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Validation of HRMSMulti-screening methods
Outline
page 2AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
� Introduction - Background
� Classification of screening methods
� Validation of Screening Methods
� Veterinary Drug Class comprehensive Method for milk samples
EU-Legislation for veterinary drugs and analytical methods
page 3Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
� COMMISSION REGULATION (EU) No 37/2010 on pharmacologically active
substances and their classification regarding maximum residue limits in
foodstuffs of animal origin
(meanwhile more than 50 amendments)
� Commission Decision 2002/657/EC implementing Council Directive 96/23/EC
concerning the performance of analytical methods and the interpretation of
result
(under revision)
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Commission Decision 2002/657/EC
page 4Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
� Contains definitions and general requirements for handling of samples, analytical methodologies, validation of analytical methods and interpretation of results
� Lays down performance criteria for screening and confirmationmethods
� Establishes the minimum required performance limits of analytical methods to be used for substances for which no permitted limit has been established.
� Defines criteria for method validation
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
GUIDELINES FOR THE VALIDATION OF SCREENING METHODS
FOR RESIDUES OF VETERINARY MEDICINES(COMMUNITY REFERENCE LABORATORIES RESIDUES (CRLs)
20/1/2010
page 5Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
� Supplements Commission Decision 2002/657/EC [1] regarding the validation
of screening methods
� Contains definitions
� Screening method classification
� Principles to be followed for the validation of screening methods
� Validation procedure
� Examples
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
GUIDELINES FOR THE VALIDATION OF SCREENING METHODS
FOR RESIDUES OF VETERINARY MEDICINES(COMMUNITY REFERENCE LABORATORIES RESIDUES (CRLs)
20/1/2010
page 6Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
� Regulatory/Action limit
• MRL, RPA, ML
� Screening Target Concentration (STC)
• The Screening Target Concentration is the concentration at which a screening test categorises the sample as “Screen Positive” (potentially non-compliant) and triggers a confirmatory test.
• STC ≤ regulatory/Action limit
• STC ≤ recommended concentrations, MRPL, RPA (CRL Guidance Paper)
• The further the Screening Target Concentration is below the Regulatory Limit, the lower the probability of obtaining a false compliant (i.e. false-negative) result in samples containing the drug at the Regulatory Limit.
� Detection Capability CCß
• CCß ≤ Regulatory/Action limit
� Cut-Off level
• The Cut-Off Level is the response or signal from a screening test which indicates that a sample contains an analyte at or above the Screening Target Concentration. If the Cut-Off Level is exceeded a subsequent confirmatory test is carried out
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Screening Methods
page 7Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
Physicochemical methods distinguish the chemical structure and molecular characteristics of analytes by separation of molecules (e.g. TLC, GC, HPLC) and the detection of signals related tomolecular characteristics (e.g. UV- DAD, Visible, FLD, FID, ECD, MS, MS/MS, Qtrap MS, QToF MS, Orbitrap). They are able to distinguish between similar molecular structures and allow thesimultaneous analysis of several analytes.
Qualitative: Peak is present or not
Semi Quantitative
quantitative
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Screening Methods – Compound Challenges
page 8Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
Large number of compounds from different drug classese.g. more than 250 compounds in milk method
Many “good” (e.g. benzimidazoles) and some “bad” guys (cephalosporins, avermectines)
Wide range of MRLs + recommended Concentrations, RPAse.g. in milk 0.1 – 170 µg/kg, 2 – 15000 µg/kg in liver
Regulated and banned or no authorised compound in one method
Standard solution with uniform concentration for all compound or standard solution with specific concentration corresponding regulatory limit, RPA etc.
