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
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� 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
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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
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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
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� 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
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�Targeting the compounds
Known compounds analysis in different sample matrices
Looking for known compounds
Target compound
� MS, MS/MS, MRM, SRM, SIM….etc..
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�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
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Biosynthesis of Flaviolin (Streptomyces griseus)
Enzyme Metabolite
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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
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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
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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
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� Penicillins (8)
� Cephalosporins (8)
� Aminoglucosides (9)
Different classes of Antibiotics
� Sulphonamides (12)
� Macrolides (9)
� Tetracyclines (4)
� Quinolones (10)
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� MS/MS database library for standard antibiotic compounds
UnderCID andHCD conditions
� Targeted Approach
� Penicillins (8)
� Cephalosporins (8)
� Aminoglucosides (9)
� Sulphonamides (12)
� Macrolides (9)
� Tetracyclines (4)
� Quinolones (10)
UnderCID andHCD conditions
� ToxID database library for antibiotic compounds
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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:
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T(min) A) 1mM HFBA + 0.5% FA B) 0.5%FA in MeOH/ACN (50:50) (V/V)
� Penicillins (8)
� Cephalosporins (8)
� Aminoglucosides (9)
� Sulphonamides (12)
� Macrolides (9)
� Tetracyclines (4)
� 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
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Capillary Temperature Effect:
�At 430° C Not identified
MS Source :
� Penicillins (8)
� Cephalosporins (8)
� Aminoglucosides (9)
� Sulphonamides (12)
� Macrolides (9)
� Tetracyclines (4)
� Quinolones (10)
Penicillin V, Oxacillin,
�At 350° C Identified
by using accurate mass
Cloxacillin and Dicloxacillin
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ToxID 2.1.1 Configuration File,,,,,,,,,,,,
� Penicillins (8)
� Cephalosporins (8)
� Aminoglucosides (9)
� Sulphonamides (12)
� Macrolides (9)
� Tetracyclines (4)
� 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
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Nafcillin
Cefalexin
Sulfaphenazole
Sulfathiazole
Antibiotics spikedPork Sample
(MRLs)Extraction
LC-LTQ-Orbitrap MS
AppliedToxID
Identifiedspiked
Antibiotics
Sulfathiazole
Erythromycin
Erythromycin
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NON-TARGETED APPROACH
�ToxID Software (Possible metabolites)
�SIEVE Software
�Fragmentation�Fragmentation
�Standard compound
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SIEVE Software
Blank samplesLCMS Data
Antibiotic treatedsamples LCMS Data
Find differences = Possible Biomarkers
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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