Screening of Veterinary Drugs by LC/MS
Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ and LC/Q-TOF Dr. Thomas Glauner Application Scientist LC/MS
Agenda Introduction
Screening by LC/QQQ
Accurate Mass MS Toolbox
All Ions MS/MS
Summary
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Veterinary Drug Residues in Food Definitions
Veterinary drugs are pharmacologically active compounds which are
used to treat and prevent diseases of animals in livestock. Their use
can result in non-desirable drug residues in the food products.
• Definitions
- Veterinary medicinal products
- Medicated feeding stuff
- Feed additives – no veterinary medicines
(Regulation (EC) No 1831/2003)
Exception: certain substances with coccidiostatic
and histomonostatic effects
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Veterinary Drug Residues in Food Current Regulations and Requirements
• Codex Alimentarius
35th Session of the Codex Alimentarius Commission (2012)
• EU regulation
- Maximum residue levels regulated in table 1 of commision regulation (EC)
37/2010 (matrix-veterinary drug combinations)
- Combinations not mentioned in table 1 are not authorized for use
- Table 2 lists completely forbidden compounds
- Minimum required performance limits (MRPLs) for
analytical methods for forbidden or not authorized
compounds are specified in Commission Decision
2003/181/EC to 1 µg/kg or below.
• US regulation
- 21CFR Part 556 Tolerances for Residues of New Animal Drugs in Food
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Screening for veterinary drugs by LCMS Requirements for screening methods
• Criteria specified in commission decision 2002/657/EC
• Method to detect the presence of a substance or substance class at the
level of interest
• Designed for high sample throughput and designed to avoid false
compliant (false negative) results
• Detection limit, Selectivity, and Ruggedness of method need to be
validated, for quantitative screening methods in addition Precision of
method has to be shown
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Analysis of Veterinary Drugs A Class Specific Approach
Group A – Substances with anabolic
effects and unauthorized
substances
A1 Stilbenes and stilbene derivatives
A2 Antithyroid agents
A3 Steroids
A4 Resorcylic acid lactones
A5 Beta-agonists
A6 Forbidden compounds included in
table 2 regulation (EC) 37/2010
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Group B – Veterinary drugs and contaminants
B1 Antibacterial substances
B2 Other veterinary drugs
B2a Anthelmintics
B2b Anticoccidials
B2c Carbamates & Pyrethroids
B2d Sedatives
B2e Non-steroidal anti-inflammatory drugs
(NSAIDs)
B2f other pharmacologically active
substances
B3 Other substances and contaminants
B3a Organochlorine compounds
B3b Organophosphorous compounds
B3c Chemical elements
B3d Mycotoxins
B3e Dyes
B3f Others
Screening for Veterinary Drugs by LC/MS A Challenging Application
• About 500 compounds are used and should be monitored
• Low LOQs need to be analyzed for not-authorized compounds
• Not yet an established generic extraction method
• Very different and difficult matrices
• Compared to pesticides, veterinary drugs contain less hetero
atoms resulting in a similar mass defect than matrix constituents
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Quinolones Sulfonamides
(e.g. Sulfamethoxazole)
Nitroimidazoles
(e.g. Metronidazole)
Multiclass Screening of Veterinary Drugs Considerations for Sample Preparation
• Generic non-selective sample preparation required and several approaches have
been published
• Promising approach: QuEChERS like extraction (buffered acetonitrile extraction
with liquid-liquid partitioning and SPE or dispersive SPE using different sorbents)
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Homogenized tissue sample
LC/MS/MS
Extraction with 1% acetic acid in acetonitrile
Addition of anhydrous Na2SO4
Dispersive SPE cleanup using BondElut NH2
(+ SCX cleanup for nitroimidazoles)
Evaporate and reconstitute
Stubbings, G. And Bigwood, T.
Anal. Chim. Acta 637 (2009) 68-78.
