Last Updated August 22, 2012

Lead Discovery References Using RapidFire
Journal Articles
2011 Bioorganic and Medicinal Chemistry Letters – Pfizer
N-benzylimidazole carboxamides as potent, orally active stearoylCoA desaturase-1 inhibitors.
Atkinson KA, Beretta EE, Brown JA, Castrodad M, Chen Y, Cosgrove JM, Du P, Litchfield J, Makowski M, Martin K, McLellan
TJ, Neagu C, Perry DA, Piotrowski DW, Steppan CM, Trilles R. Pfizer Global Research & Development, Groton Laboratories,
Pfizer Inc., Groton, CT 06340, USA.
Bioorg Med Chem Lett. 2011 Mar 15;21(6):1621-5. Epub 2011 Jan 31.
Web link: http://www.ncbi.nlm.nih.gov/pubmed/21324691
Abstract: A potent, small molecule inhibitor with a favorable pharmacokinetic profile to allow for sustained SCD
inhibition in vivo was identified. Starting from a low MW acyl guanidine (5a), identified with a RapidFire HighThroughput Mass Spectrometry (RF-MS) assay, iterative library design was used to rapidly probe the amide and tailregions of the molecule. Singleton synthesis was used to probe core changes. Biological evaluation of a SCD inhibitor(5b) included in vitro potency at SCD-1 and in vivo modulation of the plasma desaturation index (DI) in rats on a low
essential fatty acid (LEFA) diet. In addition to dose-dependent decrease in DI, effects on rodent ocular tissue werenoted. Therefore, in rat, these SCD inhibitors only recapitulate a portion of phenotype exhibited by the SCD-1
knockout mouse.

2011 Journal of Biomolecular Screening
Advances in Label-Free Screening Approaches for Studying Sirtuin-Mediated Deacetylation.
Rye PT, Frick LE, Ozbal CC, Lamarr WA.
J Biomol Screen. 2011 Sep 12. [Epub ahead of print].
Web link: http://jbx.sagepub.com/content/early/2011/09/10/1087057111420291.abstract
Abstract: The sirtuin enzymes, a class of NAD+-dependent histone deacetylases, are a focal point of epigenetic
research because of their roles in regulating gene expression and cellular differentiation by deacetylating histones and ahost of transcription factors, including p53. Here, the authors present two label-free screening methodologies to study
sirtuin activity using high-throughput mass spectrometry. The first method involves the detection of native peptides andprovides a platform for more detailed mechanistic studies by enabling the concurrent and direct measurement ofmultiple modification states. The second method obviates the need for substrate-specific assay development bymeasuring the O-acetyl-ADP-ribose co-product formed by sirtuin-dependent deacetylation. Both methodologies wereapplied to investigating the deacetylation of multiple-peptide substrates by multiple-sirtuin enzymes. Kinetic data,
including binding constants, inhibition, and, in some cases, activation, are demonstrated to correlate well, both between
the methodologies and with previous literature precedent. In addition, the ability to monitor sirtuin activity via Oacetyl-ADP-ribose production permits experimentation on whole-protein substrates. The deacetylation of wholehistone proteins by SIRT3, and inhibition thereof, is presented and demonstrates the feasibility of screening sirtuins
using more biologically relevant molecules.

2011 Journal of Biomolecular Screening
Advances in Label-Free Screening Approaches for Studying Histone Acetyltransferases.
Rye PT, Frick LE, Ozbal CC, Lamarr WA.
J Biomol Screen. 2011 Sep 9. [Epub ahead of print]
Web link: http://jbx.sagepub.com/content/early/2011/09/08/1087057111418653.abstract
Abstract: Histone acetyltransferases (HATs) catalyze the transfer of an acetyl group from an acetyl-coenzyme A donor
molecule to specific lysine residues within proteins. The acetylation state of proteins, particularly histones, is known tomodulate their intermolecular binding properties and control various cellular processes, most notably transcriptional
activation. In addition, deregulation of HAT activity has been linked to the development of a number of cancers;
therefore, compounds that affect these enzymes have strong potential as therapeutic agents. The research presented here
demonstrates three label-free HAT screening approaches, all based on the fast and direct measurement of one or more
substrate-product pairs by high-throughput mass spectrometry techniques. The first approach involves monitoring all

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Last Updated August 22, 2012
possible acetylation states of a peptide concurrently to measure HAT activity. The second approach measures
acetylation reactions, on both peptides and whole protein substrates, via direct detection of the acetyl-coenzyme A
cosubstrate and coenzyme A coproduct. Lastly, the authors demonstrate the ability to monitor directly the acetylation
state of whole histone proteins in the same high-throughput manner using time-of-flight mass spectrometry. The
generation of compound-mediated inhibition data using each of these techniques establishes mass spectrometry as aversatile, label-free, and biologically relevant screening approach to this challenging target class.

2011 Journal of Biomolecular Screening -- Pfizer/BIOCIUS
High-Throughput Screening Assay for Sphingosine Kinase Inhibitors in Whole Blood Using
RapidFire Mass Spectrometry
Maureen K. Highkin, Matthew P. Yates, Olga V. Nemirovskiy, William A. Lamarr, Grace E. Munie, John W. Rains, Jaime L.
Masferrer, Marek M. Nagiec
J Biomol Screen February 4, 2011 vol. 16 no. 2 272-277
Web link: http://jbx.sagepub.com/content/16/2/272.abstract
Note: This was also presented as a poster during APA 2010.
Abstract: To facilitate discovery of compounds modulating sphingosine-1-phosphate (S1P) signaling, the authors usedhigh-throughput mass spectrometry technology to measure S1P formation in human whole blood. Since blood contains
endogenous sphingosine (SPH) and S1P, mass spectrometry was chosen to detect the conversion of an exogenously
added 17-carbon-long variant of sphingosine, C17SPH, into C17S1P. The authors developed procedures to achieve
homogeneous mixing of whole blood in 384-well plates and for a method requiring minimal manipulations to extract
S1P from blood in 96- and 384-well plates prior to analyses using the RapidFire® mass spectrometry system.

2010 Journal of Biomolecular Screening – Schering Plough/BioTrove/Seton Hall
Univ./Merck/Idaho National Laboratory
Screening for antibacterial inhibitors of the UDP-3-O-(R-3-hydroxymyristoyl)-Nacetylglucosamine deacetylase (LpxC) using a high-throughput mass spectrometry assay.
Langsdorf EF, Malikzay A, Lamarr WA, Daubaras D, Kravec C, Zhang R, Hart R, Monsma F, Black T, Ozbal CC, Miesel L,
Lunn CAJ Biomol Screen. 2010 Jan ; 15(1): 52-61
Web link: http://jbx.sagepub.com/content/15/1/52.abstract
Abstract: A high-throughput mass spectrometry assay to measure the catalytic activity of UDP-3-O-(R-3hydroxymyristoyl)-N-acetylglucosamine deacetylase, LpxC, is described.. The results show that mass spectrometrybased screening is a valuable high-throughput screening tool for detecting inhibitors of enzymatic targets involving
difficult to detect reactions.
The Situation: Find a way to perform a label-free inhibition screen based on the native LpxC substrate in order todevelop a better anti-bacterial drug. LpxC (a deacetylase essential for endotoxin biosynthesis in gram-negative
bacteria) is a well-known anti-bacterial and anti-inflammatory drug target, but effective drugs based on inhibitors of
this enzyme have been difficult to develop. Typically, scientists have used an optical assay based on a LpxC surrogate
substrate to screen compound libraries for LpxC inhibitors. The surrogate substrate has a Km >100x that of the native
substrate and this large disparity in Km may be the reason for so many past failures to develop an effective inhibitor.
The Solution: Addressing the Challenges: In collaboration with RapidFire scientists, an assay was developed using the
RapidFire automated SPE-based system in conjunction with mass spec analysis of the native LpxC substrate. The
RapidFire team quickly performed primary screening analysis using this new LpxC assay on the client’s compressedcompound library (~250,000 wells). Secondary screens were then used to confirm several new inhibitors.
The Results: Three new patent applications and the potential of a new effective drug to treat gram-negative bacterial
infections and their associated inflammatory disease.

2009 ASSAY and Drug Development Technologies – Merck/BioTrove
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Last Updated August 22, 2012

Label-free high-throughput screening via mass spectrometry: a single cystathionine
quantitative method for multiple applications.
Holt TG, Choi BK, Geoghagen NS, Jensen KK, Luo Q, LaMarr WA, Makara GM, Malkowitz L, Ozbal CC, Xiong Y, Dufresne
C, Luo MJ
Assay Drug Dev Technol. 2009 Oct ; 7(5): 495-506
Web link: http://www.liebertonline.com/doi/abs/10.1089/adt.2009.0200
Abstract: Label-free mass spectrometric (MS) technologies are particularly useful for enzyme assay design for drug
discovery screens. ... Our results show that the HTMS method was useful for screening samples containing serum, forcell-based assays, and for liver explants. The novel extension of the in vitro analytical method, without modification, tosecondary assays resulted in a significant and advantageous economy of development time for the drug discovery
project.

