This presentation describes the investigation using Ion Mobility MS of previously reported issues with tandem quadrupole MS/MS methods for fluoroquinolone antibiotics in food of animal origin. The discovery of protomer species with different fragmentation pathways explains the variability of the tandem quadrupole MRM results.

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The use of Ion Mobility enabled Mass Spectrometry
for the development and characterisation of robust
analytical methods for Trace Residue Quantitation in
Foods of Animal Origin

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Overview
 Introduction
 Tof screening
 Ion mobility: Collision Cross Section Screening (CCS)
screening a new information point
 UPLC Ion Mobility Mass Spectrometry application for residue
analysis
 Routine screening using UPLC ion mobility
 Using ion mobility to reduce spectral complexity
 Protomers: Observation of multiple sites of intra-molecular
protonation
 The significance of protomer formation in routine surveillance
monitoring?
 Summary

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Introduction
 Over recent years TOF MS technology has gained in popularity as
a screening tool for food safety
 Ability to perform full spectral analysis
 Providing greater insight into the composition of a complex sample
 Ability to perform non-targeted analysis
 The freedom to measure compounds without prior compound specific tuning
 Increased specificity in complex matrices
 Accurate mass precursor ions, diagnostic accurate mass fragment ions…
 Ability to screen for larger number of compounds and adducts
 Compared to tandem quad screening
 Ability to perform historical (retrospective) data review
 The capability of performing structural elucidations of unknowns or suspected
compounds

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Introduction
 Parameters Typically Used for Confident Identification
– Time tolerances , Accurate mass tolerance, Multiple adducts,
Isotope Fits, Fragment ions, Ion ratios, Response thresholds.
 Mass accuracy tolerance = ≤5 ppm, Mass resolution tolerance =
≥20k (FWHM), Rt tolerance +/- 2.5%.
 Technology Advances Meet the Challenges of TOF Screening
– Xevo Interfaces, Stepwave and QuanTof
– Ion Mobility Separation (enhanced peak capacity)
– Software technology

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SYNAPT G2-S High Definition MS
(HDMS) - instrument schematic
Size
Shape
Charge
1. Increased sensitivity
2. Ion mobility 3. Accurate mass
measurement
Orthogonal acceleration QToF

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What is Collision Cross Section (CCS)
 CCS is an important
distinguishing
characteristic of an ion
which is related to:
– chemical structure
– 3-dimensional
conformation
 CCS is a robust and
precise
physicochemical
property of an ion.

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Ion-Mobility Principle
Small and compact –
rapid acceleration
Large,
extended

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Ion Mobility Separation Orthogonal
to UPLC Separation

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Fluoroquinolones are a class of antimicrobial
agents
 The fluoroquinolones are a class of antimicrobial agents that have been
administered to livestock for different purposes that include: (a)
prevention and control of infections, and (b) growth promotion.
 Due to the concerns regarding the spread of resistant microorganisms
in the human population, the U.S. Food and Drug Administration (FDA)
introduced a ban on the use of enrofloxacin and ciprofloxacin in
livestock production in September, 2005.
 The use of antibiotic growth promoting agents (AGPs) in animal
husbandry has been forbidden in the European Union (EU) since 2006,
when the final four antibiotics were banned as growth promoters.
 EU Maximum Residue Levels (MRLs) currently exist for eight (fluoro)-
quinolone compounds ranging from 10 to 1900 μg kg-1 dependant on
the species and tissue type.
Y
X N
OH
OOR
F
R
R R

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Identification of multiple sites of intra-molecular
protonation in the fluoroquinolone family
UNIFI CCS
Research Edition
New Ion
Mobility Software

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UNIFI - BPI for veterinary drug standards
fluoroquinolones, tetracyclines & macrolides
Ciprofloxacin
m/z 332
Generic gradient conditions –
mixed solvent standard containing 25
antimicrobial compounds
9 fluoroquinolones
CONVENTIONAL VIEW OF MASS SPECTROMETRY
DATA BUT WHAT IS THE TRUE EXTENT OF THE
SAMPLE COMPLEXITY?
UPLC-ION MOBILITY-MSE

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Fluoroquinolones Ion mobility: “9 become 18”. Why?
Protomer 1 CCS = 108.7Ǻ2
Protomer 2 CCS = 119.1Ǻ2
Ciprofloxacin Protomers m/z 332, rt 2.2 min
∆ =10.4Ǻ2

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Component summary for fluoroquinolone
protomers

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Ion mobility trace for ciprofloxacin protomers
F
O
OH
O
N N
N
+
H
H
F
O
O
+
O
N N
N
H
H
H
CCS = 108.7Ǻ2 CCS = 119.1Ǻ2
Site of protonation -1 Site of protonation -2
∆ =10.4Ǻ2

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Simultaneous generation of precursor &
fragment ions for all ions (MSE)
Collision Energy ramp applied
Fragment ions
No Collision Energy applied
Precursor ions
Chromatographic view
Data channel 1
Data channel 2
Spectral view
Rapid switching

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Ciprofloxacin protomers identified in
porcine muscle extracts
Observation of the
protonation on the
basic moiety
%CCS error <2
Observed vs scientific
library

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Fragmentation produced from individual
ciprofloxaxin protomers
Acid Group Protomer
MSE Fragments
Basic Group Protomer
MSE Fragments

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Utilising ion mobility
resolution to reduce
spectral complexity
Multi-residue analysis of
crude porcine tissue
extracts

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Conventional view – 1 peak
Ciprofloxacin in porcine tissue (100 μg/kg)

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Mobility trace view – 2 species
Ciprofloxacin in porcine tissue (100 μg/kg)