Multiclass Screening of Veterinary Drugs Considerations for Detection
Applicability of extraction and separation to a
variety of compound groups:
• Avermectins
• Benzimidazoles (special cleanup)
• Diaminopyrimidins
• Dinitrocarbanilide
• Fluoroquinolones
• Ionophores
• Levamisole
• Lincosamide antibiotics
• Macrolides
• Nitroimidazoles
• Pleuromutilins
• Quinolones
• Sulphonamides
• Tetracyclines
• Tranquillizers
• Also applied to:
• Amphenicoles
• Cephalosporins
• Dyes
• NSAIDs
• Penicillins
• Special methods for:
• Steroids
• Resorcylic acid lactones
• Stilbens
• Synthetic androgens
• Synthetic gestagens
• Synthetic corticosteroids
• Nitrofurans
• Aminoglycosides
Screening of Veterinary Drugs by LC/QQQ Concept
• More than 200 substances have been optimized and tested for detectability in
solvent and in matrix samples
• Concentration levels
• 10 µg/kg (Level 4)
• 1 µg/kg (Level 3)
• 0.2 µg/kg (Level 2)
• 0.05 µg/kg (Level 1)
• 180 compounds were evaluated in matrix samples
• Source parameters were optimized for highest response for low abundant
compounds
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• Tested matrices
• Muscle
• Liver
• Urine
• Honey
• Egg
Experimental UHPLC and QQQ Method Parameters
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UHPLC column Phenomenex Aqua C-18 column,
150 x 2 mm, 3 µm @ 25°C
Mobile phase A: 5 mM NH4 formate + 0.1% formic acid
B: 5 mM NH4 formate + 0.1% formic acid in methanol
Gradient program
Min % B
0 10
2 10
13.0 60
18.0 95
23.0 95
24.0 10
Stop time 27 min
Flow rate 0.30 mL/min
Injection volume 1 µL
• G6490A QQQ operated in Dynamic MRM mode with
fast polarity switching
• In total 524 MRM transitions with a fixed cycle time of 800 ms
• Unit mass resolution for both, precursor and product ion isolation
Screening of Veterinary Drugs by LC/QQQ UHPLC/MS/MS Chromatogram for Calibration Sample
• Final method includes 215 compounds with 2 transitions per compound
• Peak shapes compromised for some dyes (brilliant green, malachite green), sedatives
(promethazine), fluorquinolones (ciprofloxacin), and anticoccidials (decoquinate)
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Screening of Veterinary Drugs by LC/QQQ Example for Negative Ionization– Chloramphenicol
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Solvent
0.4 ng/mL
Liver
0.2 µg/kg
Honey
0.2 µg/kg Urine
0.2 µg/kg
Egg
0.2 µg/kg
Muscle
0.2 µg/kg
Screening of Veterinary Drugs by LC/QQQ Example for Positive Ionization– Dapsone
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Solvent
0.4 ng/mL
Liver
0.2 µg/kg
Honey
0.2 µg/kg Urine
0.2 µg/kg
Egg
0.2 µg/kg
Muscle
0.2 µg/kg
Screening of Veterinary Drugs by LC/QQQ Evaluation of Matrix Effects
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0
20
40
60
80
100
120
140
160
180
200
20 ng/ml 2 ng/ml 0.4 ng/ml 0.1 ng/ml
# o
f c
om
po
un
ds
Standard
muscle
liver
urine
honey
egg
Solvent Muscle Liver Urine Honey Egg
> LOQ* > LOD* > LOQ* > LOD* > LOQ* > LOD* > LOQ* > LOD* > LOQ* > LOD* > LOQ* > LOD*
10 µg/kg 172 6 170 5 167 9 166 6 167 6 169 7
1 µg/kg 144 20 147 19 147 15 148 16 143 16 150 11
0.2 µg/kg 102 39 97 40 98 41 111 30 92 42 100 45
0.05 µg/kg 42 56 39 50 48 52 46 57 37 52 50 50
* Evaluation based on S/N of qualifier transition
Screening of Veterinary Drugs by LC/QQQ Summary
• Sensitive target screening method has been set up which is
compliant even with most challenging MRPLs
• Due to low amounts of matrix introduced, method is robust
and shows little matrix effects in the electrospray process
• Method has the potential to make several group specific
screening tests redundant
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Data courtesy of:
• Franziska Spitzbarth, Hochschule Fresenius, Zwickau
• Dr. Günther Kempe
LC/MS Application
Kits
Method
Standards
On Site Training
LC Column
tMRM Database
tMRM LC/MS Application Kits Targeted Screening with QQQ
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Pesticides
-Test Mix: 253 compounds
-DB: 700+ compounds
-Library: 200+ compounds
Veterinary Drugs
-Test Mix: 146 compounds
-DB: 500+ compounds
-Library: 100+ compounds
Forensic Toxicology
-Test Mix: 139 compounds
-DB: 2,500+ compounds
-Library: 100+ compounds
Instrument Installation Complete
Kit Installation:
1 Day
Customized Method
Implementation: 3 Days
Begin Method
Validation
Typical LC/MS Method Implementation Timeline With LC/MS Application Kit
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Checkout Standards:
Provided
MS Conditions: Provided
LC Column: Provided
LC Method: Provided
DB or Library: Provided
Comprehensive Test Mix:
Provided
Total Method Setup Time:
~1 Week
Accurate Mass LC/MS Application Kits Untargeted Screening with TOF or QTOF
LC/MS Application
Kits
Method
Standards
On Site Training
LC Column
Accurate Mass
MS/MS Library
