Mass Spectrometry reviews 2008 v27 p20

Guest Editor: Tim Baker
MASS SPECTROMETRY ANALYSIS OF NEW CHEMICAL ENTITIES
FOR PHARMACEUTICAL DISCOVERY
Annette S. Fang,1 Xiusheng Miao,1 Peter W. Tidswell,2 Marc H. Towle,3
Wolfgang K. Goetzinger,4 and James N. Kyranos5*
1ArQule, Inc., Woburn, MA
2Pfizer Global Research and Development, St. Louis, MO
3Vertex Pharmaceuticals, Cambridge, MA
4Amgen, Cambridge, MA
5Wolfe Laboratories, 134 Coolidge Avenue, Watertown, MA 02472
Received 28 February 2007; received (revised) 20 August 2007; accepted 24 September 2007
Published online 21 November 2007 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/mas.20153
In this Section, we review the applications of mass spectrometry
for the analysis and purification of new chemical entities
(NCEs) for pharmaceutical discovery. Since the speed of
synthesis of NCEs has dramatically increased over the last
few years, new high throughput analytical techniques have
been developed to keep pace with the synthetic developments.
In this Section, we review both novel, as well as modifications
of commonly used mass spectrometry techniques that have
helped increase the speed of the analytical process. Part of the
review is devoted to the purification of NCEs, which has
undergone significant development in recent years, and the
close integral association between characterization and
purification to drive high throughput operations. At the end
of the Section, we review potential future directions based on
promising and exciting new developments. # 2007 Wiley
Periodicals, Inc., Mass Spec Rev 27:20–34, 2008
Keywords: mass spectrometry; characterization; purification;
new chemical entities
I. BACKGROUND
In recent years, the development of high throughput synthesis
techniques has significantly changed the drug discovery process.
The successful implementation of automation technology that
allows medicinal chemists to generate 10–1,000 times more
compounds for screening has augmented the traditional synthesis
of single compounds. Today’s approach for early discovery hit
identification is geared towards generating hundreds or even
thousands of new chemical entities (NCEs) every week. By
leveraging automation technology, larger numbers of compounds
can be generated with minimal additional effort. However, in
order to effectively utilize the subsequent screening information,
these compounds must be characterized to assess purity and
quantity. Moreover, screening larger collections of closely
related analogs together, yields more data per cycle and leads
to more complete structure activity relationships. By conducting
the initial cycles of design, synthesis, analysis and screening in a
high throughput manner, the typical medicinal chemistry process
is converted to a more efficient and cost effective discovery
process (Kassel, 2001).
Traditionally, every compound synthesized would be
analyzed by techniques such as nuclear magnetic resonance
(NMR), thin layer chromatography (TLC), and elemental
analysis. Although these techniques provide the medicinal
chemist with the necessary characterization information, they
are not readily amenable to unattended high throughput
automation and easily automated data interpretation. The ability
to acquire and interpret data in an automated fashion is an
important consideration to effectively handle the thousands of
NCEs being synthesized. While NMR is clearly the technique
of choice for most traditional medicinal chemists, the need for
spectral interpretation by an experienced professional limit the
overall throughput of the technique in a high throughput setting.
In addition, higher capital costs, limited automation capabilities,
large sample requirements and the use of deuterated solvents
impose further limitations on the use ofNMRfor high throughput
analysis (Haner, Llanos, & Mueller, 2000; Keifer et al., 2000;
Lindon, Nicholson,&Wilson, 2000; Louden et al., 2000;Wilson,
Lindon, & Nicholson, 2000).
II. MASS SPECTROMETRY—CENTRAL
ANALYTICAL TECHNIQUE
Simultaneous to the development of high throughput synthesis,
mass spectrometry (MS) has emerged as the key analytical
technique to characterize NCEs (Carrell et al., 1995; Enjalbal,
Martinez, & Aubagnac, 2000; Hauser-Fang & Vouros, 2000;
Su¨ssmuth & Jung, 1999; Triolo et al., 2001a,b). The inherent
high sensitivity of MS leads to small sample requirements.
Moreover, the relative ease of unattended automation and
automated data interpretation contribute toMSbeing the primary
characterization technique for high throughput analysis. Below
————
*Correspondence to: James N. Kyranos, Wolfe Laboratories, 134
Coolidge Avenue, Watertown, MA 02472.
