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11/30/2011
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High Resolution Accurate Mass Spectrometry in Clinical Toxicology
Applied to an ED HIV Study
William Clarke, PhD, MBA, DABCC
Johns Hopkins University School of Medicine
Disclosure
• William Clarke, PhD received compensation from Thermo Fisher in the form of travel support to give this talk, and has received research support from Thermo Fisher.
• All opinions expressed and implied in this presentation are solely those of William Clarke. The content of the presentation does not represent or reflect the views of the Johns Hopkins University or the Johns Hopkins Hospital and Health System.
• All potential conflicts of interest are dislosed and managed through the Johns Hopkins University Office of Policy Coordination
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Clinical vs. Forensic Toxicology
Clinical
• Blood or Urine specimens– Sometimes meconium
• Need rapid TAT
• Patient care focused
• Examples:– ED/Acute Care
– Pain Management/Rehab
– Pre‐surgical testing
– Evaluation of chronic toxic symptoms
Forensic
• Wider variety of sample types
• Different time frames for analysis
• Focused on investigation rather than patient care
• Examples:– Crime investigations
– Post‐mortem toxicology
– Workplace drug testing and pre‐employment screening
Clinical Toxicology Then & Now
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High Resolution‐Accurate Mass MS
• Often performed with FT‐ion trap instruments (FT‐ICR, orbitrap) or TOF instruments
• Resolution ranging from 20K to 140K dependent on instrument selected
• Mass accuracy should be < 1ppm
Exact Mass Ion Trap
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Exactive System
• Resolution100,000 at 1 scan per second 10,000 at 10 scans per second
• Mass accuracySub ppm
• Dynamic range>10,000 within a spectrum
• Scan speedUp to 10 scans per second
• Mass rangem/z 50 ‐ 4000
• Polarity switchingOne positive and one negative scan < 1 second (25K Resolution)
Octapole ion guiding
Orbitrap ‐ Principle of Operation
• A short ion packet of one m/z from c‐ trap enters the field tangentially• C‐trap is only used as an ion storage device• Ions are squeezed towards the central electrode by increasing voltage on the central electrode
• In the axial direction, ions are forced to move away from the narrow gap towards the wider gap near the equator. This initiates axial oscillations
• After the voltage increase stops, ion trajectories become a stable spiral
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Orbitrap – Principle of Operation
Makarov A. Anal. Chem. 2000, 72, 1156-1162.
Ion m/z separation depends on• Frequency of harmonic oscillations and is
proportional to sq root of M/Z
Three frequencies create oscillations• Frequency of rotation• Frequency of radial oscillations• Frequency of axial oscillations
Resolving power is• Inversely proportional to the square
root of M/Z• Proportional to acquisition time
Sensitivity is independent of acquisition speed
•
Red rings smallest m/z; Blue ring larger m/z; Green ring largest m/z
HRMS Video
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ED Serum Toxicology Analysis
• Our lab serves as the Toxicology Core for the HPTN Network Lab
– HPTN = HIV Prevention Trials Network
• Responsible for evaluating existing technologies and developing/validating new methods
• One project involves analyzing serum specimens characterized with respect to HIV and hepatitis infection
– 4500 JHH specimens from 2007
– Amphetamines, opiates, cocaine, marijuana
Screening Targets
Morphine Glucuronide Cannabinol
Morphine Cocaine
Hydromorphone Benzoylecgonine
Acetylmorphine Cocaethylene
Methamphetamine Normorphine
Ampetamine THC
MDMA Norcocaine
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LC Method
• Sample Prep• Cold ACN precipitation
• Evaporate, MeOH reconstitution
• Column– Hypersil GOLD PFP 150 x 2.1 mm, 5 m
• Mobile phase– A: 10 mM NH4FA, 0.1%FA in DI water
– B: 10 mM NH4FA, 0.1%FA in MeOH
– C: ACN/IPA/Acetone = 45/45/10 (v/v/v)
Ionization Parameters
• HESI probe
• Positive ionization mode
• Spray voltage: 4000 V
• Vaporizer temperature: 350oC
• Sheath gas: 25 units
• Aux gas: 35 units
• Sweep gas: off
• Capillary temperature: 320oC
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Representative Data
Initial Data Summary
• 98 HIV positive patients screened using Exactive; only positive results confirmed with SRM by QQQ were reported