2009 Combinational Chemistry and High Throughput Screening
A Case Study on Acetyl-Coenzyme A Carboxylase using RapidFire – Mass Spectrometry (RFMS)
Maxine Jonas, William A. LaMarr and Can Özbal
Abstract: In this review various technologies and approaches for the utilization of mass spectrometry in highthroughput analyses are discussed. The use of quadrupole-based mass spectrometry in the screening of chemical
libraries again stenzymatic targets for the identification of inhibitors and/or activators is highlighted. The RapidFire
mass spectrometry system, an integrated on-line solid-phase extraction system interfaced to a triple-quadrupole mass
spectrometer is described in detail, and the identification of a series of inhibitors of the acetyl-coenzyme A carboxylase(ACC) assay is described.

2008 Analytica Chimica Acta – Pfizer/BioTrove
Development of a high-throughput screening assay for stearoyl-CoA desaturase using rat liver
microsomes, deuterium labeled stearoyl-CoA and mass spectrometry.
Soulard P, McLaughlin M, Stevens J, Connolly B, Coli R, Wang L, Moore J, Kuo MS, LaMarr WA, Ozbal CC, Bhat BG
Anal Chim Acta. 2008 Oct 3; 627(1): 105-11
Web link: http://www.sciencedirect.com/science/article/pii/S0003267008006867
Abstract: Several recent reports suggest that stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme inmonounsaturated fatty acid synthesis, plays an important role in regulating lipid homeostasis and lipid oxidation inmetabolically active tissues ... High-throughput mass spec screening of over 1.7 million compounds in compressed
format demonstrated that the enzyme target is druggable ... The application of mass spectrometry to high-throughput
screening permitted the development of a high-quality screening protocol for an otherwise intractable target, SCD1.

2008 Combinatorial Chemistry & High Throughput Screening – Kalypsys(Wako)/BioTrove
Back to basics: label-free technologies for small molecule screening.
Shiau AK, Massari ME, Ozbal CC
Comb Chem High Throughput Screen. 2008 Mar ; 11(3): 231-7
Web link: http://www.benthamdirect.org/pages/content.php?CCHTS/2008/00000011/00000003/0006A.SGM
Abstract: Small molecule high-throughput screening in drug discovery today is dominated by techniques which aredependent upon artificial labels or reporter systems. While effective, these approaches can be affected by certain
experimental limitations, such as conformational restrictions imposed by the selected label or compound
fluorescence/quenching. Label-free approaches potentially address many of these issues by allowing researchers toinvestigate more native systems without fluorescence- or luminescence-based readouts. However, due to throughput
and expense constraints, label-free methods have been largely relegated to a supporting role as the basis of secondary
assays. In this review, we describe recent improvements in impedance-based, optical biosensor-based, automated patch
clamp and mass spectrometry technologies that have enhanced their ease of use and throughput and, hence, their utility
for primary screening of small- to medium-sized compound libraries. The ultimate maturation of these techniques will
enable drug discovery researchers to screen large chemical libraries against minimally manipulated biological systems.

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Last Updated August 22, 20122007 Journal of Biomolecular Screening – Bayer/BioTrove
High-throughput mass spectrometry screening for inhibitors of phosphatidylserine
decarboxylase.
Forbes CD, Toth JG, Ozbal CC, Lamarr WA, Pendleton JA, Rocks S, Gedrich RW, Osterman DG, Landro JA, Lumb KJ
J Biomol Screen. 2007 Aug ; 12(5): 628-34
Web link: http://jbx.sagepub.com/content/12/5/628.abstract
Abstract: A high-throughput mass spectrometry assay to measure the catalytic activity of phosphatidylserine
decarboxylase (PISD) is described. PISD converts phosphatidylserine to phosphatidylethanolamine during lipid
synthesis. Traditional methods of measuring PISD activity are low throughput and unsuitable for the high-throughput
screening of large compound libraries. The high-throughput mass spectrometry assay directly measures
phosphatidylserine and phosphatidylethanolamine using the RapidFire trade mark platform at a rate of 1 sample every
7.5 s. The assay is robust, with an average Z' value of 0.79 from a screen of 9920 compounds. Of 60 compounds
selected for confirmation, 54 are active in dose-response studies. The application of high-throughput mass spectrometry
permitted a high-quality screen to be performed for an otherwise intractable target.

2007 Journal of Biomolecular Screening – Bayer/BioTrove
High-throughput screening by mass spectrometry: comparison with the scintillation proximity
assay with a focused-file screen of AKT1/PKB alpha.
Quercia AK, LaMarr WA, Myung J, Ozbal CC, Landro JA, Lumb KJ
J Biomol Screen. 2007 Jun ; 12(4): 473-80
Web link: http://jbx.sagepub.com/content/12/4/473.abstract
Abstract: Mass spectrometry is an emerging format for label-free high-throughput screening. The main limitation ofmass spectrometry is throughput, due to the requirement to purify samples prior to ionization. Here the authors
compare an automated high-throughput mass spectrometry (HTMS) system (RapidFire) with the scintillation proximity
assay (SPA). The cancer therapy target AKT1/PKBalpha was screened against a focused library of kinase inhibitors
and IC50 values determined for all compounds that exhibit > 50% inhibition. A selection of additional compounds thatexhibited less than or = 50% inhibition in the primary screen was chosen as controls to confirm inactives. The selectionof compounds is expected to identify common actives, common inactives, false positives, and false negatives.
Agreement is found between HTMS and SPA in terms of primary hit identification and hit confirmation.

2004 ASSAY and Drug Development Technologies – BioTroveHigh Throughput Screening via Mass Spectrometry: A Case Study Using Acetylcholinesterase
Can C. .zbal, William A. LaMarr, John R. Linton, Donald F. Green, Arrin Katz,
Thomas B. Morrison, and Colin J.H. Brenan
Abstract: Mass spectrometry-based screening can be applied to a wide range of targets, including
those intractable targets that use substrates such as lipids, fatty acids, phospholipids, steroids,
prostaglandins, and other compounds not generally amenable to conventional screening techniques.The major
limitation to this approach is throughput, making HTS via mass spectrometry impractical.We present a mass
spectrometry-based technique and hardware for lead discovery applications. Massspectrometry enables the design oflabel-free assays using biologically native substrates for a widerange of enzymatic targets. This system can be used forthe direct quantification of analytes incomplex reaction mixtures with typical throughputs of 4Ð5 s per sample. A mass
spectrometrybasedassay was developed to identify inhibitors of acetylcholinesterase, an enzyme withclinicalimportance in AlzheimerÕs disease. The system was used to screen a small chemical library. Severalpotent
inhibitors were identified, and the IC50 values of the inhibitors were determined.

Lead Discovery Posters and Presentations
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Last Updated August 22, 20122012 SLAS – Agilent Technologies (Poster)
A Label-Free Screening Approach to Epigenetic Drug Discovery: Using Mass Spectrometry to
Monitor Protein and Nucleic Acid Modification Events
William A. LaMarr, Peter T. Rye, Lauren E. Frick, Agilent Technologies, Inc. Wakefield, MA 01880

2012 SLAS – Agilent Technologies (Poster)High-throughput Measurement of DNA Mono- and Oligo-nucleotides by RapidFire/TOF-MS
William A. LaMarr1, Deyu Li2, Vipender Singh2, John M. Essigmann2, and Peter T. Rye1 1) Agilent Technologies, Wakefield,
MA. 2) MIT, Departments of Chemistry and Biological Engineering, Cambridge, MA.

2011 SBS – BIOCIUS (poster)
Advances in Label-Free Screening Approaches for Studying Histone Acetyltransferases
Histone acetyltransferases (HATs) catalyze the transfer of an acetyl group from an acetyl-coenzyme A donor molecule
to sequence specific lysine residues within proteins The acetylation state of proteins particularly histones is known tomodulate their intermolecular binding properties and control various cellular processes, most notably transcriptional
activation. Additionally, deregulation of HAT activity has been linked to the development of a number of cancers;
therefore, compounds that affect these enzymes have strong potential as therapeutic agents. The research presented
here demonstrates three label free HAT screening approaches all based upon the fast and direct measurement of one ormore substrate/product pairs by high-throughput mass spectrometry techniques. One approach involves monitoring all
possible acetylation states of a peptide concurrently to measure HAT activity. Another approach measures cosubstrateand coenzyme A coproduct. Lastly, we demonstrate the ability to monitor directly the acetylation state of whole
histone proteins in the same high-throughput manner using time-of-flight (TOF) mass spectrometry. The generation ofcompound-mediated inhibition data using each of these techniques establishes mass spectrometry as a versatile labelfree and biologically-relevant screening approach to this challenging target class.