E-mail: james.kyranos@wolfelabs.com
Mass Spectrometry Reviews, 2008, 27, 20–34
# 2007 by Wiley Periodicals, Inc.

we will discuss some of the most common mass spectrometry-based
techniques along with advantages and disadvantages.
Depending on the focus of the investigation, a variety of
analytical data may be desired, such as overall purity, number
and type of contaminants or the identification of other com-ponents
in mixtures (Kassel, 2001). In such cases, mass
spectrometry is often used in combination with other techniques
such as ultraviolet (UV) or evaporative light scattering detectors
(ELSD) to collect a range of complementary data for higher
confidence results (Yan et al., 2000).
Adapting to the new challenges presented by modern drug
discovery, all analytical instrumentation and especially mass
spectrometry have evolved. The most notable changes in MS
have been associated with the continued refinement of ionization
techniques and the successful coupling of various separation
methods with mass spectrometry. Most analyses involving small
organic compounds to large biomolecules have benefited from
advances in ionization techniques such as electrospray ionization
(ESI), atmospheric pressure chemical ionization (APCI) and
matrix-assisted laser desorption/ionization (MALDI). The most
pronounced advantage of MS regardless of the ionization
technique noted above is that resulting spectra are simpler to
interpret with very few fragments observed compared to electron
impact (Hauser-Fang & Vouros, 2000).
In addition to generating simpler spectra, atmospheric
pressure ionization techniques allow much easier coupling of
high performance liquid chromatography (HPLC), capillary
electrophoresis (CE) or supercritical fluid chromatography
(SFC) with a mass spectrometer. Earlier publications reported
successful coupling HPLC instruments with MALDI and fast
atom bombardment (FAB) ionization sources, however, ESI and
APCI have become the dominant ionization techniques in the
field of on-line HPLC–MS.
The role of MS and HPLC–MS for the characterization of
combinatorial libraries has been described by several authors,
and rapid HPLC–MS for the analysis of small molecules has
been reviewed in the past byWehr (2003), Tiller et al. (2003), and
Hsieh, Brisson, and Wang (2003). Typically, an initial MS
characterization run provides a quick check for synthetic
success and an assessment of compound purity. The resulting
information may then be used to set up a variety of parameters for
subsequent purification or to further investigate potential
synthesis failures. Regardless of the motivation or approach,
given the increased pressure to generate more high quality
compounds in a shorter time frame, high throughput MS
methods for both characterization and purification have become
commonplace.
III. IONIZATION TECHNIQUES FOR A HIGH
THROUGHPUT ENVIRONMENT
Since mass spectrometric detection is very specific in providing
compound structural information, it is an irreplaceable tool in
drug discovery (Kleintop, Zhang, & Ray, 2004). In addition, fast
and accurate analytical methods are essential to keep pace with
compound libraries produced with high throughput synthesis
techniques. To facilitate these needs there have been many
improvements and innovations of ionization sources.
MASS SPECTROMETRY ANALYSIS &
A. MUX Source
One of the most innovative approaches to increase the effective
utilization of the mass spectrometer, and increase the overall
analysis speed, is the development of the multi-channel ESI
(MUX) source (Biasi et al., 1999;Wang et al., 1999; Fang et al.,
2002). The MUX interface consists of multiple electrospray
probes connected to separate HPLC pumps and one sampling
cone. Multi-channel electrospray sources have been designed
with up to eight individual parallel channels ending in an
electrospray tip at an angle to the sampling cone. A sampling
rotor connects the spray of each channel to the sampling cone in
rapid succession. Data acquisition occurs separately for each
channel with as many data traces as channels used (usually four or
eight). This approach leverages a single more expensive mass
spectrometer coupled with multiple separation systems to
generate an overall cost-effective solution (Sage, Little, &
Giles, 2000; Burg, 2004). However, because of the fast
acquisition times that are needed to effectively switch between
channels, this type of setup is only possible with instruments
having high scan speeds. Orthogonal acceleration time of flight
(TOF) mass spectrometer is the instrument of choice for this
application. As the speed of analysis is increased, the per-sample
capital expenses are correspondingly reduced. In order to reduce
the overall per sample analysis time, this type of system has been
applied for both pre-purification and post-purification analysis
of high throughput synthesis products (Yan et al., 2004).
