drug screening in biological samples using high resolution ... · drug screening in biological...

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)

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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


NL: 1.64E6


NVP precursor XIC

NVP product ion 3 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


Chen et al. J AIDS 2015; 69:446


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


Chen et al. PLoS One 2015; 10:e0140074

Zhang, Sivay et al. Manuscript in preparation

Zhang, Manuscript submitted


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 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.

QUESTIONS??

[email protected]

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