2010 ASMS -- Bristol-Myers Squibb (poster)Development of a High-Throughput MS-Based Bioanalytical Method for Assessment of PGlycoprotein Inhibition
Evaluation of drug-drug interactions between potential drug candidates and transporters such as P-glycoprotein (P-gp)
is an integral part of early liability screening in drug discovery. Inhibition of P-gp can be determined by performing bidirectional Caco-2 permeability studies with digoxin as a substrate probe. Existing methodologies include either
assaying H3- digoxin with liquid scintillation counting or assaying non-labeled digoxin with LC-MS detection at a
speed of several minutes per sample. Our goal was to develop an analytical method that was “label-free” and provided
ample throughput to support this potentially high-volume assay. Mass spectrometric detection was the ideal choice fordigoxin analysis because it is “label-free”, sensitive, specific, and reliable. However, it is not possible to achieve abioanalytical throughput high enough to support a screening assay using traditional LC-MS/MS. To address this, wedeveloped an ultra-fast method for digoxin analysis on the BIOCIUS RapidFire™ (RF)-MS/MS system with a cycle
time of ~9 seconds per sample. In order to validate the accuracy of the RFMS/ MS method, a traditional LC-MS/MS
method was established for results comparison. Data processing was made feasible through the development of inhouse macros that utilize XLfit for standard curve linear regression and calculation of preliminary results. The resulting
data suggests that the RF-MS/MS method generates equivalent results to both LC-MS/MS and radiolabel assays indetermining inhibitors of P-glycoprotein.

2010 ASMS – BIOCIUS (poster)

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Last Updated August 22, 2012

Evaluation of Accurate Mass TOF-MS for Use in High Throughput PAMPA & Plasma
Protein Binding Screens
Assays that measure permeability (i.e. PAMPA) and plasma protein binding (PPB) are used to screen drug compounds
early in the discovery process to eliminate poor performers and/or to identify compounds that need to be modified.
Mass spectrometry has emerged as the preferred analysis method for these assays due to its sensitivity, specificity, androbustness. Traditionally, these assays utilize tandem MS, which is limited by its requirement for compound dependent
method development. The use of accurate mass offered by time of flight (TOF) mass spectrometers, which eliminates
the need for MRM optimization, was investigated here and has implications for a wide range of other in vitro ADME
assays where MS method development is a bottleneck.
A diverse set of commercially available drug compounds with a broad spectrum of physical chemical properties wereassayed. Samples were analyzed at a rate of 6-8 seconds per sample using an ultrafast SPE–MS system consisting of a
RapidFire RF300 interfaced to an ABSciex API4000 or a RapidFire RF360 interfaced to an Agilent 6530 Q-TOF. MS
conditions for each compound were optimized on the QqQ while generic MS conditions were used for all compounds
on the Q-TOF. A single generic SPE condition was used for all analyzes.
For each assay, the results using the SPE-TOF were comparable to literature values and correlated very well withresults from the SPE-MS/MS system (R2> 0.9). Interestingly, the Q-TOF successfully analyzed compounds that could
not be analyzed on the QqQ. The tight correlation in assay values determined on the two different instruments suggeststhat the use of exact mass MS instrumentation is a valid alternative to MRM method development for the analysis of
PAMPA & plasma protein binding assays. Eliminating MRM method development significantly streamlined workflow
and improved laboratory efficiency. These generic SPE-TOF methods may also be useful for other in vitro ADME
assays that typically utilize QqQ analysis.

2009 ASMS -- GSK (poster)Label-free High-throughput Whole Protein Kinase Screening Assay
Protein kinases are involved in the regulation of multiple cellular processes by catalyzing phosphorylation of target
substrates. Dysregulation of kinase activity has been found in a variety of diseases; therefore, protein kinase inhibitors
offer an opportunity to target these diseases. To facilitate the screening and characterization of kinase inhibitors, wedeveloped a mass spec-based high throughput screening method to measure their effect on kinase activity, permitting
the use of whole protein substrates which advantageously allows detection of both competitive and non-competitive
inhibitors. In addition, this assay does not rely on site-specific antibodies, allowing detection of phosphorylated serine,
threonine, or tyrosine residues. Lastly, mass spectrometry facilitates quantitative interrogation of complex kinetics inproteins with multiple phosphorylation sites.

2009 ASMS -- OSI (poster)Discovery of Novel Inhibitors of Serine Palmitoyltransferase (SPT) by Mass Spectrometrybased High-throughput Screening (HTS)
Obesity has been linked with increased ceramide accumulation and resultant impaired insulin sensitivity. SPT may
represent an attractive enzyme to target within the ceramide synthetic pathway in order to treat type 2 diabetes. A mass
spectrometry based HTS approach to identify inhibitors of mammalian SPT was used to screen a library of >250,000
small molecules. Screening identified potent SPT inhibitors from multiple chemotypes. A mass spectrometry-based
HTS approach was developed to identify inhibitors of SPT, an enzyme that was previously intractable to conventional
screening methods. The screen successfully identified potent inhibitors of mammalian SPT activity across several
distinct chemotypes.

ASMS 2009 -- Merck (User Symposium Presentation)"The Application of RapidFire MS in a High-throughput Production Assay Lab"
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Last Updated August 22, 2012
This presentation, authored by Merck Research Labs: Deborah Zink, Tom Holt, Jonathon Lum, Marta Arocho, Anne
Vergnon, Gerica Galviz, Claude Dufresne, deals with Merck’s initially outsourced RapidFire project to develop
production assays for drug-drug interaction. They state that they went from zero to five production assays in less than 5months and, ultimately, brought the instrument in-house. They further describe they successfully tested overnight
operation of the instrument and that they expected payback on purchase cost in 5 months.

ASMS 2009 -- Boehringer-Ingelheim (User Symposium Presentation)"A Novel and Integrated Platform for Fully Automated High Throughput LC-MS/MS
Analysis of in vitro ADME Sample"
This presentation, authored by Andreas H. Luippold - Boehringer Ingelheim Pharma GmbH & Co KG , describes how
BI reduced cycle times from 80 minutes to 13 minutes for a 96 well plate using RapidFire technology.

2009 SBS -- Genzyme (poster)Discovery of Small Molecule Inhibitors of GM3 Synthase via High-throughput Mass
Spectroscopy
A label-free biochemical HTS assay using the RapidFire system was developed and HTS was performed to find small
molecule inhibitors of GM3 synthase. A collection of 250,000 compounds were screened at 10 uM in singlicate. 1816
hits have been identified which resulted in a hit rate of 0.7 %, 1217 hits were confirmed in duplicate at 10 uM, resulting
in a confirmation rate of 67%. Currently confirmed hits are under evaluation for potency and selectivity. Confirmed
potent and specific hits for GM3 synthase from the biochemical screen will help to guide medicinal chemistry efforts tofind a small molecule inhibitor of GM3 synthase. RapidFire system enables measurement of enzymatic activity ofproteins which are difficult to measure by other known conventional methods. We have shown that RapidFire system
can be used for multiple enzyme assays such as GM3 Synthase and ST6Gal-1.

2009 SBS – BIOCIUS (poster)High-throughput Mass Spectrometric Detection of Histone 3 Demethylation
Here we illustrate the utility of RapidFire-MS to study epigenetic changes directly, using reactions between LSD1
enzyme and a dimethylated Histone3 peptide (H3K4me2) as an example. The RapidFire platform enables highthroughput mass spectrometric analysis of native molecules from in vitro reactions by performing on-line desalting inseconds, as opposed to HLPC, which requires minutes.
Early Phase Identification of Candidates for Epigenetic Drug Targets
The Situation: A biopharmaceutical company focused on the emerging field of epigenetic-based drug targets sought a
faster, more efficient way to advance their screening programs. Traditionally, epigenetic targets were screened using
conventional screening methods; however, this company was interested in a native analyte-based technology that could
provide more biologically relevant data from assays measuring slight modifications in protein structure.
Addressing the Challenges: Working with BIOCIUS in-house scientists, assays for methylases, acetylases,
phosphorylases and other modifying enzymes involved in histone and chromatin modulation were developed andoptimized. The RapidFire mass spec platform had the ability to investigate reactions that are not practical using HPLC,
fluorescence, or luminescence screening methodologies. The technology avoids some confounding effects (such as
auto-fluorescence) and provides fast, high capacity sample analysis throughput that is required in early drug discovery.
Results: With the RapidFire system, the client was able to use biologically relevant assays earlier in the discovery
process, ultimately providing rapid and accurate data analysis, reducing R&D time, and enabling faster identification ofpromising new lead compounds for epigenetic drug development.

