Drug Screening in Biological Samples using High Resolution Mass
Spectrometry
William Clarke, PhD, MBA, DABCC
Johns Hopkins University School of Medicine
Disclosures
• Grant/Research Support: Thermo Fisher, Shimadzu, Nova Biomedical, Instrumentation Laboratories, miDiagnostics
• Consultant/Advisory Board: Thermo Fisher, Shimadzu, Nova Biomedical, Roche Diagnostics, Instrumentation Laboratories, BD Diagnostics
• Equity: miDiagnostics
• Honorarium/Expenses: None
• Intellectual Property/Royalty Income: None
RAPID ANTIRETROVIRAL (ARV) SCREENING
Rapid Analysis with Q Exactive and LC-MS
• Preparation: protein precipitation plates on a Tecan Evorobotic station
• Two minute chromatographic method for 20 compounds
• Full scan MS-data dependent MS2 (ddMS2): fragmentation is triggered if a compound of interest is detected above a threshold; exact mass for analysis of fragments
• Positive mode electrospray ionization; resolution = 17.5K at m/z 200
• Detection utilizes 1-3 product ions per compound; verification possible through data query for precursor exact mass
Nevirapine (NVP) 20 ng/mL in serumRT: 0.00 - 2.00 SM: 11G
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0
Time (min)
0
20
40
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Re
lative
Ab
un
da
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0
20
40
60
80
100
Re
lative
Ab
un
da
nce
0
20
40
60
80
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Re
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Ab
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Ab
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1.29 1.291.29
1.281.31
1.281.32
1.271.33
1.27 1.34
1.351.26
1.25 1.371.39 1.461.23 1.520.00 1.100.21 1.060.350.29 1.600.16 0.38 0.910.850.46 0.790.730.52
1.29 1.291.29
1.281.31
1.281.32
1.27
1.331.27
1.341.26
1.351.25
1.371.401.23 1.471.21
1.29 1.291.29
1.281.31
1.281.32
1.271.27 1.33
1.27 1.34
1.351.26
1.25 1.371.40 1.471.22
1.291.29 1.29
1.28
1.311.28
1.321.271.27 1.33
1.27 1.34
1.351.26
1.25 1.371.40 1.471.22
NL: 1.64E7
m/z= 267.1212-267.1266 F: FTMS + p ESI Full ms [120.00-750.00] MS Four_Channel_test_19
NL: 4.05E5
m/z= 80.0493-80.0509 F: FTMS + p ESI d Full ms2 [email protected] [50.00-290.00] MS Four_Channel_test_19
NL: 4.29E5
m/z= 107.0597-107.0619 F: FTMS + p ESI d Full ms2 [email protected] [50.00-290.00] MS Four_Channel_test_19
NL: 1.64E6
m/z= 226.0825-226.0871 F: FTMS + p ESI d Full ms2 [email protected] [50.00-290.00] MS Four_Channel_test_19
NVP precursor XIC
NVP product ion 3 XIC
NVP product ion 2 XIC
NVP product ion 1 XIC
High Throughput Screening Assay• Automated sample preparation
• 30 min/96-well plate (active run time); 0.3 min/sample
• 4 min to first result by LC-MS• 2 min sample to sample
• Approx. 3h/plate (172 min for subjects + QC)
• Overnight runs (18h) = 6 plates per instrument
• 2 instruments = 972 specimens/day
• LOD = 2-20 ng/mL for all ARV drugs
Applications of the multi-drug assay in HPTN studies
Cross-sectional HIV incidence (as part of a multi-assay algorithm)
HPTN 043: 6.7% of MAA-positive individuals had ARV drugs and were
excluded from incidence assessments Laeyendecker et al. PLoS One. 2013; 8:e68349
Discrepant HIV diagnostic test results
HPTN 043: Most HIV-infected adults with discordant rapid tests were
virally suppressed without ARV drugsFogel et al. J AIDS. 2015; 69:446
Transmitted HIV drug resistance
HPTN 061: Analysis of ARV drug resistance in seroconverters; estimation of
transmitted drug resistance was reduced (23%12%) after accounting for
ARV drug use
Applications of the multi-drug assay in HPTN studies
Chen et al. J AIDS 2015; 69:446