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Using a multi-class screening method (e.g. an LC-ToF-MS screening method), if analyteshave a different retention time (Rt), they may undergo different effects (ion suppression or ionenhancement) because of different amounts of co-eluting matrix compounds. For that reason, it isadvisable to test for all analytes and not for a subselection even if the analytes have very similarphysicochemical properties
Screening Methods – Compound Challenges
page 9Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
1 complete mixture
1 µg/ml
4 working mixtures
10 µg/ml31 Working mixtures 100 µg/ml
8 - 9 compounds
Stock solution 1 mg/ml
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Screening Methods – Method Challenges
page 10Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
� Generic enough to apply to several different matrices and species, e.g. meat, egg, milk, poultry, fish, mammalians
� Sample prep must minimize loss of analytes and be simple and cost-effective
Non-selective samplePreparation
(Quadrature of the circle)
Direct injection
Liquid-liquid extraction
QuEChERS
SPE-Extraction
Combinations of extraction processes
and defattening
sele
ctivity
Matrix d
isturb
ance
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Screening Methods – Evaluation Challenges
page 11Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
� Non target Screening
� e.g. Molecular feature extraction
� Qualitative semi target screening
� Find by formula algorithm
� Quantitative target screening
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Screening Methods – Validation approaches
page 12Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
� Quantitative Screening methods
� Have to fulfil requirements of Commission Decision 2002/657/EC for quantitative methods
� Validation like a confirmatory method
� Confirmatory method Screening
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Screening Methods – Validation approaches
page 13Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
� Semi Quantitative Screening methods
� Semi-quantitative methods give an approximate indication of the concentration of the putative analyte.
� Have to not fulfil requirements of Commission Decision 2002/657/EC with respect to e.g. reproducibility or trueness
� Validation according alternative or classical approach
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Screening Methods – Validation approaches
page 14EURL-Workshop, 7-9 May 2019Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
� multi-residue screening method in muscle and liver1
1: EURL Workshop 2015 Berlin
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Screening Methods – Validation approaches
page 15EURL-Workshop, 7-9 May 2019Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
244 Componds:
47 Anthelmintics (Benzimidazoles, Avermectines, Salicylicacid derivates)
35 Coccidiostats (Ionophores, chemical Coocidiostats, Nitroimidazoles)
41 NSAIDs (basic and acidic, Coxibes)
103 Antibiotics
18 Sedatives
Screening Methods – Validation approaches
page 16EURL-Workshop, 7-9 May 2019Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
� Validation according alternative approach
• Application of experimental design
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Screening Methods – Validation approaches
page 17Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
� Validation like a confirmatory method• Validation results of a multi-residue screening method in
muscle and liver, EURL workshop 2015 Berlin• Application of experimental design
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Screening Methods – Validation approaches
page 18Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
� 16 Runs
� Each run contains 10 samples ( 8 spiked , 1 matrix and 1 reagent blank
� Spike level: 2, 5, 10, 25, 50, 100, 150, 200 µg/kg
� Lowest MRL/ML – range• 2 µg/kg (e.g. – for Monensin in muscle)
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Screening Methods – Validation approaches
page 19Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
� Validation according to classical approach1, 2
� For qualitative and semi quantitative methods
� Comparison between blank samples and spiked samples (e.g 0.1 – 0.5 regulatory limit
� Calculation of blank Threshold T, Cut-off level Fm and and CCß
� Fm > T CCß < Regulatory Limit
1: E. Dubreil et al. Validation approach for a fast and simple targeted screening method for 75 antibiotics in mat and aquaculture productsusing LC-MS/MSFood Additives & Contaminants: Part A, 2017, Vol.34, No. 4, 4532: Guidelines for the Validation of Screening Methods for Residues of Veterinary MedicinesCommunity Reference Laboratories residues 20/1/2010
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Multi residues screening in milk
page 20Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
Compounds
262 Compounds: 31 Mixes 100 ng/µl
� 47 Anthelmintics (Benzimidazoles, Avermectines, Salicylic acid derivates)
� 35 Coccidiostats (Ionophores, chemical CoocidiostatsNitroimidazoles)
� 7 Nitroimidazoles A6
� 41 NSAIDs (basic and acidic, Coxibes)
� 103 Antibiotics
� 18 Sedatives
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Multi residues screening in milk
page 21Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
Compounds
262 Compounds:
111 thereof with a MRL
0,05 µg/kg (clazuril) – 200 µg/kg (spiramycin)