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Last Updated August 22, 20122009 ELRIG – BIOCIUS (poster)Consistent, High-throughput Metabolic Stability Screening Across Mass Spectrometry
Platforms
The metabolic half-life or stability of a drug candidate has important pharmacokinetic and clinical significance in vivo,
because it influences both oral bioavailability and plasma concentration of a compound, which in turn, affect its
efficacy. Therefore, a high-throughput metabolic stability assay is a valuable tool in the go/no-go decision-making
process in drug discovery. However, conventional LC-MS requires several minutes per sample processing time. Using
the RapidFire system, we have developed analytical techniques which lower analysis times to only a few seconds per
sample, allowing metabolic stability assays to be performed at throughputs consistent with other drug discovery
screening platforms. In the present study we evaluated the compatibility of the RapidFire system with 3 MS platforms(ABI/Sciex, ThermoFisher, and Agilent) by measuring half lives using conventional assay methods. The values
measured across all MS platforms tested were within a 2-fold window. This data shows that the RapidFire systemproduces consistent, high-throughput, metabolic stability data across multiple MS platforms enabling performance of
these critical assays in a typical drug discovery environment.

2008 Protein Society – Amgen/BioTrove (poster)
Screening for ATP Citrate Lyase Inhibitors Using Mass Spectrometry
Used RapidFire to identify potential ACL inhibitors, screened more than 43,600 compounds in single point and morethan 300 IC50 follow-ups. Showed that the RapidFire platform is ideal for identifying and verifying lead compounds
such as Compound A and Compound B. RapidFire technology was also used to look at SAR around these most potent
compounds as well as investigate reversibility and probe the mechanisms of inhibition.
Using both RapidFire and FT- Orbitrap® mass spectrometry to characterize PKA treated ACL researchers phosphoresidue mapped and assayed under Vmax conditions, though report only marginally lower activity with theunphosphorylated ACL enzyme as opposed to Potapova et.al. who report a 6-fold increase in ACL phosphorylated by
PKA. In addition, the high resolution of FT-Orbitrap enabled the direct analysis of ACL activity in cells anddemonstrates compensatory increase in cellular AcetylCoA levels upon inhibition.

2008 SBS -- Pfizer (poster)
The Use of High Throughput Mass Spectrometry (HTMS) in Drug Discovery
The 1st generation of RapidFire instrument was delivered to Pfizer Groton HTS Center of Emphasis (CoE) in Sept.
2006. In 2007, the HTS CoE has applied the HTMS system to: Assay development and Subset screening for DGAT;
Hit to Lead exploration with Stearoyl CoA Desaturase (SCD); Assay Development for SirT1. In 2008, we have
expanded our impact by supporting 7 drug targets at various discovery stages.

2007 SBS -- Merck (poster)Identification of Inhibitors for the Phosphatidylethanolamine N-methytransferase Enzyme by
High-Throughput Mass Spectrometry Detection of Reaction Products
Phosphatidylethanolamine N-methyltransferase (PEMT) is the enzyme that transfers methyl groups from SAM tophosphatidylethanolamine (PE), to generate phosphatidylcholine (PC) and SAH. It has been reported that in PEMT -/mice there is a decrease in plasma PC and cholesterol found in high density lipoproteins when compared to wild typemice. Thus it might be of interest to see how PEMT inhibitors could modulate the levels of plasma lipoproteins. Two
inhibitors were identified, with potency equivalent or higher than SAH, and could represent interesting lead molecules
for the development of potent and selective inhibitors of PEMT. Such compounds could be used in a relevant animalmodel of atherosclerosis to evaluate the role of PEMT in this disease

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Last Updated August 22, 20122007 SBS -- Bayer (poster)High-Throughput Mass Spectrometry Screening for Inhibitors of Phosphatidylserine
Decarboxylase
Abstract: A high-throughput mass spectrometry assay to measure the catalytic activity of phosphatidylserine
decarboxylase (PISD) is described. PISD converts phosphatidylserine (PS) to phosphatidylethanolamine (PE) during
lipid synthesis. Traditional methods of measuring PISD activity are low throughput and unsuitable for the highthroughput screening of large compound libraries. The high throughput mass spectrometry assay measures
phosphatidylserine and phosphatidylethanolamine directly using the RapidFire platform at a rate of one sample every
7.5 s. The assay is robust, with an average Z′ value of 0.79 from a screen of 9,920 compounds. Of 60 compounds
selected for confirmation, 54 are active in doseresponse studies. The application of high-throughput mass spectrometry
permitted a high-quality screen to be performed for an otherwise intractable target

ADME References Using RapidFire-MS
Journal Articles
2012 Future Science Ltd. – Agilent Technologies
SPE-MS analysis of absorption, distribution, metabolism and excretion assays: a tool toincrease throughput and streamline workflow
Vaughn P. Miller, Agilent Technologies, 11 Audobon Road, Wakefield MA 01880

2011 Analytical Chemistry – J&J/Agilent
Evaluation of a high-throughput online solid phase extraction-tandem mass spectrometry system for in vivo
bioanalytical studies. Jian W1, Romm MV2, Edom RW1, Miller VP2, LaMarr WA2, Weng N1. 1Johnson & Johnson
Pharmaceutical Research & Development, Raritan, New Jersey 08869, United States; 2Agilent Technologies, Inc. 11
Audubon Rd., Wakefield, MA 01880, USA. Anal. Chem. 83, 8259-8266 (2011).
Web Link:
http://pubs.acs.org/doi/abs/10.1021/ac202017c?mi=scbaih&af=R&pageSize=20&searchText=cyclodextrin



Evalution of RapidFire/MS for in vivo (PK) bioanalysis: RF-MS/MS correlates with LC-MS/MS

2011 Journal of Biomolecular Screening – Boehringer Ingelheim
Application of a Rapid and Integrated Analysis System (RIAS) as a High-Throughput Processing
Tool for In Vitro ADME Samples by Liquid Chromatography/Tandem Mass Spectrometry. Andreas
H. Luippolda,Thomas Arnholda, Wolfgang Jörga , B Krüger , and Roderich D. Süssmuthb a Department of Drug
Discovery Support, Boehringer Ingelheim Pharma GmbH & Co KG, Birkendorfer Str. 65, 88397 Biberach an derRiss, Germany; b Institut für Chemie, Technische Universität Berlin, Strasse des 17. Juni 124, 10623 Berlin,
Germany. Journal of Biomolecular Screening, 16(3):370-7 (2011).
Web Link: http://jbx.sagepub.com/content/16/3/370.abstract
RF-MS/MS correlates with LC-MS/MSo CYP inhibitiono Caco-2
o PAMPA2011 Rapid Communications in Mass Spectrometry – BMS
Ultrafast mass spectrometry based bioanalytical method for digoxin supporting an in vitro Pglycoprotein(P-gp)
inhibition screen. Andrew D. Wagner1, Janet M. Kolb1, Can C. Özbal2, John J. Herbst1,
Timothy V. Olah1, Harold N. Weller1, Tatyana A. Zvyaga1 and Wilson Z. Shou1* 1Applied Biotechnology, Bristol‐Myers Squibb,
5 Research Parkway, Wallingford, CT 06492, USA; 2BIOCIUS Life Sciences, Inc. 10P Gill St., Woburn, MA 01801, USA. Rapid
Communications in Mass Spectrometry, 25, 1231-1240 (2011).
Web Link:
http://onlinelibrary.wiley.com/doi/10.1002/rcm.4984/abstract;jsessionid=C35156AFB1BA0CE1C759126FB74A49DF.d01t01?systemMessage=Wiley+Online+Library+will+be+dis

11 Audubon Road I Wakefield, MA 01880 I T: 781.928.2700 I www.Agilent.com

Last Updated August 22, 2012rupted+14+May+from+10-12+BST+for+monthly+maintenanceP-gp digoxin inhibition assay in Caco-2 cells: RF-MS/MS correlates with LC-MS/MS

