Validation of HRMS Multi-screening methods...Wolfgang Radeck: Development and Validation of a...

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


� 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



GUIDELINES FOR THE VALIDATION OF SCREENING METHODS

FOR RESIDUES OF VETERINARY MEDICINES(COMMUNITY REFERENCE LABORATORIES RESIDUES (CRLs)

20/1/2010


� 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



GUIDELINES FOR THE VALIDATION OF SCREENING METHODS

FOR RESIDUES OF VETERINARY MEDICINES(COMMUNITY REFERENCE LABORATORIES RESIDUES (CRLs)

20/1/2010


� 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



Screening Methods


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



Screening Methods – Compound Challenges


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.



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


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



Screening Methods – Method Challenges


� 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



Screening Methods – Evaluation Challenges


� Non target Screening

� e.g. Molecular feature extraction

� Qualitative semi target screening

� Find by formula algorithm

� Quantitative target screening



Screening Methods – Validation approaches


� Quantitative Screening methods

� Have to fulfil requirements of Commission Decision 2002/657/EC for quantitative methods

� Validation like a confirmatory method

� Confirmatory method Screening





� 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




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




page 15EURL-Workshop, 7-9 May 2019Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS AOAC Europe – NMKL – NordVal International


244 Componds:

47 Anthelmintics (Benzimidazoles, Avermectines, Salicylicacid derivates)

35 Coccidiostats (Ionophores, chemical Coocidiostats, Nitroimidazoles)

41 NSAIDs (basic and acidic, Coxibes)

103 Antibiotics

18 Sedatives



� Validation according alternative approach

• Application of experimental design





� 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





� 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)





� 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



Multi residues screening in milk


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





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.





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



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;


page 23Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS AOAC Europe – NMKL – NordVal International




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





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





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





Resolution (QExactive)

35000

70000

140000

Fenbendazol


Wolfgang Radeck: Development and Validation of a Multi-screening method with HRMS page 28

Qualitative Data Evaluation: Mass Hunter Find by formula



Dimetridazol, 1 µg/kg









� 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)





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





Quantitative Data Evaluation: Trace Finder





Quantitative Data Evaluation: Trace Finder





Evaluation Validation



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!


[email protected]

The financial support by theEuropean Commission isgratefully acknowledged.



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