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

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

July 28, 2014

Webinar Separation Science - Vet Drugs

2

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)

http://upload.wikimedia.org/wikipedia/commons/3/3e/Chinolone.png

http://upload.wikimedia.org/wikipedia/commons/8/8b/Sulfamethoxazole.png

http://upload.wikimedia.org/wikipedia/commons/0/05/Nitroimidazoles.svg

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


-DB: 500+ compounds


Forensic Toxicology


-DB: 2,500+ 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


-DB: 1600+ compounds


Veterinary Drugs




Forensic Toxicology




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


Documents

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