2010 International Journal of Mass Spectrometry – Boehringer Ingelheim
An integrated platform for fully automated high-throughput LC–MS/MS analysis of in vitro
metabolic stability assay samples. Andreas H. Luippolda,Thomas Arnholda, Wolfgang Jörga and Roderich D.
Süssmuthb a Department of Drug Discovery Support, Boehringer Ingelheim Pharma GmbH & Co KG, Birkendorfer
Str. 65, 88397 Biberach an der Riss, Germany; b Institut für Chemie, Technische Universität Berlin, Strasse des 17.
Juni 124, 10623 Berlin, Germany. International Journal of Mass Spectrometry, 296:1-9 (2010).
Web Link: ftp://ftp.ni.com/pub/branches/germany/artikel/2010/11_nov/ni_10_IJMS.pdf
Metabolic stability with HLM: RF-MS/MS correlates with LC-MS/MS
Abstract: The evaluation of ADME properties contributes importantly to drug candidate selection and therefore is
crucial for the drug discovery process. Providing a sufficiently high-throughput capability in laboratories dedicated toearly pharmacokinetic studies will thereby shorten the entire drug discovery process. In this paper an integrated andfully automated LC MS/MS-based platform is described, which enables the assessment of in vitro metabolic stability, a
key ADME parameter. An ultra-rapid injection system was coupled to a triple quadrupole mass spectrometer and hardand software were customized to perform sample identification, cleanup, MS compound optimization and sample
measurement without manual interaction. Conventional chromatography was initially evaluated but was later replacedby solid phase extraction as the only purification step. Ultra-fast robotics, combined with a generic step gradientenables analysis times of 8 sec/sample. Reproducibility and quality of data has been found fully comparable to data
generated by validated LC-MS/MS methods. The system handles data for mass spectrometric compound optimizationand MRM (Multiple Reaction Monitoring) analytics in a fully automated way and exhibits great potential for ageneralized use in ADME replacing currently applied conventional LC-MS/MS approaches.

2010 Journal of Biomolecular Screening – Takeda/BIOCIUS
Development of a High Throughput On-Line Solid Phase Extraction/Tandem Mass Spectrometry
Method for Cytochrome P450 Inhibition Screening. Journal of Biomolecular Screening, 15 (4), 447452 (2010). Kheng B. Lim1, Can C. Ozbal2, Daniel B. Kassel1 (1Takeda San Diego, Inc., 2BIOCIUS Life Sciences).
Web Link: http://jbx.sagepub.com/content/15/4/447.abstract
CYP inhibition: RF-MS/MS correlates with LC-MS/MS

2010 Rapid Communications in Mass Spectrometry – Novartis
High-Throughput Analysis of In-Vitro Cytochrome P450 Inhibition Samples Using Mass
Spectrometry Coupled with an Integrated Liquid Chromatography/Autosampler System.
Ann Brown*, Shari Bickford, Panos Hatsis, Jakal Amin, Leslie Bell, and Shawn Harriman. Metabolism andPharmacokinetics, Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue Cambridge, MA 02139
USA. Rapid Commun Mass Spectrom. 2010 Apr 30;24(8):1207-10.
Web Link: http://onlinelibrary.wiley.com/doi/10.1002/rcm.4461/full
CYP inhibition: RF-MS/MS correlates with LC-MS/MS

ADME Posters and Presentations
2012 ISSX/MDO – Agilent Technologies
Evaluation of Ultrafast Online SPE/MS for the Efficient Screening of Cytochrome P540
Inhibition Using Cassette Analysis
Nikunj Parikh1, Michelle V. Romm1, Vaughn P. Miller1, Moritz Wagner2 and William A. LaMarr1 1. Agilent Technologies
Inc., Wakefield, MA 2. Agilent Technologies, Waldbronn, Germany

11 Audubon Road I Wakefield, MA 01880 I T: 781.928.2700 I www.Agilent.com

Last Updated August 22, 2012
Abstract: The Cytochrome P450 (CYP) enzymes are involved in many drug metabolism transformations and accountfor a significant number of bioactivation and metabolic reactions. The presence of some drugs can interfere with themetabolic activity of CYP enzymes causing drug-drug interactions. Analysis of potential drug-drug interactions caused
by inhibition of the enzymatic activities of CYP enzymes plays an important role in the drug discovery process. The
ever increasing need to evaluate a large number of samples and the need to eliminate weaker candidates in the initial
phase of the drug discovery process, has created a bottleneck at LC/MS/MS analysis. We evaluated the use of an ultrafast online SPE/MS system (the Agilent RapidFire High-throughput Mass Spectrometry system) coupled to an Agilent
QqQ and Agilent QTOF for the analysis of pooled samples across several CYP450 isoforms in a single cassette
injection mode to provide an even faster and more cost-effective screen.

2010 AAPS – BIOCIUS (poster)Streamlining ADME Workflow Using SPE-TOF Analysis
The throughput of in vitro ADME analysis is gaining an almost equal footing with data quality as a determinant oflaboratory workflow. These assays have traditionally utilized tandem MS, which is limited by its requisite MRM
method development that requires several minutes per sample of processing time.
The use of accurate mass offered by high resolution (time of flight: TOF) mass spectrometers, which eliminates theneed for MRM optimization, in conjunction with a high throughput solid phase extraction (SPE) system wasinvestigated to assess its ability to enable a faster and more efficient assay analysis workflow.
We compared the assay results for a panel of in vitro ADME assays (CYP inhibition, metabolic stability, PAMPA andplasma protein binding) using ultrafast SPE-MS/MS and SPE-TOF systems for analysis. A chemically diverse set of 50
compounds was used for each assay and all analyses utilized the same generic SPE conditions. All SPE-TOF analyses
were performed using the same generic MS conditions with a RapidFire 360 interfaced to an Agilent 6530 Q-TOF run
in ESI-TOF mode. The experimental results obtained for all four of the ADME assays by each analysis system werecomparable (R2 >0.9).
Using a generic SPE condition, the ultrafast SPE-MS systems consistently produced sample analysis cycle times of 7
seconds/sample with no changes to standard laboratory workflow and no significant sample carryover. Using a genericMS method, the TOF analysis gave comparable results to conventional triple quadruple analysis. Assay results with the
SPE-TOF system were comparable to the SPE-MS/MS system for a variety of ADME assays indicating that MRM
method development could be eliminated for these assays providing a significant increase in efficiency of laboratory
workflow. The SPE-TOF system enabled faster and more efficient assay data analysis across a panel of ADME assays
without compromising the quality of results.

2010 AAPS -- Novartis (poster)
Ultra-fast Solubility Sample Analysis Using SPE-MS/MS
We investigated the utility of solid phase extraction (SPE) coupled with mass spectrometry for improving sample
throughput of the solubility assay. The current method for solubility sample analysis employs HPLC with ultraviolet
absorbance detection (UV). We investigated a SPE-TOF system consisting of an ultra-fast SPE system (RapidFire®)
linked to an Agilent 6530 Q-TOF mass spectrometer. The two analytical methods were compared using a set ofcompounds from Novartis’ drug discovery programs that covered a wide range of chemical space and properties.
Solubility was measured in triplicate in pH 6.8 buffer and in FASSIF buffer. A four point calibration curve wasprepared to quantify the solubility of each sample. The SPE-TOF method had a cycle time of eight seconds comparedto two minutes for the HPLC-UV method. This represents a 15-fold improvement in cycle time, which can be used toincrease the capacity of this assay. The SPE-TOF method was able to maintain data quality as evidenced by linear
standard curves (r2 ≈ 0.99) and back-calculated standards within 30%. Moreover, a correlation plot of the solubility
determined using SPE-TOF vs. HPLC/UV was linear with a slope of approximately one and an r2 > 0.8.

2010 APA – J&J/BIOCIUS (poster)
Evaluation of RapidFire Ultra-fast Online SPE-MS/MS System for Analysis of in vivo Bioanalytical
Samples. Wenying Jian1, Michelle V. Romm2, Richard W. Edom1, Vaughn P. Miller2, William A. LaMarr2, NaidongWeng1; 1Johnson & Johnson Pharmaceutical Research & Development, Raritan, NJ 2BIOCIUS Life Sciences,
Wakefield, MA.

11 Audubon Road I Wakefield, MA 01880 I T: 781.928.2700 I www.Agilent.com

Last Updated August 22, 2012


Human & animal plasma bioanalysis: RF-MS/MS correlates with LC-MS/MS

2010 ISSX – BIOCIUS (poster)
The End of MRM: Ultrafast SPE-TOF Analysis Streamlines Workflow and Increases Throughput of
ADME Assays. Michelle V. Romm, Nikunj Parikh, Vaughn P. Miller, William A. LaMarr, Can C. Ozbal.
RF-TOF correlates with RF-QqQ
o CYP inhibitiono Microsomal stabilityo PAMPAo Plasma protein binding

2010 DDI Conference – BD Gentest/BIOCIUS (poster)
Evaluation of SPE-MS/MS (RapidFire®) for Relative Quantitation of Hydroxy-testosterone
Metabolites in Cytochrome P450 3A4 Inhibition Assays In Vitro. David M. Stresser, Eric T. Gangl, Andrew
K. Mason, Andrew P. Blanchard, Elke S. Perloff, Charles L Crespi and Vaughn P. Miller 1. BD Biosciences Discovery
Labware, Woburn, MA 01801, USA 1BIOCIUS Life Sciences, Wilmington, MA 01880.
RF-MS/MS produces robust, quantitative outcomes for the 6B-hydroxytestosterone assay.