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Impact of matrix on the site of intra-
molecular protonation – replicate 1
Different ratio of basic vs acidic protomers

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Impact of matrix on the site of intra-
molecular protonation – replicate 2
Different ratio of basic vs acidic protomers

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Impact of protomer formation & differential
fragmentation pathways
 FQs are routinely monitored using tandem MS (MRM)
 Literature search has shown all major fragmentation transitions
are used
 Fragmentation pathways are found to be affected by;
– pH
– Matrix (composition & age)
– Cone voltage
– Capillary voltage
 Is MRM alone reliable?
– Potential for poor assay repeatability / reproducibility and False
Negative results

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Spectral Clean Up
Fragments are Retention time
aligned
Ion mobility resolution spectral “clean up”, more specificity

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Spectral Clean Up
Fragments are Mobility Aligned
And Retention time aligned
Ion mobility resolution spectral “clean up”, more specificity

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Richard J. Fussell
Food and Environment Research Agency (York, UK)
www.fera.defra.gov.uk

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Analytical Issues
• Variable results for fluoroquinolone compounds
• Enrofloxacin in extracts of fish
• Ciprofloxacin in extracts of honey
ciprofloxacin
enrofloxacin

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Ciprofloxacin in honey
• On-going AQC
Concentration
µg kg-1
IS corrected
‘Apparent ‘
Recovery’ (%)
RSD
(%)
n
25 112 n/a 2
50 108 n/a 1
100 111 8 21
250 106 6 9
500 109 11 3
1,000 107 9 10
2,500 100 5 12
10,000 99 6 22

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Biological variability?
0 50 100 150 200 250 300
4
5
6
7
8
9
10
Days after dosing
Log(Concentration)(g/kg)
Hive 87
Hive 89
Hive 92
Hive 93
Hive 96
Hive 109
Hive 121
Hive 128
Hive 148
Hive 221
Prediction (black solid line) and 95% prediction intervals (black dashed lines) for
log(concentration) of ciprofloxacin over time for the 10 hives
green lines
(super box 1)
red lines
(super box 2)
blues lines
(super box 3)
R J. Fussell et al, (2012) Drug Testing and Analysis, 4, S1,
118-124

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Stability of observed CCS values
Ciprofloxacin (Ǻ2) Ciprofloxacin_1 (Ǻ2)
25 106.3 117.5
50 106.2 117.3
100 106.4 117.3
200 105.3 116.4
400 105.3 116.6
25 105.4 116.8
50 105.5 116.8
100 106.2 117.4
200 106.0 117.1
400 105.9 117.1
Incurred honey AS12-028284 77 106.5 117.2
Incurred honey AS12-030416 110 106.4 117.3
Incurred honey_AS12-030415 * 160 106.8 117.7
Incurred honey_AS12-030415* 240 106.6 117.3
Mean 106.1 117.2
SD 0.5 0.3
RSD 0.5 0.2
Solvent standards
Observed CCS
Sample identity
Ciprofloxacin
concentration (ng/ml)
Matrix matched standard

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Variation of protomer formation
Matrix- matched standard
Ciprofloxacin
equiv.100 ng/g

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Variation of protomer formation incurred
honey sample
110 ng/g ciprofloxacin

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Ratio of acidic/basic protomers
25 0.23
50 0.22
100 0.27
200 0.23
400 0.21
25 0.19
50 0.14
100 0.36
200 0.31
400 0.28
Incurred honey AS12-028284 77 0.39
Incurred honey AS12-030416 110 0.36
Incurred honey_AS12-030415 * 160 0.39
Incurred honey_AS12-030415* 240 0.55
Mean 0.33
SD 0.12
RSD 36.7
Sample identity
Ciprofloxacin
concentration
(ng/ml)
Matrix matched standard
Solvent standards
Ratio cipro_cipro_1

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Difloxacin 4 Protomers Observed

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UNIFI CCS
Research Edition
New Ion
Mobility Software

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Observed residue indoxacarb with
retention time aligned fragments
One chromatographic
peak
Two fragments
peaks
M
R
M
E
Q
U
I
V
A
L
E
N
T

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Observed MSE spectra for indoxacarb in EU
RL sample FV-13
Two mobility
separated species
Ion mobility protomer resolution and even more specificity
Data processed to target two protomers

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Observed HDMSE spectra for indoxacarb
protomers in EU RL sample FV-13

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Protomers of Fenproximate
Two mobility
separated
species

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Fragmentation spectra of Fenproximate
protomers

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Prednisone Negative Ion Mode 2 Drift
Times Observed 3.48 and 3.83 ms
(Deprotonation)
OH
O OHO
O

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Tetrahydrocortisone Negative Ion Mode 2 Drift
times Observed 3.71 and 3.90 ms.
(Deprotonation).
OH
O
OH
O
OH

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Summary
 Using UPLC IMS MSE it has been possible to observe;
• Separation of different intra-molecular protonated species
• Different fragmentation routes; the site of protonation affects the
fragmentation process
• UNIFI software has been used to routinely screen for fluoroquinolone
protomers.
• Formation of protomers of ciprofloxacin is likely contribute to
observed variability in results
• Further work
- re-analysis of samples by triple quadrupole MS after re-
optimisation of acquisition parameters - re-analysis by Ion
mobility to improve separation of protomers
• Other compound classes also known to form protomers.

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Acknowledgements
Waters, Manchester, UK
Michael McCullagh
Sara Stead
David Eatough
Kieran Neeson
Jeff Goshawk
Food and Environment Research Agency (Fera), York, UK
Monica Garcia Lopez
Richard Fussell
RnAssays
Aldert Bergwerff and Wouter de Keizer - Utrecht, Netherlands