The general approach, with appropriate modification made for
preparative HPLC, has also been used for high throughput
purification of diastereomers (Irving et al., 2004).
B. Combination Sources
It is estimated that about 80% of small molecular weight
compounds that are of the same structural class readily ionize
using a similar electrospray ionization method. For samples that
do not ionize well using a typical acid modified mobile phase and
ESI, one can investigate increasing the pH of the mobile phase or
change the ionization technique. Gallagher et al. (2003) have
developed a new combined ESI–APCI source (ESCi) for use
with on-line HPLC applications. This combined source can
generate clearly differentiated and reproducible ESI and APCI
spectra. Furthermore, positive and negative spectra can readily be
obtained by switching polarity. The qualitative performance of
the combined source has been compared to existing ESI and
APCI interfaces. The ESI mode of the new source has been found
to be equivalent to conventional ESI and the APCI mode
produces less thermal fragmentation. This new source operates
effectively at flow rates from 50 to 1,000 mL/min and can be used
with a variety of separation techniques. By eliminating the need
to repeat chromatographic separations in order to generate
data using different ionization modes or polarity, the overall
analysis time is reduced and throughput is increased.
Similarly, in order to gain an advantage in the ionization of
low polarity compounds, an APCI/APPI (atmospheric pressure
chemical ionization/atmospheric pressure photoionization)
source has been developed. The advantage of this approach, for
example, is that this source can easily be coupled with normal
Mass Spectrometry Reviews DOI 10.1002/mas 21

phase HPLC systems for the analysis of very nonpolar
compounds such as steroids (Robb, Covey, & Bruins, 2000).
C. MALDI
MALDI–TOF has become a widely used and powerful tool
for the analysis of large peptides, biomolecules, and synthetic
polymers. However, the analysis of small molecules by MALDI
has remained a challenge. This is primarily due to the technique’s
relatively poor ionization efficiency and the presence of a variety
of abundant matrix-related ions in the low mass range. In
addition, proper sample preparation requires significantly more
operator experience and thus is not amenable to open access
walk-up systems that are available to the medicinal chemists.
Regardless of the potential limitations, MALDI has many
advantages including low detection limits that are in the
femtomole range. This level of sensitivity is ideal for the analysis
of resin bound compounds obtained from individual beads.
MALDI characterization of single beads has been successfully
used for characterization of split-and-mix combinatorial libra-ries.
(Egner, Cardno, & Bradley, 1995; Egner, Langley, &
Bradley, 1995). By using appropriate photocleavable linkers
during synthesis, products can be analyzed directly off the solid
support (Fitzgerald et al., 1996). The MALDI laser irradiation
provides enough energy to cleave the compound off the bead and
ionize it at the same time.
Although low molecular weight interference produced by
conventional matrices, has limited the utility of MALDI for NCE
characterization (Cohen & Gusev, 2002; Krutchinsky & Chait,
2002), recent introductions of new matrices are beginning to
overcome this issue (Iijima, 1991; Iijima & Ichihashi, 1993;
Dale, Knochenmuss,&Zenobi, 1996;Ayorinde et al., 1999;Wei,
Buriak, & Siuzdak, 1999; Han & Sunner, 2000; Kinumi et al.,
2000; Cuiffiet al., 2001; Shen et al., 2001; Zhang et al., 2001; Guo
et al., 2002; Cai et al., 2003; Fu & Sun, 2003; Lewis et al., 2003;
Peng et al., 2003; Reyzer et al., 2003; Xu et al., 2003; Pan et al.,
2004; Go et al., 2005; Gobey et al., 2005; Pan et al., 2005; Sunner,
Dratz, & Chen, 1995). Stolzberg and Patel (2004), for example,
were able to produce clean negative ion mass spectra of
molecules in the 100–300 Da range using a light-absorbing
electrically conductive polymer matrix. Peterson et al. (2004)
used porous polymer monoliths for MALDI analysis and were
able to transfer sufficient energy to the analyte to induce
desorption and ionization. The performance of the monolith
hydrophobic porous surfaces was demonstrated with several
small molecules, including drugs, explosives and acid labile
compounds.
