Alturas Analytics, Inc Presentation

1
HPLC/MS/MS Used in Homeland Security Efforts
Quantitative Bioanalysis of Drugs Used to Protect
Against Chemical Warfare Agents
Shane Needham1, Binying Ye1 and J. Richard
Smith2 1) Alturas Analytics, Inc. Moscow, ID 2)
US Army Medical Research Institute. Aberdeen
Proving Ground, MD
December 2002
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Chemical Warfare Agents

• Sarin gas, Soman gas organo-phosphorus nerve
  gas agents
• Extremely Toxic
• Active acetylcholinesterase inhibitor
  covalently bound, irreversibly
• Lethal dose lt0.01 mg/kg (0.5 mg per adult)
• Easy to make
• Death can occur in less than 10 minutes upon
  exposure
• Easy to deliver - Respiratory or contact exposure
• Build-up of acetylcholine causes nerve impulses
  to misfire and bodily functions shut down

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Structure of Nerve Gas Agent, Sarin
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Protection from Nerve Gas Agents

• Gas mask and protective clothing
• Pre-exposure antidotes (pyridostigmine)
• Cholinergic
• Take orally before possible exposure to nerve gas
• Post-exposure antidotes (atropine, oximes, etc.)
• Anticholinergic
• Take IM injection after suspected exposure

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Atropine

• Immediate action upon exposure to nerve agents
• Atropine becomes bound to the receptors for
  acetylcholine, which are present in the
  cholinergic synapse. The signal is transmitted
  but if atropine has become bound to the receptor,
  then no such transmission takes place.

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Pyridostigmine Bromide (PB)

• Pretreatment compound to protect against chemical
  warfare nerve agents
• Enhances the effects of atropine/oxime treatments
  for exposure to nerve gas agents
• Pyridostigmine inhibits acetylcholinesterase and
  protects the enzyme against inhibitory effects of
  nerve agents. The pyridostigmine-inhibited
  enzyme is continuously released to active state
  and thereby can reasonably effectively maintain
  the transfer of nerve impulses despite injury
  caused by nerve agents

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Nerve Cell
Nerve Cell
Muscle
Muscle
Acetylcholinesterase
Muscle Receptor
Acetylcholine
Nerve Gas Agent
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Nerve Cell
Nerve Cell
Muscle
Muscle
Acetylcholinesterase
Muscle Receptor
Pyridostigmine
Acetylcholine
Nerve Gas Agent
Atropine
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Homeland Security Efforts Post 9/11

• Anti-nerve gas agents distributed to metro areas
• Hospitals, fire departments, police departments
• Early warning systems in the event of an attack

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PB Analysis Needs

• Measure low level concentrations in guinea pig
  plasma
• lt 500 pg/mL
• Extract less than 30 ?L of plasma

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PB Analysis Challenges

• PB is highly polar thus difficult to retain with
  reversed-phase HPLC without ion-pair reagents
• Ion-pair reagents may reduce ESI-MS signal
• PB is a quaternary amine thus secondary
  interactions cause tailing and broad peaks with
  silica based reversed-phase HPLC columns

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Pyridostigmine Structure
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Method Development

• Will standard methods without ion-pair reagents
  (TFA, etc.) and typical reversed-phase columns
  work?
• Does TFA significantly decrease ESI-MS signal for
  the analysis of PB?

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HPLC/MS/MS Chromatogram from the Analysis of
Pyridostigmine On a PRP Column without the
addition of TFA
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HPLC/MS/MS Chromatogram from the Analysis of
Pyridostigmine On a PRP Column with the addition
of TFA
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Experimental

• Mass spectrometry
• Sciex API3000-LC/MS/MS
• Turboionspray (400 C)
• MRM Transitions- 181 ? 124 (PB), 187 ? 130
  (PB-D6)
• HPLC
• Two Shimadzu HPLC pumps for delivery of mobile
  phase
• Column PRP 1 poly(styrene-divinylbenzene)
  2.1x50 mm
• Mobile phase 2 ACN in water with 0.1 TFA and
  1 mM ammonium acetate
• Flow rate 0.6 mL/min

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HPLC/MS/MS Chromatogram from the Analysis of a
Pyridostigmine Standard (0.50 ng/mL) from Guinea
Pig Plasma.
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HPLC/MS/MS Chromatogram from the Analysis of a
Pyridostigmine Sample from Mouse Plasma (Calc.
Conc. 3.6 ng/mL).
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Standard Curve Results
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Recent Developments

• Full GLP project
• US Army needs GLP validation in guinea pig plasma
• Need even lower limits of quantitation
• Improved chromatography and extraction thus able
  to detect gt 5x lower levels (0.1 ng/mL)

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(No Transcript)
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Improved Method

• HPLC
• Strong-cation exchange (SCX)-poly(2-sulfoethyl
  aspartamide)
• Better peak shape and the use of a higher
  concentration of organic (70 ACN)
• gt 5 X better signal
• Extraction
• Direct injection after precipitation
• High organic in HPLC method allows direct
  injection of precipitate

