCβ = CCα +1,64 σRW

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MRL CC

Signal orConcentration

CC

=

+1.64MRL +1.64sample

Detection CapabilityCase of permitted limit

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Criteria for Detection Capability (CCβ)2002/657/EC:

- Banned Substances (no permitted limit):

1. Calibration curve: analysis of blank samples fortified below and at the MRPL

CCβ = CC α +1,64 σRW

OROR

2. Analysis of 20 blank materials fortified at CCα

CCβ = CC α +1,64 σRW

OROR

3. Analysis of 20 blank materials fortified at and above CCα:

Less than 5% of false compliant

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Detection CapabilityCase of no established permitted limit or banned substance

XblankCC

Signal orConcentration

CC

≠

+2.33blank +1.64sample

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Validation of corticosteroids in liver

0 MRL 0,5 MRL 1 MRL 1.5 MRL 2 MRL

Day 1 5 3 3 3 3

Day 2 5 3 3 3 3

Day 3 5 3 3 3 3

Day 4 5 3 3 3 3

1. Analysis of mix of liver (pork, beef, horse, sheep)

MATRIX EFFECTS: DILUTION EFFECT

Alternative validation strategy

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0 MRL 0,5 MRL 1 MRL 1.5 MRL 2 MRL

Day 1 4 3 3 3 3

Day 2 4 3 3 3 3

Day 3 4 3 3 3 3

Day 4 4 3 3 3 3

2. Analysis of bovine liver

+ calibration curve in matrix (0 - 0,25 - 0,5 - 1 - 1,5 - 2 MRL/MRPL)

+ calibration curve in mobile phase (0 - 0,25 - 0,5 - 1 - 1,5 - 2 MRL/MRPL)

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Flexible scope

Criteria that should be fulfilled

by ISO 17025 accredited laboratories

working with the principle of flexible scope for analyses

concerning EC Decision 96/23/EC – 2002/657/EC

BELAC 2 – 105 rev1-2006

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Flexible scope- Continuous changes of normes flexibility

- Classes of matrices for flexible scope:

- eggs and protein-rich tissues (muscle, kidney, liver, thyroid,fish, aquaculture, eggs)

- fat

- urine, bile water

- plasma, blood, milk

-Faeces

-Feed

-Preparations

-Hair

-Others (retina, honey, ...)

Secondary validation:- Qual methods: 3 blank samples, 3 fortified samples at or below MRL/MRPL

- Quant methods: 3 blank samples, 6 fortified samples at or below MRL/MRPL (CC – repeatability – trueness - recovery)

completion of validation: routine analyses

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0 MRL/MRPL

0,5 MRL/MRPL

1 MRL/MRPL

1.5

MRL/MRPL

2 MRL/MRPL

pork 1 3 3

horse 1 3 3

Sheep day1

3 3 3 3 3

Sheep day2

3 3 3 3 3

3. Mini validation: Analysis of other species (horse, porcine, ovine)

ovine: 2 series of analyses because of additional extraction step (hexane)

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Qualitative validation parametersRelative retention time (RRT)

RRT = absolute retention time target absolute retention time internal std

2002/657/EC: RRT (LC-MSn) = ± 2,5 % RRT reference std or control sample

Diagnostic ions - identification points (IPs)

- Flm,TrmAt (banned)

2002/657/EC: min. 4 IPs

- Dxm, Btm, Prolon, Mprolon

2002/657/EC: min. 3 (MRL bovine) or 4 IPs (horse, sheep)

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RRT - IPs

monsternr. precursor ion 379(%) ion 409(%) RRT20070330refstd03 469 100,00 57,39 0,80 0.25MRPL-afwijking 80,00 45,91 0,78+afwijking 120,00 68,87 0,82I.P. 1 1,5 1,5

monsternr. precursor ion 379(%) besluit ion 409(%) besluit RRT besluit IP20070330spike01 469 100,00 ok 62,88 ok 0,79 ok 4,0 0,5MRPL

Identification Points: - Precursor Ion → 1 IP- Transition Product Ion → 1.5 IP

1 + 1.5 + 1.5 = 4 IPs

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Linearity

Mandel’s Fitting Test :

linear (y = ax + b) or quadratic (y = ax2 + bx + c) regression?