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Pesticides
-Test Mix: 253 compounds
-DB: 1600+ compounds
-Library: 700+ compounds
Veterinary Drugs
-Test Mix: 146 compounds
-DB: 1000+ compounds
-Library: 600+ compounds
Forensic Toxicology
-Test Mix: 139 compounds
-DB: 7500+ compounds
-Library: 2500+ compounds
The Toolbox for Accurate Mass Screening The Complete Solution from Agilent
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iFunnel technology
(sensitivity)
Dual-stage
ion mirror
(resolution)
Ion Beam Compression
Technology (resolution +
mass accuracy)
Orthogonal spray
source (signal-to-noise)
Ion acceleration in hexapole
collision cell (faster MS/MS spectra)
Longer flight tube
(resolution)
INVAR flight tube
(mass accuracy)
ADC (dynamic range)
4 GHz electronics
(resolution, mass
accuracy, sensitivity,
dynamic range)
Screening workflow using LC/Q-TOF MS Instrumentation
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Screening of Vet Drugs by LC/Q-TOF MS Concept
• Comprehensive multi-residue screening using accurate mass MS including
different groups of veterinary drugs
- Comprehensive list of target compounds
- Reference standards might not be available or not at hand
- Databases and libraries with spectral data are available
- Retrospective data processing possible
- Challenges: Efficient data analysis and review, elimination of potential false positives
• Verification of results
- Retention times
- Accurate masses and Isotope pattern matching
- MS/MS spectal comparison (auto MS/MS and target MS/MS)
- All Ions MS/MS functionality
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Experimental UHPLC and Q-TOF method parameters
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UHPLC column Agilent ZORBAX EclipsePlus-C18 RRHD
2.1 x 100 mm, 1.8 µm @ 40°C
Mobile phase A: 0.1% formic acid
B: 0.1% formic acid in acetonitrile
Gradient program
Min % B
0 12
2.0 12
3.0 50
10.0 90
12.0 100
12.1 12
Stop time 15.0 min
Flow rate 0.40 mL/min
Injection volume 20 µL
• G6550 iFunnel Q-TOF operated in 4GHz High Resolution Mode
• Acquisition rate 3 scans/sec in MS
and 3 scans/sec in All Ions MS/MS
Screening of Vet Drugs by LC/Q-TOF MS UHPLC/TOF MS Chromatogram for Calibration Sample
• Sample includes 100 veterinary drugs and internal standards
• Database included more than 200 compounds with retention time information
• Data evaluation using the Find-by-Formula data mining algorithm
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Screening Workflow using LC/Q-TOF MS Innovative Workflow– Find by Formula
TIC chromatogram of spiked
muscle extract
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• Fully automatic compound identification based on formula information
included in database and definition of ion species
Automatic EIC extraction for all adducts
and isotopes
Extraction of MS and MS/MS spectra and
visual comparison of isotope patterns
Results scoring based on RT, mass
accuracy, isotope abundance and spacing
Dapsone
Find-by-Formula Data Mining Algorithm Spiked Muscle Extract
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(1) Ion mass
(2) Resolution
Dapsone
Chlorotetracycline
(1) Ion mass
(2) Resolution
Tylosin A
(1) Ion mass
(2) Resolution
Erythromycin A /
Erythromycin A-[C13]2
(1) Ion mass
(2) Resolution
Screening of Vet Drugs by LC/Q-TOF MS Spectral resolution using LC/Q-TOF MS
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Muscle spike
0.5 µg/kg
Muscle spike
1.0 µg/kg
Muscle spike
5.0 µg/kg
Muscle spike
25.0 µg/kg
Screening of Vet Drugs by LC/Q-TOF MS Example Chromatograms and Spectra for Avermectin B1a
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CompoundMuscle spike
0.5 µg/kg
Muscle spike
1.0 µg/kg
Muscle spike
5.0 µg/kg
Muscle spike
25.0 µg/kg
Avermectin B1a -0.41 -0.64 0.28 -1.96
Doramectin 0.28 0.14 -0.64 -0.53
Emamectin B1a 1.63 1.14 1.82 0.31
Eprinomectin B1a -0.37 -0.02 6.27 -0.31
Ivermectin B1a 0.47 0.32 0.27 -0.13
Laidlomycin-propionat -0.3 0.16 0.93 0.57
Lasalocid 1.27 0.08 0.71 0.09
Maduramycin 0.3 0.48 -0.04 -0.13
Monensin -0.24 -0.52 -0.78 -0.56
Moxidectin -2.31 1.25 0.24 -0.84
Narasin A n.n. n.n. n.n. 1.84
Salinomycin -1.84 -6.41 -2.83 0.51
Semduramycin 5.86 0.31 1.2 -0.56
Nemadectin (ISTD) -1.01 -0.35 -0.59 0.12
Nigericin (ISTD) -0.54 -1.23 -0.36 -0.97
Selamectine (ISTD) -0.08 -0.09 -0.75 -0.5
Screening of Vet Drugs by LC/Q-TOF MS Results Table for Avermectins and Ionophores
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• Data extraction with Find-by-Formula algorithm, mass difference to
database given in ppm (weighted average for all detected species)
• Green: automatically found, low mass error
• Orange: automatically found, inspection required
due to low score and high mass error
• Red: not found due to high mass error
• n.n.: not detected, no EIC peaks
