Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by...
Transcript of Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by...
![Page 1: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/1.jpg)
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
![Page 2: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/2.jpg)
Agenda Introduction
Screening by LC/QQQ
Accurate Mass MS Toolbox
All Ions MS/MS
Summary
July 28, 2014
Webinar Separation Science - Vet Drugs
2
![Page 3: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/3.jpg)
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
July 28, 2014
Webinar Separation Science - Vet Drugs
3
![Page 4: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/4.jpg)
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
July 28, 2014
Webinar Separation Science - Vet Drugs
4
![Page 5: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/5.jpg)
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
July 28, 2014
Webinar Separation Science - Vet Drugs
5
![Page 6: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/6.jpg)
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
July 28, 2014
Webinar Separation Science - Vet Drugs
6
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
![Page 7: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/7.jpg)
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
July 28, 2014
Webinar Separation Science - Vet Drugs
7
Quinolones Sulfonamides
(e.g. Sulfamethoxazole)
Nitroimidazoles
(e.g. Metronidazole)
![Page 8: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/8.jpg)
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)
July 28, 2014
Webinar Separation Science - Vet Drugs
8
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.
![Page 9: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/9.jpg)
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
![Page 10: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/10.jpg)
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
July 28, 2014
Webinar Separation Science - Vet Drugs
10
• Tested matrices
• Muscle
• Liver
• Urine
• Honey
• Egg
![Page 11: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/11.jpg)
Experimental UHPLC and QQQ Method Parameters
July 28, 2014
Webinar Separation Science - Vet Drugs
11
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
![Page 12: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/12.jpg)
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)
July 28, 2014
Webinar Separation Science - Vet Drugs
12
![Page 13: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/13.jpg)
Screening of Veterinary Drugs by LC/QQQ Example for Negative Ionization– Chloramphenicol
July 28, 2014
Webinar Separation Science - Vet Drugs
13
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
![Page 14: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/14.jpg)
Screening of Veterinary Drugs by LC/QQQ Example for Positive Ionization– Dapsone
July 28, 2014
Webinar Separation Science - Vet Drugs
14
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
![Page 15: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/15.jpg)
Screening of Veterinary Drugs by LC/QQQ Evaluation of Matrix Effects
July 28, 2014
Webinar Separation Science - Vet Drugs
15
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
![Page 16: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/16.jpg)
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
July 28, 2014
Webinar Separation Science - Vet Drugs
16
Data courtesy of:
• Franziska Spitzbarth, Hochschule Fresenius, Zwickau
• Dr. Günther Kempe
![Page 17: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/17.jpg)
LC/MS Application
Kits
Method
Standards
On Site Training
LC Column
tMRM Database
tMRM LC/MS Application Kits Targeted Screening with QQQ
July 28, 2014
Webinar Separation Science - Vet Drugs
17
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
![Page 18: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/18.jpg)
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
July 28, 2014
Webinar Separation Science - Vet Drugs
18
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
![Page 19: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/19.jpg)
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
July 28, 2014
Webinar Separation Science - Vet Drugs
19
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
![Page 20: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/20.jpg)
The Toolbox for Accurate Mass Screening The Complete Solution from Agilent
July 28, 2014
Webinar Separation Science - Vet Drugs
20
![Page 21: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/21.jpg)
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
July 28, 2014
Webinar Separation Science - Vet Drugs
21
![Page 22: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/22.jpg)
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
July 28, 2014
Webinar Separation Science - Vet Drugs
22
![Page 23: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/23.jpg)
Experimental UHPLC and Q-TOF method parameters
July 28, 2014
Webinar Separation Science - Vet Drugs
23
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
![Page 24: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/24.jpg)
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
July 28, 2014
Webinar Separation Science - Vet Drugs
24
![Page 25: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/25.jpg)
Screening Workflow using LC/Q-TOF MS Innovative Workflow– Find by Formula
TIC chromatogram of spiked
muscle extract
July 28, 2014
Webinar Separation Science - Vet Drugs
25
• Fully automatic compound identification based on formula information
included in database and definition of ion species
![Page 26: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/26.jpg)
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
July 28, 2014
Webinar Separation Science - Vet Drugs
26
![Page 27: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/27.jpg)
(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
July 28, 2014
Webinar Separation Science - Vet Drugs
27
![Page 28: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/28.jpg)
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
July 28, 2014
Webinar Separation Science - Vet Drugs
28
![Page 29: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/29.jpg)
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
July 28, 2014
Webinar Separation Science - Vet Drugs
29
• 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
![Page 30: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/30.jpg)
Screening Workflow using LC/Q-TOF MS Alternative Approach to Verification: Auto MS/MS
July 28, 2014
Webinar Separation Science - Vet Drugs
30
• 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)
![Page 31: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/31.jpg)
Screening Workflow using LC/Q-TOF MS Personal Compound Database and Library with Auto MS/MS
July 28, 2014
Webinar Separation Science - Vet Drugs
31
![Page 32: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/32.jpg)
Screening Workflow using LC/Q-TOF MS Verification of Albendazole Sulfone by MS/MS Library Matching
July 28, 2014
Webinar Separation Science - Vet Drugs
32
MS results (5 Hz)
MS/MS results (5 Hz)
Library match score 85.1
![Page 33: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/33.jpg)
Efficient Data Review and Quantitation Automatic Creation of Quantitation Method
July 28, 2014
Webinar Separation Science - Vet Drugs
33
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
![Page 34: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/34.jpg)
Efficient Data Review and Quantitation MS Quantitation of Doramectin
July 28, 2014
Webinar Separation Science - Vet Drugs
34
![Page 35: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/35.jpg)
Efficient Data Review and Quantitation MS Quantitation of Acetopromazine and Ronidazole
July 28, 2014
Webinar Separation Science - Vet Drugs
35
+ 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
![Page 36: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/36.jpg)
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
July 28, 2014
Webinar Separation Science - Vet Drugs
36
![Page 37: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/37.jpg)
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
July 28, 2014
Webinar Separation Science - Vet Drugs
37
![Page 38: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/38.jpg)
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
July 28, 2014
Webinar Separation Science - Vet Drugs
38
![Page 39: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/39.jpg)
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.
![Page 40: Screening of Veterinary Drugs by LC/MS - Agilent · PDF fileScreening of Veterinary Drugs by LC/MS Solutions and Workflows for Screening, Identification and Quantitation by LC/QQQ](https://reader030.fdocuments.in/reader030/viewer/2022020411/5aa673937f8b9a1d728e6fec/html5/thumbnails/40.jpg)
July 28, 2014
Webinar Separation Science - Vet Drugs