70 Not for use in animals from which milk are produced for human consumption.
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Multi residues screening in milk
page 22Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
Matrices, Operator, Instruments
� bovine, goat and sheep
� 2 operators
� 2 instruments (QTOF, Q-Exactive)
� 10 runs within 3 month
� Concentration level
� Blank, 1, 5, 20, 50, 100 µg/kg
� 24 samples/conc. level
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
If the Screening Target Concentration is set at half the Regulatory/Action Limit or lower, the occurrence of one or no false-compliant results following the analysis of at least 20 “Screen Positive” Control Samples is sufficient to demonstrate that CCβ is less than the Regulatory/Action Limit (MRL) and less than or equal to the ½ MRL;
Multi residues screening in milk
page 23Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Multi residues screening in milk
page 24Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
Chromatographic conditions (Agilent HP1290, Vanquish):Mobile phases
Mobile phase A: water/acetonitrile = 95/5 +0.1% formic acid Mobile phase B: water/acetonitrile = 5/95 +0.1% formic acid
Gradient0 – 0.5 min 100% A- 19 min 2% A- 24 min 2% A- 26 min 100 % A- 30 min 100 % A
Flow: 300 µl/minHPLC column: Luna® 3 µm C18(2) 100Å 150 x 2 mmColumn temperature: 40 °CInjection volume: 10 µl
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Multi residues screening in milk
page 25Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
MS-conditions (Agilent QTOF 6550)Ion source: Dual AJS ESIMeasurement mode: MS All Ion mode (for positive and negative mode)CE: 0, 20, 30, 40 VMass range (positive mode): 100 - 1500 m/zMass range (negative mode): 100 – 1100 m/zResolution: 2 GHz extended dynamic range
Reference masses (positive mode): 121.05087, 922.0098Reference masses (negative mode): 119.0371, 1033.9881Source parameters (positive and negative mode):
Gas temp.: 150 °CGas flow: 18 l/minNebulizer: 30 psigShealth gas temp.: 400 °CShealth gas flow.: 12 l/minVCap: 4500Nozzle voltage: 500 V
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Multi residues screening in milk
page 26Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
MS-conditions (QExactive)Ion source: Measurement mode: Full MS mode (for positive and negative mode)
Microscans 1 Resolution 35,000
AGC target 1e6 Maximum IT 110ms Number of scan ranges 1 Scan range 100 to 1500m/z Spectrum data type ProfileLock mass off
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Multi residues screening in milk
page 27EURL-Workshop, 7-9 May 2019Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS
Resolution (QExactive)
35000
70000
140000
Fenbendazol
Multi residues screening in milk
Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS page 28
Qualitative Data Evaluation: Mass Hunter Find by formula
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Dimetridazol, 1 µg/kg
Multi residues screening in milk
Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS page 29
Qualitative Data Evaluation: Mass Hunter Find by formula
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Multi residues screening in milk
Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS page 30
Qualitative Data Evaluation: Mass Hunter Find by formula
� Semi Target Screening, based of a spectra database
� Reference Standards required for spectra recording and Rt determination
� Time consuming depending of PC performance, number found analytes and acquisition mode
e.g. 23 min. for 1 sample, all Ion, 200 compounds
1 min for blank sample or real sample
� False negative during validation > 5% (means not detected by Find by Formula but by EIC)
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Multi residues screening in milk
Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS page 31
Quantitative Data Evaluation: Mass Hunter
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Multi residues screening in milk
Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS page 32
Quantitative Data Evaluation: Mass Hunter
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Multi residues screening in milk
Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS page 33
Quantitative Data Evaluation: Mass Hunter
� Target approach
� Very fast, < 1 min for a batch (12 samples, blanks and calibration)
� Time consuming step: manual integration check
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Multi residues screening in milk
Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS page 34
Quantitative Data Evaluation: Trace Finder
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Multi residues screening in milk
Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS page 35
Quantitative Data Evaluation: Trace Finder
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Multi residues screening in milk
Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS page 36
Evaluation Validation
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019
Regulatory limit Dimetridazol
recommended conc. 3 µg/kg
Screening target Concentration 1 µg/kg
Blank Spike level
0 1
No µg/kg µg/kg
1 18,606 103,873
2 13,692 88,337
3 40,937 94,055
4 41,824 81,536
5 15,269 136,372
6 55,153 80,990
7 15,199 76,988
8 23,523 68,936
9 0 133,224
10 2,646 126,879
11 627 121,723
12 524 122,700
13 15,451 256,540
14 14,471 164,199
15 153 153,978
16 13,338 165,675
17 24,311 266,022
18 25,385 225,612
19 25,208 243,907
20 45,256 242,864
21 2,454 205,506
22 662 196,755
23 3,517 185,216
24 2,553 230,393
Mean (B) 16,698 157,178
SD 16,021.91 63,631.54
Blank Threshold value T: 42,974
Cut off Level (Fm): 52,823
Area
Contact:
Acknowledgement:
Thank you for your attention!
Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS page 37
The financial support by theEuropean Commission isgratefully acknowledged.
AOAC Europe – NMKL – NordVal International
symposium , 3 – 4. June 2019