The Use of LCMS for

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The Use of LC-MS for Systematic Toxicological
Analysis (STA)
Michelle Wood Jan Bohuslavek, PhD
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Outline

• Background to Systematic Toxicological Analysis
  (STA)
• Review of existing screening techniques
• Waters approach to STA
• Details of methodology

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Background
The Daily Challenge analysis of biological
specimens

• The known (or suspected toxic analyte)
• Class identified by screening assay
• Routine TDM/adverse effects to medication
• History of misuse of prescription medication or
  illicit drugs
• Evidence at scene (drug paraphernalia, tablets)

• The unknown
• Typical situation presentation to ER
• illicit compounds?
• Misuse of prescribed drugs (accidental/intentional
  )?
• Or basically anything else!

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Techniques for Toxicology Screening Different
Approaches

• Traditional screening methods
• Immunoassays (FPIA, EMIT, RIA)
• Screening quantitation
• Cross reactivity, cost (10/panel)
• Not available for all drugs
• LC/UV
• Identification in complex mixtures
• UV libraries exist
• Sensitivity/specificity issues
• GC/MS (gold standard)
• Relatively inexpensive
• EI existing libraries (PMW, NIST, Wiley)
• Derivatisation, labourious
• Not suitable for ALL compounds

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LC-MS ChromaLynx Approach

• Current method combines LC-MS with searchable
  database (in source CID)
• Benefits of using LC
• large number of samples can be analysed by LC
• Good alternative for the analysis of polar,
  thermally labile, and semi- and non-volatile
  compounds
• no derivatisation
• Benefits of multiple identification criteria
• Collected data is matched to a searchable (and
  appendable) library

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Toxicology Screening LC/MSInformation
available from one LC/MS Analysis
HPLC
PDA
MS

• Polarity Information
• RT
• (1)

• Absorption Information
• UV spectrum
• (2) Currently not used

• Mass Spec Info
• Parent Ion (3)
• (CID) Fragment Ions (4-15!)

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Mass Spectra Database Library BuildingLC/MS
Experimental Conditions

• Instrumentation
• ZQ, Quattro Micro
• Alliance 2695
• Liquid Chromatography
• Generic gradient based on water/acetonitrile
  mobile phase with ammonium formate and formic
  acid ( 30 min run)
• 2.1 mm id - 3. 5µm XTerra C18 MS column _at_ 35C
  with pre-column
• LC/MS allows collection of multi-spectral data
• Pos/neg mass spectra recorded at different CV
• One analyte might be characterised by up to 12
  mass spectra plus retention time

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LC/MS and In-Source CID Generation of Fragments

• Ions formed in the atmospheric region are
  sampled through the sample cone.
• These ions are entering a relatively high
  pressure region (1 mBar) and can be accelerated
  by applying a voltage between the sample cone and
  the extractor cone.
• These ions gaining speed will hit residual
  neutral molecules in that region and will
  subsequently gain internal energy.
• At a certain level depending on the ion nature,
  internal energy will reach a level to the point
  that the ions fragment into specific ions.

Extractor
Sample Cone Cone Gas
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LC/MS and In-Source CID Generation of Fragments
Library Concept
No Fragmentation
MH
M-H-
15 Volts
-15 Volts
30 Volts
-30 Volts
Exhaustive Structural Information
45 Volts
-45 Volts
60 Volts
-60 Volts
75 Volts
-75 Volts
90 Volts
-90 Volts
F
F-
Total Fragmentation
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In-Source CID - LC/MS Library Some Examples
TIMOLOL Free Base C13H24N4O3S MW 316.4 (Nominal
316)CAS 91524-16-2
ESI_at_90V
ESI_at_75V
ESI_at_60V
ESI_at_45V
ESI_at_30V

• Results in getting 6 specific m/z values with
  different fragmentation patterns for
• each voltage value m/z _at_ 317 MH,
• 261,244,188,144 113

ESI_at_15V
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In-Source CID - LC/MS Library Some Examples
ESI-_at_90V
BUTOBARBITAL C10H16N2O16 MW 212.25 (Nominal
212)CAS 77-28-1
ESI-_at_75V
ESI-_at_60V
ESI-_at_45V
ESI-_at_30V

• Barbiturates give a good response to negative
  ESI. The ion _at_ m/z 155 is generally observed for
  most of the barbiturates.

ESI-_at_15V
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In-Source CID - LC/MS Library Some Examples
NITRAZEPAM C15H11N3O3 MW 281.27 (Nominal
281)CAS 146-22-5
ESI_at_90V
ESI_at_70V
ESI_at_50V
ESI-_at_90V
ESI-_at_70V

• Some molecules may give a good response in both
  positive negative ESI thus allowing
  unambiguous identification.