2010 DDI Conference – BD Gentest/BIOCIUS (poster)
Validation of an Automated Cytochrome P450 Inhibition Assay Using SPE/MS/MS (RapidFire®)
Analysis. Elke S. Perloff, Andrew K. Mason, Michael S. Shanler, Sudarshan Kapadnis, Vaughn P. Miller, William A.
LaMarr, and David M. Stresser. BD Biosciences (ESP, AKM, MSS, SK, & DMS), BIOCIUS Life Sciences (VPM &WAL).

CYP inhibition: RF-MS/MS correlation to LC-MS/MS

2010 ASMS – Novartis/BIOCIUS (podium talk)
Reducing Bottlenecks in ADME Sample Analysis using Solid Phase Extraction with a Quadrupole
Time-of-Flight Mass Spectrometer. Panos Hatsis1, Michelle Romm2, Vaughn Miller2, Jakal Amin1, William
LaMarr2, Can Ozbal2 and Shawn Harriman1 1Novartis Institutes for Biomedical Research 2BIOCIUS Lifesciences.

RF-TOF correlates with LC-MS/MSo CYP inhibitiono Microsomal stabilityo PAMPA2010 ASMS – BMS/BIOCIUS (poster)Development of a High-Throughput MS-Based Bioanalytical Method for Assessment of PGlycoprotein
Inhibition. Andrew Wagner¹, Janet Kolb¹, John Herbst¹, Charlie Conway¹, Tatyana Zvyaga¹, Harold
Weller¹, Wilson Shou¹, Can “Jon” Ozbal²; ¹Bristol-Myers Squibb Company; Applied Biotechnology ²BIOCIUS Life
Sciences, Inc.

High-throughput P-glycoprotein inhibition assayo RF-MS/MS correlates with LC-MS/MS

2010 ASMS – Novartis (poster)Comparison of LC/MS/MS and a Fully Integrated Autosampler/Solid Phase Extraction System for
the Analysis of Permeability Samples; Adam C Amaral; Christopher Caldwell; Panos Hatsis; Jakal Amin;
Shawn Harriman; Novartis, Cambridge, MA.
RF-MS/MS correlates with LC-MS/MSo PAMPA2010 ASMS – BIOCIUS (poster)
Evaluation of Accurate Mass TOF-MS for Use in High Throughput PAMPA & Plasma Protein

11 Audubon Road I Wakefield, MA 01880 I T: 781.928.2700 I www.Agilent.com

Last Updated August 22, 2012
Binding Screens. Michelle V. Romm, Nikunj Parikh, Vaughn P. Miller, William A. LaMarr, Can C. Ozbal.
RF-TOF correlates with RF-QqQ
o PAMPAo Plasma protein binding

2010 SBS – BIOCIUS (poster)
A High-Throughput In Vitro ADME Assay Panel: Incorporation of SPE-MS/MS Into Standard
Laboratory Workflow. Michelle V. Romm, Nikunj Parikh, Vaughn P. Miller, William A. LaMarr, Can C. Ozbal.
RF-TOF correlates with RF-QqQ
o CYP inhibitiono Microsomal stabilityo PAMPAo Plasma protein binding

2009 ISSX – BioTrove (poster)
Evaluation of accurate mass TOF-MS for use in high throughput metabolic stability screening.
Michelle V. Romm, Nikunj Parikh, Vaughn P. Miller, William A. LaMarr, and Can C. Özbal.
Metabolic Stability: TOF correlates with QqQ2009 ISSX – AstraZeneca/BioTrove (poster)Comparison of High-Throughput Fluorescence, Traditional LC/MS/MS and BioTrove RapidFire
uHTMS Analysis to Assess Cytochrome P450 Inhibition for Use in Drug Discovery. Reema Harish1,
Garrett Ainslie1, Michael Rooney1, Patrick Brassil1, Simon Roberts1, Vaughn Miller2 and Caroline Rivard1*.
1Drug Metabolism and Pharmacokinetics, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451.
2BioTrove Inc, 12 Gill St, Ste 4000, Woburn, MA 01801.

CYP inhibition: RF-MS correlation to LC-MS/MS

2009 ISSX – BD Gentest/BioTrove (poster)
Evaluation of High Throughput Screening Methods for Time-Dependent Inhibition of CYP3A4
Utilizing RapidFire LC/MS/MS Technology. Vaughn P. Miller1, Can C. Ozbal1, Elke S. Perloff2, Shangara S.
Dehal2, Andrew K. Mason2, Andrew P. Blanchard2, Charles L. Crespi2, David M. Stresser2, and William A. LaMarr1
1 BioTrove, Inc., 10P Gill St. Woburn, MA 01801, USA; 2 BD Biosciences, 6 Henshaw Street, Woburn, MA 01801,USA.

CYP inhibition: RF-MS/MS correlation to LC-MS/MS

2009 ISSX – BD Gentest/BioTrove (poster)Comparison of RapidFire® Ultra High Throughput LC/MS/MS with Traditional LC/MS/MS for
Cytochrome P450 Inhibition Testing. Elke S. Perloff1, Shangara S. Dehal1, Andrew K. Mason1, Andrew P.
Blanchard1, William A. LaMarr2, Can C. Ozbal2, Vaughn P. Miller2, Charles L. Crespi1, and David M. Stresser1
1BD Biosciences Discovery Labware, BD GentestSM Contract Research Services, Woburn, MA 01801, USA
2BioTrove, Inc. 10P Gill St. Woburn, MA 01801, USA.

CYP inhibition: RF-MS/MS correlation to LC-MS/MS

2009 ELRIG – BioTrove (poster)Consistent High-throughput Metabolic Stability Screening Across Mass Spectrometry Platforms.
Michelle V. Romm, Nikunj Parikh, Vaughn Miller, William A. LaMarr, Selena Larkin, Can C. Ozbal.
Metabolic stability data correlates with RF using 3 different MS QqQ vendor platforms

11 Audubon Road I Wakefield, MA 01880 I T: 781.928.2700 I www.Agilent.com

Last Updated August 22, 20122009 ASMS – Merck (BioTrove Users Group presentation)
The application of RapidFire MS in a high-throughput production assay lab. Merck Research Labs:
Deborah Zink, Tom Holt, Jonathon Lum, Marta Arocho, Anne Vergnon, Gerica Galviz, Claude Dufresne. BioTrove:
William LaMarr, Can Ozbal, Maxine Jonas, Arrin Katz.

CYP inhibition: RF/MS/MS correlation to LC-MS/MS

2009 ASMS – BMS (poster)
Application of the BioTrove RapidFireTM Ultra-fast Online SPE-MS/MS System for CompoundspecificAnalysis of in vitro ADME Samples. Anthony Paiva; Andrew Wagner; Xianmei Cai; Ying Li; Shu Li;
Janet Kolb; Aaron Walker; John Herbst; Charlie Conway; Harold Weller; Wilson Shou; Bristol-Myers Squibb,
Wallingford, CT.

RF-MS/MS correlation to LC-MS/MSo Caco-2 permeability

2009 ASMS – BioTrove (poster)
Evaluation of accurate mass TOF-MS for use in high throughput CYP450 inhibition screening.
William A. LaMarr, Michelle V. Romm, Nikunj Parikh, Lauren Frick, Can C. Ozbal.
CYP Inhibition using RF/TOF

2009 ASMS – Boehringer Ingelheim (podium talk)
A Novel and Integrated Platform for Fully Automated High-Throughput LC-MS/MS Analysis of In
Vitro ADME Samples. Andreas H. Luippold, Thomas Arnold, Wolfgang Jorg, Klaus Klinder & Kurt
Schumacher, Boehringer Ingelheim Pharma GmbH & Co KG, Biberach an der Riss, Germany.


RF-MS correlation to LC-MS/MSo CYP Inhibitiono Metabolic Stabilityo PAMPAo Caco-2

2009 SBS – BioTrove (poster)Consistent High-throughput CYP Inhibition Data Across Mass Spectrometry Platforms.
William A. LaMarr, Michelle V. Romm, Nikunj Parikh, Vaughn Miller, Can C. Ozbal.
CYP Inhibition data correlates with RF using 3 different MS QqQ vendor platforms

2008 ASMS - Takeda (poster)
An Integrated Approach for an Ultra-High Throughput On-Line SPE-MS/MS System and its
Applications to ADME Assays. Rongda Xu, Marianne T. Quintos, Melinda Manuel, Joshua E. Cramlett, Kheng
B. Lim, and Daniel B. Kassel; Takeda San Diego, Inc., San Diego, CA 92121.
RF-MS/MS correlation to LC-MS/MSo Metabolic Stability
o Plasma Protein Bindingo PAMPA2008 SBS – Exelixis (poster)
CYP450 Inhibition Profiling Using High Throughput RapidFire Mass Spectrometry Assays.
Sean Wu, Lory Tan, Jing Wang, Stefan Engst, Wentao Zhang* and Kirk McMillan
Exelixis, Inc., South San Francisco, CA 94083.