• None were positive for THC
• 32.5% were positive for cocaine
• 21.5% were positive for opiates
• The remainder of the specimens are to be screened for the same compounds
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Additional Information (HIV Drugs)
• Performing the full scan with an Exactive mass analyzer (or TOF MS) detects all ions and allows for retrospective data queries
• Performed an ‘in silico’ screening for HIV medications (database matching based on exact mass measurement +/‐ 5 ppm)– Retention time not a factor
• Screened for 36 HIV drugs and metabolites
• 28.5% of HIV positive patients demonstrated detectable concentrations of HIV medication
Example of patient sample data
Emtricitabine
Hydroxy‐Emtricitabine
Ritonavir
Darunavir
Atazanavir
Hydroxy‐Atazanavir
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Emtricitabine:Theoretical isotopic pattern
Patient sample: Emtricitabine peak and the
experimental isotopic pattern
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Emtricitabine: Three most abundant fragments predicted by Mass Frontier software
m/z=130.0411
m/z=87.0353
m/z=113.0146
Emtricitabine: chromatograms of parent ion and fragments and all ions spectrum
Parent ion
Fragment m/z=130.0411
m/z=248.0500 in full scan
m/z=248.0500 in SCID full scan
m/z=130.0411 in SCID full scan
m/z=87.0357 in SCID full scan
m/z=113.0146 in SCID full scan
Reconstructed
chromatogram
s of SCID fragm
ents
SCID all ion spectrumChromatograms reconstructed with mass accuracy of 5 ppm
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Why is this relevant to HPTN??
• Once type of HIV prevention trial is based on pre‐exposure prophylaxis (PrEP trials)
• In the majority of these trials, the trial subject must be HIV negative, and both the subject and partner must be ARV naïve
• Self‐report is not always reliable …
HPTN 052
• >1,500 HIV‐serodiscordant couples (97% heterosexual)
• 13 sites in Africa, Asia, & the Americas
• 2 randomized groups– Immediate administration of drug
– Delayed administration of drug (CD4 < 250)
• Immediate administration group say 96% reduction in risk of HIV transmission
• Question: were the study drugs the only drugs involved?
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Antiretroviral (ARV) Screening
• Same chromatography protocol as for the ED drug screening study
• Injection of 15 standard ARV compounds to build library
• Screen tested on previously analyzed (SRM) ED specimens for method validation
• Next step: testing blinded 052 specimens
– Currently both US and non‐US specimens are being pulled for analysis
ARV Screening Menu
Nevirapine
Saquinavir
Ritonavir
Atazanavir
Nelfinavir
Amprenavir
Efavirenz
Indinavir
Lopinavir
Tipranavir
Darunavir
Emtricitabine
Zidovudine
Tenofovir
Lamivudine
Stavudine
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Nevirapine
Ritonavir
Atanazavir
Nelfinavir
Amprenavir
Efavirenz
Indinavir
Saquinavir
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ARV Screening Grid
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ARV Screening Results
• SRM Analysis from the Clinical Pharmacology core designated as reference method
• No false negatives; 3 false positives (relative to SRM)
• NPV = 100%; PPV = 93%
What’s Next for HRMS at JHH?
• Pain management/rehab toxicology panel
• Investigation of immunoassay interferences and cross reactivity
• Study with Moore Clinic (HIV) examining veracity of patient self‐report
• General unknown screening??
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Menu for Pain Management/Rehab
Challenges for Pain Management Support
• Detection of prescribed drugs the patient is supposed to be taking
• Detection of drugs (illicit and prescribed) the that aren’t part of the treatment
• Clinical labs are not typically experienced in evaluating for specimen adulteration
• Wide distribution regarding level of understanding with respect to limitations of laboratory testing
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Final Thoughts
• High resolution accurate mass spectrometry provides a relatively rapid approach to targeted toxicology screening
• An automated analyzer with an HRAMS ‘engine’ could drastically change the approach to clinical toxicology in the hospital environment
Acknowledgements
• Johns Hopkins– Autumn Breaud– Sabitha Schools– Nkechinyere Emezienna– Robert Harlan– Andrew Stolbach
• Thermo Fisher– Marta Kozak
• NIH U01‐AI‐068613 (PI: Susan Eshelman)