ESI-_at_50V
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In-Source CID - LC/MS Library Some Examples
Acepromazine aceprometazine UV versus MS

• Same molecular formula
• Similar retention times
• Same UV spectra

Identical mass spectra _at_ 15 30 V ? Differences
appear around 45 V ? Differentiation possible ?
The probability to have 2 compounds of the same
mass, that have the same fragmentation patterns
(over different CVs), in both positive and
negative ion polarities, eluting at the same
retention time (and having the same absorption
characteristics) is ...very low!
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Toxicology Screening LC/MSAnalysis of the
Unknown Challenges

• Challenges
• In-depth examination detection of the
  components
• Minor peaks
• Hidden peaks
• Relating to identification of the analytes
• Mass spectrum extraction/refining process
• Algorithm used in Library search (NIST) and
  specific filters
• Mass spectra database
• Content of the library
• Sufficient spectral information

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Chromatogram Examination and Peak Detection Do I
really see what I should see ?
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Chromatogram Examination and Peak Detection Mass
Spectra Extraction
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ChromaLynx for Toxicological Screening Background

• Especially developed for screening using ESI/MS
  mass spectral libraries
• Data processing software based on chromatographic
  deconvolution techniques
• Chromatogram examination
• Component detection
• Candidate proposal

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ChromaLynx for Toxicological Screening Chromatogra
m Examination How does it work ?

• Virtually, all scans (all mass spectra) from
  each function (each TIC chromatogram) can be
  examined and extracted ion chromatograms (EIC)
  are generated for the most intense ions.

Automated Ion Chromatogram Generation
Automated Mass Spectrum Extraction _at_ scan 237 for
Function 5
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ChromaLynx for Toxicological Screening Component
Detection and Mass Spectrum Extraction

• Peaks in extracted ion chromatograms should be
  within a limit in terms of scan position to be
  considered as a sole peak. Mass spectrum is then
  extracted.

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A Example of Polyintoxication - Suicide attempt
by massive ingestion of various medication

• The context of the intoxication
• A depressive woman, 65 years old, was brought to
  the emergency area of the hospital of Lille
  (France, Nord) after emergency medical
  technicians were alerted and took care of her.
  She mentioned that she had taken different pills
  from among her regular medications. Urine samples
  were immediately analyzed for toxicants.
  Toxicologists were looking to both molecules
  coming from the regular treatment and unexpected
  active substances that may have been absorbed by
  the patient. The regular known medications taken
  by the patient are the following

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A Example of Polyintoxication - Suicide attempt
by massive ingestion of various medication

• Experimental conditions

Analytical Equipment and Instrumentation Waters
Micromass Toxicology Screening LC/MS System
constituted of - ZQTM Single Quadrupole Mass
Spectrometer - Alliance 2695 Separations
Module - MassLynxTM 4.0 Data Station -
ChromaLynxTM 4.0 Application Manager Sample
Preparation Liquid/liquid extraction at 2 pH (4.5
9.0) using dichloromethane/ether/hexane
305020 0.5 isoamyl alcohol. HPLC
Separation Method Waters XTerraTM MS Column
Precolumn C18, 3.5 µm, 2.1mm id x 150 mm (10 mm
for precolumn) _at_ 30C Mobile Phase based on
Water/Acetonitrile with Ammonium Formate 5 mM _at_
pH 3 Gradient 5 organic to 90 organic from 2
min to 16 minutes MS Operating
Conditions Capillary 3.5 kV in both positive and
negative ion modes Source Temperature _at_120C
Desolvation Temperature _at_ 250C Desolvation Gas
Flow Rate _at_ 350 l/h Cone Gas Flow Rate _at_ 100
l/h Function 1 Full Scan - Negative ESI from 100
to 650 amu in 250 ms _at_ 30 Volts Functions 2 to 7
Full Scan - Positive ESI from 100 to 650 amu in
250 ms _at_ from15 Volts to 90 Volts
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A Example of Polyintoxication - Suicide attempt
by massive ingestion of various medication
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A Example of Polyintoxication - Suicide attempt
by massive ingestion of various medication

• Results discussion
• All expected molecules were found excepted
  Tramadol
• Additional molecules not mentioned were
  identified as the main cause of the intoxication
• Meprobamate (not detected in UV) - Mepronizine
• Aceprometazine - Mepronizine
• Bromazepam
• Additional processing using 6 functions instead 3
  led to identical results.

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ChromaLynx for Toxicological Screening Conclusion

• From a technical point of view
• LC/MS constitutes an excellent additional tool
  for Toxicological Screening
• The library contains exhaustive structural
  information for each compound
• ChromaLynx utilises a very efficient data
  Deconvolution process which
• Avoid the risk of missing peaks hidden in the
  baseline or difficult coelution
• Allows to detect small amount of compounds in
  Full Scan
• Includes multi-functions examination
• Includes Retention time control
• Potentially useable on any Waters-Micromass
  Quad-Based Instrument

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Acknowledgements
M. Lhermitte and L. Humbert, Biochemistry and
Molecular Biology Laboratory, Calmette Hospital,
Lille (France)
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Thanks for your attention Questions ?