CYP inhibition: RF/MS/MS correlation to LC-MS/MS

2007 AAPS - AstraZeneca (poster)Comparison of BioTrove RapidFire uHTMS and Traditional LC/MS/MS Analysis to Assess
Cytochrome P450 Inhibition Utilizing Clinically Relevant Probe Substrates. Ethan Hoffmann1, Caroline
Rivard1, David Ayres1, Can Ozbal2, Steve Good1 and Patrick Brassil1 AstraZeneca R&D Boston, 35 Gatehouse Drive,

11 Audubon Road I Wakefield, MA 01880 I T: 781.928.2700 I www.Agilent.com

Last Updated August 22, 2012
Waltham, MA 02451 U.S.A 2 BioTrove Inc, 12 Gill St, Ste 4000, Woburn, MA 01801.
 CYP inhibition: RF-MS/MS correlate with LC-MS/MS

2007 ASMS – Takeda (podium talk)
Development of an Ultra High Throughput MS/MS CYP Inhibition Assay.
Daniel B. Kassel1; Kheng Lim1; Can C Ozabal2; William A LaMarr2
1Takeda San Diego, Inc, San Diego, CA; 2BioTrove, Inc., Woburn, MA.

CYP inhibition: RF-MS/MS correlation to LC-MS/MS

Clinical/Forensic Toxicology References
Using RapidFire-MS2012 AACC – Agilent Technologies (Poster)
Ultrafast Analysis of Drugs of Abuse using SPE/MS/MS
Nikunj R. Parikh1, Kari Schlicht1, Michelle V. Romm1, Vaughn P. Miller1, and William A. LaMarr1, 1Agilent Technologies Inc.,
Wakefield, MA

2012 AACC – Agilent Technologies (Poster)High-Throughput Analysis of Immunosuppressant Drugs in Whole Blood Using Ultra-fast
SPE/MS/MS
Kari E. Schlicht1, Pierre Wallemacq2 , Vaughn P. Miller1, and William LaMarr1 1. Agilent Technologies Inc, Wakefield, MA
2. Cliniques universitaires St-Luc, UCL, Brussels, Belgium

2012 ASMS – Agilent Technologies (Poster)High-Throughput Analysis of Drugs of Abuse in Urine Using Ultra-fast SPE/MS/MS for
Forensic Toxicology
Michelle Romm; Kari Schlicht; Matthew Woodcock; Vaughn Miller; Can "Jon" Ozbal; William A. Lamarr

2012 ASMS – Agilent Technologies (Poster)
Automated High-Throughptu Analysis of Proteins in Blood-based Matrices Combining
Immunoaffinity Purification and Ultra-fast SPE/MS/MS (AssayMAP)
Michelle V. Romm1, Zachary Van Den Heuvel2, Nikunj Parikh1, Vaughn P. Miller1 and William A. LaMarr1 1.
Abstract: Measuring biomarkers in plasma is challenging due to the complexity of the matrix. Currently,
immunoaffinity purification followed by LC/MS/MS is often employed to isolate and quantify proteins or peptides.
However, both steps are limited by inefficiency; immunoaffinity purification is labor intensive, while LC/MS/MS
analysis requires minutes per sample. Automation of the immunoaffinity purification step, allowing for simultaneous
preparation of 96 samples, followed by online SPE/MS/MS analysis at a rate of seconds per sample could greatly
improve the throughput of this workflow. We investigated the feasibility of using such an automated high-throughput
workflow to analyze both proteins and peptides from blood-based matrices.

2012 ISSX/MDO – Agilent Technologies (Poster)High-Throughput Analysis of 1’-Hydroxymidazolam in Plasma Using Ultra-fast SPE/MS/MS
11 Audubon Road I Wakefield, MA 01880 I T: 781.928.2700 I www.Agilent.com

Last Updated August 22, 2012
Michelle V. Romm1, Nikunj Parikh1, Vaughn P. Miller1, Moritz Wagner2, and William A. LaMarr1 1. Agilent Technologies,
Wakefield, MA 2. Agilent Technologies, Waldbronn, Germany

Abstract: There is an increasing demand for greater throughputs and efficiencies of mass spectrometry-based
bioanalysis by DMPK researchers. We evaluated the ability of an ultra-fast SPE/MS/MS system to analyze small
molecule analytes in a plasma based matrix with much faster sample cycle times and similar analytical resultscompared to LC/MS/MS methods. Critical bioanalytical parameters were systematically investigated using the small
molecule analyte 1’-hydroxymidazolam spiked into plasma. 1’-Hydroxymidazolam is the primary CYP3A4 metaboliteof the benzodiazepine midazolam and is often used for clinical drug-drug interaction studies. Comparable accuracy,
precision, and linearity were achieved at rates 10-30 fold faster than traditional LC/MS/MS methods.
Hydroxymidazolam calibration curves and quality control standards were prepared in a wide dynamic range by spiking
blank matrix with 1’-hydroxymidazolam. The samples were then precipitated with acetonitrile containing an
isotopically labeled internal standard, followed by dilution. Sample analysis was performed at a rate of <10 seconds per
sample using an Agilent RapidFire ultra-fast autosampler/online SPE system (reverse phase) coupled to an Agilent
6460 QQQ mass spectrometer. SPE methods were optimized for the analyte and the SPE/MS/MS results werecompared to LC/MS/MS analysis. Data analysis was performed using RapidFire Integrator software.
Hydroxymidazolam had excellent linearity within the measured range of 10-1000 ng/ml with an R^2 value greater than
0.995. Intra- and interday accuracies and precision for all concentrations were within 10%. A robustness test
comprising of 1500 sequential injections of the same hydroxymidazolam concentration in protein precipitated plasma
revealed a CV of less than 3% with no changes to peak signal or SPE cartridge pressure. Ionization suppression waspresent, but it could be effectively managed by using the stable isotope labeled internal standard (IS). Carryover was
found to be < 1% of the low standard. A very strong correlation of SPE/MS/MS results from the same samples
analyzed by traditional LC/MS/MS was seen (correlation coefficient = 0.999). The ultra-fast SPE/MS/MS system had
comparable accuracy, precision and linearity results to LC/MS/MS for 1’hydroxymidazolam in plasma. Thismethodology is capable of throughputs >400 samples per hour. The SPE/MS/MS system may be useful for the efficientbioanalysis of 1’-hydroxymidazolam clinical drug-drug interaction studies and for analysis of similar small moleculesin plasma from animal and human research studies.

2012 MSACL – Agilent Technologies (Poster)
Rapid Analysis of Drug Analytes in Uring for Forensic Toxicology Using Ultra-fast Online
SPE/MS/MS
Michelle V. Romm1, Kari Schlicht1, Matthew Woodcock2, Vaughn P. Miller1 and William A. LaMarr1 1. Agilent Technologies
Inc., Wakefield, MA 2. Dominion Diagnostics, LLC, North Kingstown, RI
Abstract: Forensic drug testing has traditionally utilized GC/MS as the analytical method of detection. In the present
study, we evaluated the ability of an ultra-fast SPE/MS/MS system to analyze metabolites of some of the drugs ofabuse including benzoylecgonine (BE), the major metabolite of cocaine, and 11-nor-9-delta-9-tetrahydrocannabinol(THCCOOH), the major metabolite of marijuana, in urine with much faster sample cycle times (under 15 seconds per
sample) and similar analytical results compared to GC/MS or LC/MS assays.

2012 MSACL – Agilent Technologies (Poster)
Ultra-fast analysis of Small Molecule Analytes in Urine and Blood-based Matrices using an
SPE/MS/MS System
Michelle V. Romm, Nikunj Parikh, Kari Schlicht, Vaughn P. Miller and William A. LaMarr; Agilent Technologies Inc.,
Wakefield, MA
Abstract: Mass spectrometry-based analysis in clinical research and forensic toxicology has emerged as a viable
analytical method due to it’s sensitivity, specificity, and robustness. We evaluated the ability of an ultra-fast
SPE/MS/MS system to analyze small molecule analytes in human urine or blood-based matrices with much fastersample cycle times and similar analytical results compared to LC/MS/MS assays. Hydroxymidazolam was investigatedin plasma and the major metabolite of cocaine, benzoylecgonine, was investigated in urine for forensic toxicology.

