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The LTQ-Orbitrap mass spectrometer: Identification of antibiotic residues in

biological matrices

AOAC-ASFILAB International Workshop, 23-24 November, PARIS

Thota Jagadeshwar Reddy, Dominique Pessel & Eric VerdonAFSSA-LERMVD, Fougères, France

CRL for antibiotic/dye residues in food from animal origin

Titre du diaporamaTitre du diaporama

� Different classes of antibiotics are used extensively in both humans and food- producing animals to treat infections

Antibiotics

� Antibiotic-resistant

Antibiotics

2

� Antibiotic-resistant bacterial strains that

may cross from livestock to humans

� Identification

� Control of Antibiotics Usage


Analysis by biological and physico-chemical methods

� Biological method: Especially Microbiological methods

1) Premi test (Bacteria: stearothermophilus)• Less time• On site analysis• Wide range of antibiotic compounds

3

o No structure determination

� Identification


2) 4 plate test:

• Time consuming process• Different media• Wide range of antibiotic compounds

Sample(+Ve Antibiotic activity)

Sample (-Ve Antibiotic activity)

Sample(+Ve Antibiotic activity)

Bacterial Growth Media

o No structure determination

� Identification


� Physico-chemical methods:

• Spectrophotometric detectors

• Mass spectrometry (Specificity and Sensitivity)

Mass spectrometryplaysan important role for the traceMass spectrometryplaysan important role for the traceanalysis of compounds in different sample matrices

� Identification and structure determination


�Targeting the compounds

Known compounds analysis in different sample matrices

Looking for known compounds

Target compound

� MS, MS/MS, MRM, SRM, SIM….etc..


�Non-targeting compounds

� In biological system some compounds undergo chemical

modification (Drug Metabolism)

Ex. Hydroxylation, Oxydation, Reduction, Dehydration..etc…

Oxidation involving cytochrome P450 (the microsomal mixed-function Oxydase)

Aneasthetic and Antidysrhythmic Drug

CH3

CH3

HN C

H2C N

C2H5

C2H5

O CH3

CH3

HN C

H2C N

C2H5

C2H5

O

OHLignocaine 3-Hydroxylation of lignocaine

Cytochrome P450

Protein

Drug


Biosynthesis of Flaviolin (Streptomyces griseus)

Enzyme Metabolite


Electron Ionization: universal conditions, databases are available

Electrospray ionization:Collision activation dissociation mass spectra of electrosprayed ions are

heavily instrument dependant

• Quadrupole

• Sector instruments

Many analyzers are available today

M +.M-e

70 eV

• Sector instruments

• Q-Tof

• Iontrap

• FT-ICR

Technology diversity Huge work to create reliable databases for ESI-MS


Linear ion TrapC-Trap

HCDCollision Cell

Orbitrap

API Source

LTQ-Orbitrap Mass Spectrometer(Consecutive

Fragmentation)(Structure elucidation)MSn

LTQ Orbitrap XL Instrument Diagram

Dr. Alexander Makarov


Why we need High Resolution?

Ex. NH3 has a mass of 17.0265 and OH 17.0027.

� Mass of proton : 1,6726 x 10^(-27) kg� Mass of neutron: 1,6749 x 10^(-27) kg

� Calculating Error in ppm

• (Exact mass found - Exact mass calculated)/Exact mass calculated∆M/M x 106 Error in ppm


� Penicillins (8)

� Cephalosporins (8)

� Aminoglucosides (9)

Different classes of Antibiotics

� Sulphonamides (12)

� Macrolides (9)

� Tetracyclines (4)

� Quinolones (10)


� MS/MS database library for standard antibiotic compounds

UnderCID andHCD conditions

� Targeted Approach

� Penicillins (8)




� Macrolides (9)


� Quinolones (10)

UnderCID andHCD conditions

� ToxID database library for antibiotic compounds


LC Method :

� Column: LichroCART C18, 125x3 mm, 5 µm

� Injection Volume: 25 µL

� Sample concentration: 250 ppb and 125 ppb

� Flow Rate: 500 µL/min

� Gradient:

__________________________________________________________________

T(min) A) 1mM HFBA + 0.5% FA B) 0.5%FA in MeOH/ACN (50:50) (V/V)

� Penicillins (8)




� Macrolides (9)


� Quinolones (10)

T(min) A) 1mM HFBA + 0.5% FA B) 0.5%FA in MeOH/ACN (50:50) (V/V)

0 90 10

12 5 95

15 5 95

16 90 10

22 90 10


Capillary Temperature Effect:

�At 430° C Not identified

MS Source :

� Penicillins (8)




� Macrolides (9)


� Quinolones (10)

Penicillin V, Oxacillin,

�At 350° C Identified

by using accurate mass

Cloxacillin and Dicloxacillin


ToxID 2.1.1 Configuration File,,,,,,,,,,,,

� Penicillins (8)




� Macrolides (9)


� Quinolones (10)

Antibiotic compounds mixture

250/125 ppbLCMSanalysis

ToxID 2.1.1 Configuration File,,,,,,,,,,,,,,,,,,,,,,,,Index,Compound Name,Elemental Composition,Polarity,Analyte Type,Expected RT,Intensity Threshold,Adduct1,Adduct2,Adduct3,Fragment1,Fragment2,Fragment31,1-(3-Chlorophenyl)-Piperizine,C10H13ClN2,+,parent drug,4.7,1000,1,1,1,154,,2,"1,1-Dimethylbiguanide",C4H11N5,+,parent drug,1,1000,1,1,1,60.2,,,,,,,,,,,,,,3,11-hydroxy-delta-9-THC,C21H30O3,+,metabolite,5.8,1000,1,1,1,295.3,,4,Amoxicillin,C16H19N3O5S,+,parent drug,5.05,1000,1,1,1,,,5,Ampicillin,C16H19N3O4S1,+,parent drug,6.19,1000,1,1,1,,,

ToxIDIdentification of all Antibiotic compounds


Nafcillin

Cefalexin

Sulfaphenazole

Sulfathiazole

Antibiotics spikedPork Sample

(MRLs)Extraction

LC-LTQ-Orbitrap MS

AppliedToxID

Identifiedspiked

Antibiotics

Sulfathiazole

Erythromycin

Erythromycin


NON-TARGETED APPROACH

�ToxID Software (Possible metabolites)

�SIEVE Software

�Fragmentation�Fragmentation

�Standard compound


SIEVE Software

Blank samplesLCMS Data

Antibiotic treatedsamples LCMS Data

Find differences = Possible Biomarkers


Conclusions:

� Opened new area for the identification of untargeted antibiotic residuesin meat and milk

� Identification of these compounds using high resolution mass spectrometersuch as LTQ-Orbitrap mass spectrometer

� MS/MS database library generation (CID and HCD)

� Application of ToxID and SIEVE software

� Comparison with synthesized chemical standard compound

� FUTURE : same procedure applied to NRCP samples


Thank you all

21

Thank you all

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