Calibration curves linear

Linear regression

y = 0,2488x + 0,0288R2 = 0,9928

-0,4

-0,2

0

0,20,4

0,6

0,8

1

1,2

-2 -1 0 1 2 3 4 5

X

Y

Dexamethasone0 – 4 µg/ kg

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Matrix effect

-0,5

0

0,5

1

1,5

2

2,5

3

0 5 10 15 20 25

addition

standard

Matrix effect

Calibration curve’s slope: comparison mobile phase – matrix (T-test)

matrix effect

prednisolone

-0,5

0

0,5

1

1,5

2

2,5

0 1 2 3 4 5

addition

standard

flumethasone

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Quantitative validation parametersCCα - CCβ

ISO 11843 is used to calculate CCα and CCβ

Cost Normes (µg/ kg)

CC (µg/ kg)

CC (µg/ kg)

Btm 2 2.5 3.0

Dxm 2 2.4 2.7

Flm 2 0.8 1.3

MProlon 10 13.7 17.4

Prolon 10 13.6 17.2

TrmAt 2 0.4 0.8

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Repeatability

recovery (70-110%) and CVr (max. 2/3 CVrw Horwitz)

Cost CVr (%) Recovery (%)

Btm 17.4 99

Dxm 2.6 96

MProlon 11.9 104

Prolon 9.5 105

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Repeatability


Reproducibility

CVrw

Cost CVrw (%)

Btm 19

Dxm 9

MProlon 11.9

Prolon 25.2

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Repeatability


Reproducibility

CVrw

Selectivity and specificity

LC-MSn: selective and specific

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LC-MSn: selectivity - specificityC:\Xcalibur\data\20070314spike11 03/20/2007 08:18:30 AMsp1: 22-2006-03544+ 1m rpl

RT: 0.00 - 20.06 SM : 5G

0 2 4 6 8 10 12 14 16 18 20Time (min)

0

5

10

15

20

25

30

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40

45

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100

Re

lati

ve

Ab

un

da

nc

e

TrmAt

5.10 NL: 4.13E6m/z= 374.50-375.50+412.50-413.50 F : - c ESI Full ms2 [email protected] [ 300.00-496.00] M S 20070314spike11

20070314spike11 #281 RT: 5.10 AV: 1 SB: 43 2.00-3.54, 7.52-9.73 NL: 3.57E6F: - c ESI Full ms2 [email protected] [ 300.00-496.00]

300 320 340 360 380 400 420 440 460 480

m/z

0

5

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45

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Re

lativ

e A

bu

nd

an

ce

375

413

433

357491

415337493

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Repeatability

recovery (70-120%) and CVs (max. 2/3 CV Horwitz)

Reproducibility

CV rw

Selectivity and specificity

LC-MSn: selective and specific

Stability

under current development

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Stability in solution

2002/657/EC:- Analyte in solution at each concentration level

Practical work:- Analysis:

- Cost in Meoh: 1 mg/ ml, 100 µg/ml, fortification mix- Cost in mobile phase: reference std mix

- Analysis at time 0, after 1 week, 2 weeks, … until instability

- 20 °C + 4 °C + 20 °C

Dark 10 aliquots 10 aliquots 10 aliquots

Light 10 aliquots

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Stability in solution

concentration Solvent conservation

1 mg/ ml Meoh 12 months (- 20 °C)

100 µg/ ml Meoh 12 months (- 20 °C)

Fort mix Meoh 3 months (8 °C)

100 µg/ ml Mobile phase 1 week (8 °C)

Ref std mix Mobile phase 1 week(8 °C)

reference std in mobile phase with fortification mix in meoh

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Stability in matrix

2002/657/EC:

- Analyte in matrix at detection capability level

Practical work:

- Preparation of fortified blank samples, conservation at min. – 20 °C

- non-compliant samples: conservation for max. 6 months

under development

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Conclusions

All 2002/657/EC validation criteria for the determination of corticosteroids in liver using LC-MSn are fulfilled

CONCLUSION

The analytical procedure is suitable for identification and quantification of Betamethasone, Dexamethasone, Flumethasone, Methylprednisolone, Triamcinolone-Acetonide in bovine, porcine, horse and ovine liver.

Documents

FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 1 Validation of confirmatory methods for MRPL and MRL substances in matrices of