2012 SOFT – Agilent Technologies (Poster)
Ultra-Fast Online SPE/MS/MS Confirmation Analysis of THCCOOH in Urine for Forensic
Toxicology
11 Audubon Road I Wakefield, MA 01880 I T: 781.928.2700 I www.Agilent.com

Last Updated August 22, 2012
Michelle V. Romm1, Lawrence J. Andrade2, Matthew Woodcock2, Vaughn P. Miller1 and William A. LaMarr1 1. Agilent
Technologies Inc., Wakefield, MA 2. Dominion Diagnostics, LLC, North Kingston, RI
Abstract: Forensic drug testing has traditionally utilized GC/MS and more recently LC/MS as the analytical method of
detection. Steady increases in the need for greater analytical capacity and throughput have placed demands on
traditional technologies. We evaluated the ability of an ultra-fast SPE/MS/MS system to accurately measure 11-nor-9delta-9-tetrahydrocannabinol (THCCOOH, the major metabolite of marijuana) and metabolites of synthetic
cannabinoids in urine with much faster sample cycle times (under 15 seconds per sample) and similar analytical resultscompared to LC/MS/MS assays.

2012 SOFT – Agilent Technologies (Poster)
Screening for Drugs of Abuse in Forensic Toxicology Using an Ultra-Fast Online
SPE/MS/MS System
Nikunj R. Parikh1, Kari Schlicht1, Michelle V. Romm1, Vaughn P. Miller1, and William A. LaMarr1, 1Agilent Technologies
Inc., Wakefield, MA.
Abstract: Forensic drug screening is extensively used by Law Enforcement officials; employers and pathologists today.
Traditionally the screening for these drugs involves analysis by immunoassays followed by a confirmatory test by
GC/MS detection and more recently by LC/MS assays. The steady increase in sample size has created a bottleneck inscreening for these drugs across different classes and longer turnaround times for confirmatory tests. Whileimmunoassays are convenient and well established; they are not as sensitive or specific as LC/MS which leads to many
false negatives and positives. In the present study; we evaluated the ability of an ultra-fast SPE/MS/MS system toscreen across different classes of drugs of abuse in urine while approaching the sensitivity and accuracy of LC/MS andmaintain the speed and efficiency of a screen (sample cycle times <15 seconds per sample).

2012 US HUPO – Agilent Technologies (Poster)Development of Automated SISCAPA Assays for High-Throughput Quantitation of Protein
Biomarkers
Christine Miller3, Leigh Anderson1, Matt Pope2, Morteza Razavi2, Terry Pearson2
Abstract: SRM-MS allows specific, internally-standardized measurement of protein biomarkers and can achieve subnanogram/mL detection levels when specific anti-peptide antibodies are used to enrich target peptides from the plasma
digests (SISCAPA). We have developed an automated protocol for implementing this immunoaffinity enrichment ofbiomarker peptides. The effectiveness of the protocol was evaluated using a multiplex SISCAPA panel based on 11
rabbit monoclonal antibodies to specific peptides from 10 proteins spanning a wide plasma concentration range.
Parameters for each of the 11 target peptides and cognate labeled standards have been optimized, permitting use ofretention-time scheduled MRM data collection and rapid (3 min) analysis times. Results will be presented
demonstrating the performance of the workflow for high-throughput quantitationof protein biomarkers.

2011 AACC – Agilent Technologies (Poster)High-Throughput Analysis of Levetiracetam in Serum Using Ultra-fast SPE/MS/MS
Michelle V. Romm, Eric W. Korman, Vaughn P. Miller, Christine L. Snozek, Frank W. Crow, Loralie J. Langman and William
A. LaMarr
Abstract: Mass spectrometry-based assays have emerged as a viable analytical method due to their sensitivity,
specificity, and robustness. We evaluated the ability of an ultra-fast SPE/MS system (Agilent RapidFire Highthroughput Mass Spectrometry System) to analyze levetiracetam in human serum with much faster sample cycle times
and similar analytical results compared to HPLC or LC/MS/MS assays.

2011 AACC – Agilent Technologies (Poster)
Ultra-fast, Simultaneous Analysis of a Panel of Benzodiazepines in Human Urine using an
SPE-TOF System
Vaughn P. Miller1, Michelle V. Romm 1, Nikunj Parikh1, and William A. LaMarr1 1. Agilent Technologies Inc., Wakefield,MA

11 Audubon Road I Wakefield, MA 01880 I T: 781.928.2700 I www.Agilent.com

Last Updated August 22, 2012
Abstract: Benzodiazepines are widely prescribed drugs for the treatment of anxiety, sleep disorders and other illnesses.
Because addiction and abuse can occur with these drugs, efficient screening methods are critical to clinical, forensic
and toxicology laboratories. We evaluated the ability of an ultra-fast SPE-MS system (RapidFire) to analyze a panel ofbenzodiazepines in human urine with much faster sample cycle times and similar analytical results compared to
traditional LCMS systems.

2011 AACC – Agilent Technologies (Poster)High-Throughput Analysis of Tacrolimus in Whole Blood Using Ultra-Fast SPE/MS/MS
Kari E. Schlicht 1, Eric W. Korman2, Vaughn P. Miller1, Christine L. Snozek2, Frank W. Crow2, Loralie J. Langman2 andWilliam A. LaMarr1 1. Agilent Technologies Inc., Wakefield, MA 2. Mayo Clinic, Rochester, MN
Abstract: In many clinical research laboratories, liquid chromatography-mass spectrometry (LC/MS) methods ofanalysis of immunosuppressant drugs have proven superior because of their increased sensitivity and selectivity. We
have evaluated the ability of an ultrafast SPE/MS/MS system (Agilent RapidFire High-throughput Mass Spectrometry
System) to analyze the immunosuppressant drug tacrolimus in whole blood. The results demonstrate the superior speedof SPE/MS/MS to complement the superior sensitivity and selectivity of MS with significantly faster sample cycle
times than LC/MS while yielding similar analytical results.

2011 ASMS – Agilent Technologies (Poster)
Ultra-fat Analysis of Benzodiazebines in Human Urine using Dilute and Shoot Methodology
and SPE/MS/MS
Lauren E. Frick, Michelle V. Romm, Vaughn P. Miller, and William A. LaMarr
Abstract: Liquid-liquid extraction and protein precipitation represent time-consuming and cumbersome steps in clinicalsample preparation, the elimination of which would greatly speed the rate with which labs can analyze clinical samples.
Dilute and shoot has been proposed as a preparation-free alternative, though its utility can be limited by matrix effects
resulting from the lack of pre-analysis sample cleanup. Here, we apply the method to the detection of a panel ofbenzodiazepines in human urine using an ultra-fast online SPE cleanup prior to MS/MS analysis. With a cycle time of~9 seconds per sample, the system offers a considerable speed advantage over chromatography-based dilute and shootmethodologies while maintaining data quality.

2011 ASMS – Agilent Technologies (Poster)
Ultrafast SPE Integrated with TOF-MS Increases the Throughput of Metabolic Stability
Assays and Enables Analysis of Metabolites
Nikunji Parikh1, Michelle V. Romm1, Yuqin Dai2, Vaughn P. Miller1 and William A. LaMarr1 1. Agilent Technologies Inc.,
Wakefield, MA 2. Agilent Technologies Inc. Santa Clara, CA

2011 ASMS – Johnson and Johnson (Poster)
Bioanalysis without chromatography: the Evaluation of RapidFire Ultra-fast online SPEMS/MS System for Quantitation of Drugs in Biological Matrixes
Wenying Jian1, Michelle V. Romm2, Richard W. Edom1, Vaughn P. Miller2, William A. LaMarr2, Naidong Weng1 1. Johnson& Johnson Pharmaceutical Research & Development, Raritan, NJ 2. Agilent Technologies, formerly BIOCIUS Life Sciences,
Wakefield, MA
Abstract: The RapidFire Mass Spectrometry platform is a fully automated system that integrates on-line SPE sample
preparation and mass spectrometry analysis, and is capable of conducting analysis at a speed of 5-10 s per sample. Its
existing applications mostly involve in vitro high-throughput ADME or therapeutic target screening studies. In thecurrent work, the feasibility of utilizing RapidFire for analysis of in vivo bioanalytical samples was evaluated for the
first time. Key potential analytical liabilities of RapidFire, such as matrix suppression (caused by matrix components
and dosing vehicles) and carryover, were systematically investigated using structurally diverse commercial compounds.
More importantly, quantitation data obtained from the RapidFire system was compared with those from conventional
LC-MS/MS analysis to evaluate its performance for bioanalysis.

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Last Updated August 22, 2012

11 Audubon Road I Wakefield, MA 01880 I T: 781.928.2700 I www.Agilent.com