Separation combined with MALDI–MS for small molecule
analysis has been reported by several authors using ultra-thin
layer chromatography (TLC–MALDI) (Busch, 1992; Somsen,
Morden,&Wilson, 1995;Weins& Hauck, 1996; Chen, Shiea,&
Sunner, 1998; Wilson, 1999; Gusev, 2000; Mehl & Hercules,
2000; Hauck et al., 2001; Ka´lasz & Ba´thori, 2001; Hauck &
Schulz, 2002;Wu&Chen, 2002; Crecelius, Clench,&Richards,
2003; Salo et al., 2003; Santos et al., 2004). In this approach,
analytes were separated on TLC plates, which were then coated
with matrix and subsequently introduced into theMALDIsource.
Salo et al. (2003) used an atmospheric pressure MALDI (AP-MALDI)
source in order to reduce possible contamination of the
mass spectrometer from introducing chromatographic material
into a vacuum. All analyte spots were successfully ionized
directly from the TLC plate.
Gobey et al. (2005) have shown that the use of a high
repetition rate laser can increase sample throughput while
filtering out matrix interferences through the use of tandem mass
spectrometry. The authors especially emphasize that this type of
MALDI analysis is the ultimate high-throughput technique
with an analysis time limited only by the speed of the plate
readers of the MALDI instrument rather than the solvent
handling capacity of an HPLC–MS system. Limitations include
ion suppression effects from complex matrices and the need for
additional SPE cleanup procedures for complex and impure
samples.
In a comparison of MALDI and ESI using 14 compounds
that failed ESI analysis, only four of them were successfully
analyzed by MALDI. These data suggest that MALDI and ESI
are generally not orthogonal techniques. In a recent publication
by Corra et al. (2006), ESI and MALDI were compared with
regards to several operating considerations, including low-molecular
weight sensitivities, analysis speed, throughput and
instrument robustness. They found that although MALDI has a
faster acquisition speed, detection limits are higher compared to
HPLC/ESI–MS. However, by developing methods to concen-trate
samples on the target, detection limits can be significantly
improved.
Lee, Chen, and Gebler (2004) have shown that MALDI–MS
can be used both as a qualitative or quantitative technique.
MALDI can certainly be used in an automated high-throughput
mode with individual compounds loaded onto the target.
However, the typical spot-to-spot sample reproducibility and
matrix interferences are still limiting factors, especially for small
molecule analysis. Sleno (2005) have reviewed the fundamental
and technical aspects of MALDI and its limitations in greater
detail. If the limitations of the technique can be overcome,
MALDI analysis might one day provide extremely fast analysis
of NCEs while consuming only small sample quantities.
D. Nano and Chip Based Techniques
In a unique approach Pan et al. (2004, 2005) used oxidized and
nonoxidized carbon nanotubes as a matrix for small molecule
MALDI analysis (Iijima, 1991; Iijima & Ichihashi, 1993; Sunner
et al., 1995; Dale, Knochenmuss,&Zenobi, 1996; Han&Sunner,
2000; Cai et al., 2003; Fu&Sun, 2003; Peng et al., 2003;Xu et al.,
2003). Compared to conventional matrices, they observed
dramatically reduced matrix interferences for both types of
nanotubes. Oxidized carbon nanotubes enhanced signal intensity
compared to nonoxidized ones. The advantages of oxidized
carbon nanotubes seem to be associated with greater water
solubility that leads to a more homogeneous matrix mixture.
By using silicon nanowires (Go et al., 2005), the sensitivity
of the technique was increased down to the attomole level.
Moreover, the 40 nm silicon nanowires were used to conduct a
chromatographic separation by taking advantage of their high
surface area and fluid wicking capabilities. The nanowires
provide a unique platform for both separation and mass
spectrometric analysis.
& FANG ET AL.
22 Mass Spectrometry Reviews DOI 10.1002/mas

Zhang et al. (2001) described quantitative analysis of small
molecules using chip-based nano ESI–MS/MS. This system is
reported to have advantages in limiting carryover, sample
consumption, cycle time and the ability to be fully automated.
Chip-based nano-electrospray that is integrated into a micro
purification/collection system has also been reported and will be
discussed further in the purification section.
IV. SEPARATIONS COUPLED WITH
MASS SPECTROMETRY
Speed is an important consideration in modern drug discovery.