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Improved Method Experimental

• HPLC
• Two Shimadzu HPLC pumps for delivery of mobile
  phase
• Column SCX 2.0 x 35 mm (PolyLC)
• Mobile phase 70 ACN in water 100 mM ammonium
  formate
• Flow rate 0.6 mL/min
• Extraction
• 25 ?L of plasma and 50 ?L ACN
• Inject supernant

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0.51
2260 CPS
0.1 ng/mL Pyridostigmine From Plasma Using New
SCX Method
5X Lower Limit of Quantitation!!
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Time (Min.)
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QC Results with New GLP Method (0.10 t0 50 ng/mL)
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Other HPLC/MS/MS Results

• Bare silica column (weak cation exchanger) did
  not give good results poor peak shape
• Hydrophilic interaction liquid chromatography
  (HILC) did not give good peak shape
  Poly(hydroxyethyl A)
• Increasing buffer concentrations did not decrease
  MS signal (5 to 100 mM)
• Increasing pH on SCX column increased retention

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Pyridostigmine Summary

• Developed method for HPLC/MS/MS analysis of
  pyridostigmine from plasma
• HPLC method development important
• Improved detection limits gt5x with SCX HPLC
  method
• SCX HPLC method allows direct injection of
  supernant from precipitation
• Fully validated GLP method

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Atropine Analysis Needs

• Limits of quantitation as low as possible in
  monkey serum for new formulation studies
• lt 10 pg/mL
• Extract less than 300 ?L of serum

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Atropine Structure
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Method Development

• Conventional 96-well plate liquid-liquid
  extraction gave extraction recoveries lt30
• Off-line solid phase extraction (SPE) gave
  recoveries gt50 but not reproducible
• Used polymer solid-phase material Waters Oasis
  like

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Method Development

• In-house Universal extraction method, buffered
  brine (pH 11) with acetonitrile
• Not a typical acetonitrile protein precipitation
• Aqueous layer (plasma) and organic layer separate
• More selective than conventional acetonitrile
  precipitations/extractions
• Gave reproducible recoveries gt65

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Experimental

• Mass spectrometry
• Sciex API3000-LC/MS/MS
• Turboionspray (400 C)
• MRM Transitions- 290 ? 124 (Atropine), 293 ? 127
  (Atropine-D3)
• HPLC
• Two Shimadzu HPLC pumps for delivery of mobile
  phase
• Column Advantage Armor C8 2.1x30 mm
• Mobile phase gradient in 2 minutes with 2 mM
  ammonium formate, 0.1 formic acid and MeOH
  mixtures
• Flow rate 0.6 mL/min

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Atropine Standard Curve Results
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Max. 2520.0 cps.
1.31
2520
Chromatogram from the Analysis of 0.01 ng/mL
Atropine Standard
2400
2200
2000
1800
1600
1400
Intensity, cps
1200
1000
800
600
400
2.07
2.00
0.87
1.91
200
1.84
1.75
1.47
0.84
1.17
0.89
1.50
1.08
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Time, min
35
Max. 1300.0 cps.
0.87
1300
Chromatogram from the Analysis of a Blank
1200
1100
Atropine Carryover? Peak
1000
900
1.30
800
700
Switching Valve Artifact
Intensity, cps
600
500
400
300
200
1.69
1.71
2.07
1.99
100
0.95
1.11
1.53
1.83
1.49
1.24
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Time, min
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Max. 3.3e5 cps.
1.32
3.3e5
Chromatogram from the Analysis of a Monkey
Serum Sample 320 min. Post Dose
3.2e5
3.0e5
2.8e5
2.6e5
2.4e5
2.2e5
2.0e5
1.8e5
Intensity, cps
1.6e5
1.4e5
1.2e5
1.0e5
8.0e4
6.0e4
4.0e4
2.0e4
1.50
1.59
1.74
0.85
1.80
1.88
1.99
0.90
1.17
1.08
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Time, min
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Atropine Assay Facts

• Standard curve ranges from 0.01 to 1 ng/mL
• Correlation coefficients gt0.99
• Limits of Quantitation of 0.001-0.002 ng/mL
  achievable if blank problem solved only
  occurs in extracted samples
• Limits of Quantitation of 0.01 ng/mL are overly
  sufficient for monkey studies

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Atropine Blank Problem

• Only occurs in extracted injected samples
• Not reproducible
• A group at the US Army has seen similar blank
  problem using totally different method
• Early method development to minimize blank
  problem
• Area of standard preparation and dilution must be
  performed in separate lab than sample preparation

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Conclusions

• PB and Atropine drugs are both used to counter
  act nerve gas agents
• HPLC/MS/MS assays provide rapid and selective
  methods for analysis of PB and atropine
• In collaboration with US Army, future work
  includes HPLC/MS/MS support of new chemical
  entities, better formulations and improved
  delivery for anti-chemical warfare agents