Applications of the multi-drug assay in HPTN studies
Undisclosed ART use among HIV-infected participants
HPTN 052: 45 (46.9%) of 96 “ARV naïve” index participants who had a
VL<400 at enrollment were on ART; many continued off-study ART after
enrollment Fogel et al. J Infect Dis. 2013; 208:1624
Marzinke et al. Clin Infect Dis. 2014; 58:117
Undisclosed knowledge of HIV status
HPTN 061: >40% of 155 men initially characterized as “newly diagnosed,
ARV naïve” were on ART at enrollment; many had unusual patterns of ARV
drugs detected
Use of ARV drugs in HIV-uninfected cohorts
HPTN 064: 2% of 1,806 HIV-uninfected women had ARV drugs detected at
enrollment (15% in Baltimore; 5% in Bronx; NNRTIs and PIs; 1-4
drugs/sample)
HPTN 068: None of >2,000 HIV-uninfected young women had ARV drugs
detected at enrollment
HPTN 073: Two of 208 HIV-uninfected Black MSM were taking off-study
TDF/FTC at enrollment
Applications of the multi-drug assay in HPTN studies
Chen et al. PLoS One 2015; 10:e0140074
Zhang, Sivay et al. Manuscript in preparation
Zhang, Manuscript submitted
Applications of the multi-drug assay in HPTN studies
Population-level ARV drug use
HPTN 043: ARV drug use was analyzed in a large community-randomized
clinical trial; ARV drug use was associated with sex (women>men),
pregnancy, older age, and study site; increased ARV drug use was
associated with reduced HIV incidence at one study site
Fogel et al. J AIDS. 2017; 74:158
UNTARGETED TOXICOLOGY SCREENING
Untargeted LC-HRMS Screening
• Samples preparated by simple protein precipitation and dilution; 30-minute mixed mode chromatography
• Mass peaks are selected based on minimum intensity threshold (ion current in quadrupole)
• Selected peaks are fragmented and analyzed by high-resolution orbitrap
• Data-dependent fragmentation and analysis
• Resulting pattern matched to stored mass spectra patterns• Curated spectra: MZ cloud• Theoretical spectra: ChemSpider
Analysis of known cocaine positive specimen
Analysis of known cocaine positive specimen
Analysis of known cocaine positive specimen
Analysis of known cocaine positive specimen
Analysis of urine toxicology negative control
Analysis of urine toxicology negative control
Analysis of urine toxicology negative control
Analysis of urine toxicology negative control
Next Steps
• Post-analysis data processing is necessary to exclude endogenous metabolites
• Analyze “drug-free” urines to set exception list
• Analyze known pain management samples to optimize algorithm
• Analyze blinded samples from external reference lab
Next Steps
• Repeat workflow optimization with serum samples
• Analyze known serum/plasma TDM samples
• Analyze serum toxicology samples (collaboration with Medical Examiner?)
• Validation of both urine and serum workflows and sample preparation
Specimen preparation and
LC-HRMS
Remove excluded compounds
Generate compound list from library
match
Analyze & build local library
Preliminary Report
Obtain compounds from
prelim report
Final Report (confirmed)
• JHU Advanced Clinical Chemistry Diagnostics Lab• Autumn Breaud• Sabitha Schools• Abed Pablo• Branch Coleman
• JHU School of Medicine• Susan Eshleman, MD, PhD• Mark Marzinke, PhD• Claire Knezevic, PhD• Matthew Olson, MD
• Thermo Fisher Scientific• Marta Kozak• Kristine Van Natta
• Funding and Support: NIH (UM1AI068613), Thermo Fisher Scientific
Acknowledgements
Acknowledgements
The HIV Prevention Trials Network is sponsored by the
National Institute of Allergy and Infectious Diseases,
the National Institute of Mental Health, and the National
Institute on Drug Abuse, all components of the
U.S. National Institutes of Health.