The more time that is reduced from the discovery and develop-ment
cycle, the more patent protection the company can enjoy
once the drug is on the market. Extending the patent protection by
6 months to a year translates into hundreds of millions of dollars
in total revenue. It is these types of incentives that drive the
industry to greater speed and efficiency. For high throughput
analysis, the need for speed usually means that large numbers of
compounds have to be analyzed in a matter of days and the results
have to be interpreted quickly thereafter.
For practical purposes, high throughput analysis is usually
further divided into high throughput characterization and
purification. In characterization, the focus lies in determining
the contents and/or assessing the purity of a large number of
samples. Characterization analysis is used as an initial check for
synthesis completion, quality control purposes or to collect
important data about samples that need to be purified later. For
purification, the data obtained in characterization is typically
used to optimize the subsequent preparative analyses. Given the
robustness of the single quadrupole systems and the lower cost
relative to the high end mass spectrometers, low-resolution
HPLC–ESI–MS instruments have become the industry standard
for high throughputNCEcharacterization. There have been some
examples of high throughput high resolution work but usually
this process is significantly more complex and in most cases
unnecessary (Walk et al., 1999).
A. FIA–MS
Flow injection analysis mass spectrometry (FIA-MS) is the
simplest form of rapid sample introduction into the mass
spectrometer. This approach was one of the earliest examples
of characterization and has been widely used in the analysis of
high throughput synthesis products. If separation is not needed,
FIA can be used to confirm synthesis by the presence of the mass-to-
charge ratio (m/z) of the expected compound ions. Since flow
injection analysis is not limited by separation run times, it has real
speed advantages. FIA–MS provides the highest throughput and
greatest ease of automation and use for large numbers of samples
(Yu & Balogh, 2000). A major drawback to FIA–MS is ion
suppression due to co-eluting sample components. The ion
suppression can be quite significant. In many cases there is
significant chemical interference and the expected molecular ion
of the desired compounds is not observed, even though the
synthesis may have been successful. Moreover, without separa-tion
of the synthesis mixture components, there is no reliable
purity determination.
MASS SPECTROMETRY ANALYSIS &
One of the early issues arising in FIA–MS analysis was
sample to sample carry-over. This issue can be a serious concern
for any analysis, however, it is of particular issue when running
large number of samples very quickly, where wash cycles may be
abbreviated to gain cycle time speed. Earlier instrumentation was
limited in its control of injector parameters and thus more prone
to carryover when trying to maximize analysis speed. Richmond
(2000) and Richmond and Go¨rlach (1999a) described minimiz-ing
sample carryover in reaction monitoring using FIA–MS.
Morand et al. (2001) reported an improved method with a
throughput of 4 sec/sample by using a parallel sampling system
with an eight-probe injector. Others have worked on improving
speed or the analytical design (Felten et al., 2001) and also on
theoretically analyzing inter-sample carryover (Richmond &
Go¨rlach, 1999b; Richmond, 2000). The increasing numbers of
samples for analysis required a further reduction in the sample
measurement duty cycle while maintaining the carry-over below
1%. Lazar et al. (1999) introduced high-speed autosamplers with
duty cycles that could be reduced from 168 to 44 sec. Further
optimization of syringe and loop wash steps could reduce the
median inter-sample carry-over to 0.01%. Wang et al. (1998)
shortened the sample injection duty cycle by using a multiprobe
autosampler for flow-injection analysis of eight samples within
1 min. Using this type of autosampler, it has been shown that
more than eight flow-injection peaks can be generated in 1 min,
which translates into analyzing one whole 96-microtiter well
plate in less than 12 min (Wang et al., 1998).
Shah et al. (2000) described a quantitation method with
FIA–MS and chemiluminescence nitrogen detection (CLND)
for simultaneous proof of structure and estimation of purity,
respectively. Characterization of one compound was completed
every 60 sec, allowing for more than 1,000 compounds analyzed
in a single day. However, when large amounts of impurities were
present or the compound of interest was present at a very low
concentration, purity estimation was less reliable. In these types
of cases, HPLC–CLND and HPLC–MS may be required.
Although, as described above, there have been many
publications focusing on high-throughput injections and the
reduction of carryover in FIA–MS, most commercial systems
have been optimized for approximately one injection per minute.
This injection speed is adequate for typical HPLC analysis but
not high throughput FIA–MS operations. The approaches
described above that were used for multiple injections per
minute, require extensive modifications of injector hardware and
software.