Screening Workflow using LC/Q-TOF MS Alternative Approach to Verification: Auto MS/MS
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• Peak purity algorithm based on isotope model
• Variation of scan speed based on precursor abundance
• Directed Auto MS/MS (triggers only on masses included in the preferred list)
Screening Workflow using LC/Q-TOF MS Personal Compound Database and Library with Auto MS/MS
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Screening Workflow using LC/Q-TOF MS Verification of Albendazole Sulfone by MS/MS Library Matching
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MS results (5 Hz)
MS/MS results (5 Hz)
Library match score 85.1
Efficient Data Review and Quantitation Automatic Creation of Quantitation Method
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Compound results
- Major adducts
- Accurate mass EICs for
quantifier and qualifier
- Relative responses
- Retention times
Quantitation method
-Automatic selection of quantifier
and qualifier(s)
-Reporting of qualifier ratios
- Isotope pattern comparison and
accurate mass metrics as QC
Efficient Data Review and Quantitation MS Quantitation of Doramectin
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Efficient Data Review and Quantitation MS Quantitation of Acetopromazine and Ronidazole
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+ EIC(327.1526) Scan Sample_2.d
Acquisition Time (min)
9.6 9.7 9.8 9.9 10 10.110.210.310.410.5
Cou
n ts
5x10
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Acetopromazine10.060 min.
Acetopromazine - 5 Levels, 5 Levels Used, 5 Points, 5 Points Used, 0 QCs
Concentration (ng/ml)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Resp
onse
s 7x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
y = 569768.149845 * x - 5236.878993R^2 = 0.99917533Type:Linear, Origin:Ignore, Weight:None
Ronidazole - 5 Levels, 5 Levels Used, 5 Points, 5 Points Used, 0 QCs
Concentration (ng/ml)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Resp
onse
s 6x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2y = 60786.006393 * x - 50129.337644R^2 = 0.99550981Type:Linear, Origin:Ignore, Weight:None
+ EIC(201.0618) Scan Sample_2.d
Acquisition Time (min)
3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6
Cou
n ts
4x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
3.2 Ronidazole3.911 min.
Acepromazine
Ronidazole
N
S
O
N
CH3
CH3
CH3
NN+
N
O-
O
OO
NH2
CH3
Qualitative Screening using All Ions MS/MS A New Approach
Acquisition
- Easy method setup
- All Ions is faster than MS/MS
- Chromatography is monitored in 2 channels (or more if desired)
• A low energy channel ensures optimum molecular ion generation
• A high enery channel produces fragments
- support of TOF (fragementor) as well as Q-TOF(collison cell)
Finding?
- As with TOF screening compounds are found using the same find by formula algorithm already well
proven
Verifying
- verification of results by using PCDL MS/MS library
- Chromatographic verification of fragment ions uses a unique
co-elution score
- Allows retrospective data interogation, growing libraries can be applied
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Qualitative Screening using All Ions MS/MS All Ions MS/MS Workflow • Modified “Find by Formula Algorithm” searches compound based on database entry in the
low energy domain
• Uses PCDL spectra as source of fragments to look for in the high energy domain and
compares elution profiles
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Qualitative Screening using All Ions MS/MS Results overview
• Mebendazole was found
with 2 valid qualifier
fragments from the PCDL
spectrum
• Overlay of the EICs with
the Co-elution Plot shows
excellent agreement
between the products and
precursor ion
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Summary
• Modern accurate mass Q-TOF instruments allow comprehensive screening and sensitive
quantitation of veterinary drugs in complex matrices.
• Higher instrument sensitivity and higher resolution, both improve the detection of
veterinary drugs at the MRPL
• UHPLC for improved chromatographic resolution, short run times and highest sensitivity
• Innovative data mining tools for identification of trace residues in complex matrices
• Verification of suspects by true MS/MS acquisition and library matching
• All Ions MS/MS integrated workflow for enhanced qualification of suspected compounds by
additional fragment ions in qualitative screening
• Seamless integration of compound identification and quantitation for easy and quick batch
review
• Application kit including accurate mass database and library, methods, columns, and
comprehensive stdard available to help you to get started with the application.
July 28, 2014
Webinar Separation Science - Vet Drugs