ion mobility ms for the troubleshooting of methods for trace residue quantitation - waters...

©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

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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

©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

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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

©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

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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

©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

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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 ProtomerMSE Fragments

Basic Group ProtomerMSE 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

©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

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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

©2015 Waters Corporation 34

Difloxacin 4 Protomers Observed

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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

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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).

OHO

OH

OOH

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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.

©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