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©2015 Waters Corporation 1
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
©2015 Waters Corporation 2
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
©2015 Waters Corporation 3
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
©2015 Waters Corporation 4
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
©2015 Waters Corporation 5
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
©2015 Waters Corporation 6
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.
©2015 Waters Corporation 7
Ion-Mobility Principle
Small and compact – rapid acceleration
Large, extended
©2015 Waters Corporation 8
Ion Mobility Separation Orthogonal to UPLC Separation
©2015 Waters Corporation 9
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
©2015 Waters Corporation 10
Identification of multiple sites of intra-molecular protonation in the fluoroquinolone family
UNIFI CCS Research Edition
New Ion
Mobility Software
©2015 Waters Corporation 11
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
©2015 Waters Corporation 12
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
©2015 Waters Corporation 13
Component summary for fluoroquinolone protomers
©2015 Waters Corporation 14
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
©2015 Waters Corporation 15
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
©2015 Waters Corporation 16
Ciprofloxacin protomers identified in porcine muscle extracts
Observation of the protonation on the
basic moiety
%CCS error <2 Observed vs scientific
library
©2015 Waters Corporation 17
Fragmentation produced from individual ciprofloxaxin protomers
Acid Group ProtomerMSE Fragments
Basic Group ProtomerMSE Fragments
©2015 Waters Corporation 18
Utilising ion mobility
resolution to reduce
spectral complexity
Multi-residue analysis of
crude porcine tissue
extracts
©2015 Waters Corporation 19
Conventional view – 1 peak Ciprofloxacin in porcine tissue (100 μg/kg)
©2015 Waters Corporation 20
Mobility trace view – 2 species Ciprofloxacin in porcine tissue (100 μg/kg)
©2015 Waters Corporation 21
Impact of matrix on the site of intra-molecular protonation – replicate 1
Different ratio of basic vs acidic protomers
©2015 Waters Corporation 22
Impact of matrix on the site of intra-molecular protonation – replicate 2
Different ratio of basic vs acidic protomers
©2015 Waters Corporation 23
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
©2015 Waters Corporation 24
Spectral Clean Up
Fragments are Retention time aligned
Ion mobility resolution spectral “clean up”, more specificity
©2015 Waters Corporation 25
Spectral Clean Up
Fragments are Mobility Aligned And Retention time aligned
Ion mobility resolution spectral “clean up”, more specificity
©2015 Waters Corporation 26
Richard J. Fussell
Food and Environment Research Agency (York, UK)
www.fera.defra.gov.uk
©2015 Waters Corporation 27
Analytical Issues
• Variable results for fluoroquinolone compounds • Enrofloxacin in extracts of fish • Ciprofloxacin in extracts of honey
ciprofloxacin
enrofloxacin
©2015 Waters Corporation 28
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
©2015 Waters Corporation 29
Biological variability?
0 50 100 150 200 250 3004
5
6
7
8
9
10
Days after dosing
Lo
g(C
on
ce
ntr
atio
n)
(g
/kg
)
0 20 40 60 80 100 120 140
5
5.5
6
6.5
7
7.5
8
8.5
9
9.5
10
Days after dosing
Log
(Con
cen
tratio
n)
(g/k
g)
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
©2015 Waters Corporation 30
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 CCSSample identity
Ciprofloxacin
concentration (ng/ml)
Matrix matched standard
©2015 Waters Corporation 31
Variation of protomer formation Matrix- matched standard
Ciprofloxacin equiv.100 ng/g
©2015 Waters Corporation 32
Variation of protomer formation incurred honey sample
110 ng/g ciprofloxacin
©2015 Waters Corporation 33
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
©2015 Waters Corporation 34
Difloxacin 4 Protomers Observed
©2015 Waters Corporation 35
UNIFI CCS Research Edition
New Ion
Mobility Software
©2015 Waters Corporation 36
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
©2015 Waters Corporation 37
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
©2015 Waters Corporation 38
Observed HDMSE spectra for indoxacarb protomers in EU RL sample FV-13
©2015 Waters Corporation 39
Protomers of Fenproximate
Two mobility separated species
©2015 Waters Corporation 40
Fragmentation spectra of Fenproximate protomers
©2015 Waters Corporation 41
Prednisone Negative Ion Mode 2 Drift Times Observed 3.48 and 3.83 ms (Deprotonation)
OH
O OHO
O
©2015 Waters Corporation 42
Tetrahydrocortisone Negative Ion Mode 2 Drift times Observed 3.71 and 3.90 ms. (Deprotonation).
OHO
OH
OOH
©2015 Waters Corporation 43
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.
©2015 Waters Corporation 44
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