B. HPLC–MS
Flow–injection analysis provides the shortest run time for a high
throughput system but does not yield confident purity determi-nation.
In order to assure the highest possible data quality with
more complex samples, chromatographic separation is neces-sary.
There is significant opportunity to develop the appropriate
analytical methods by coupling HPLC to mass spectrometry,
however, usually at the expense of longer analysis times. In an
effort to decrease HPLC–MS run times, rapid mobile phase
gradients and/or modifiers, as well as packing materials and
column size are among the more flexible components that have

been adjusted to fit individual analysis. All these parameters have
been used to develop effective high quality and rapid character-ization
and purification methods. Of course, chromatographic
analysis times can also be dramatically shortened by using
different techniques such as capillary electrophoresis (CE),
capillary electrochromatography (CEC) or supercritical fluid
chromatography (SFC). Some of these techniques will be
reviewed further in subsequent sections.
In general, modifiers are compounds that alter the
physicochemical properties of the mobile phase. Modifiers have
been used quite extensively to optimize specific chromatographic
separations. Although a variety of modifiers have been developed
and reported, only those that are volatile are compatible with the
needs of the mass spectrometer. This requirement has limited
the use of modifiers primarily to trifluoroacetic acid (TFA) and
formic acid (FA) for acidic methods and ammonium buffers for
basic separations.
In addition to optimizing chromatographic resolution,
mobile phase additives can be used to improve MS sensitivity
in both ESI and APCI detection modes. Mallet, Lu, and Mazzeo
(2004) evaluated ion suppression/enhancement in negative-ion
mode by using several additives and ion-pairing agents at
different concentrations for test compounds. The intensity of a
novel indolocarbazol compound increased by a factor of four
in negative-ion mode when the pH of the mobile phase was
increased from 8 to 10 using 2mMammonium hydroxide (Wang
et al., 2002). Similarly, the sensitivity of detection was increased
up to tenfold for isorhamnetin with a decrease in pH of the mobile
phase ranging from 4.5 to 2.3 in positive-ion mode (Rauha,
Vuorela, & Kostiainen, 2001). The addition of alkali metal salts
to enhance the intensity of molecular ion adducts by forming
stable Liþ, Naþ, or Kþ complexes of organic compounds has
been observed in FAB, MALDI, and ESI mass spectrometry of
various compounds (Williams et al., 2003). The extreme form of
mobile phase modification can be considered micelle formation
used with electrophoresis, which affects the fundamental
separation processes. There have been reports of various
approaches, such as micellar electrokinetic chromatography
in combination with mass spectrometry (Goetzinger & Cai,
2005).
Chromatographic resolution and mass spectrometric sensi-tivity
are important considerations for all analytical methods.
However, the real speed limitation for any high throughput
analysis using HPLC–MS is the chromatographic separation
time. Analysis speed has been enhanced by reducing the column
size from typical column dimensions of 4.6150 mm to
4.630 mm. These shorter columns can be used with higher
flow rates without exceeding the operating pressures limits.
HPLC instrumentation has also been adapted to enable high-speed
gradients. Presently, gradients of 5–95% acetonitrile/
aqueous buffers within 1–5 min with fast column regeneration
are quite common. These developments have increased the
throughput from 24 to 48 samples per day using a 30–60 min
analysis to more than 250 samples per day using a 5 min analysis.
One to five minute run times have become common for many
laboratories. For our operations, run times of 2.5 min are the
standard with no apparent negative effects on peak shape or
resolution (Kyranos et al., 2001a). Such short run times make
high throughput HPLC–MS analysis a practical and effective
analytical solution to support high throughput synthesis.
Goetzinger and Kyranos (1998) investigated packing
materials, gradient methods and sample solvents to allow
FIGURE 1. QC of combinatorial library sample with rapid HPLC. a: 1504.6 mm, 5 mm Zorbax SB-C8,
1.5 mL/min, 15–95% acetonitrile in 16 min; (b) 504.6 mm, 3.5 mm Zorbax SB-C8, 2 mL/min, 15–95%
acetonitrile in 4 min; (c) 304.6 mm, 3.5 mm Zorbax SB-C8, 4 mL/min, 15–95% acetonitrile in 0.6 min.
FANG ET AL.

ultra-fast gradient HPLC–MS methods down to 1 min per
sample. Similarly, Hsieh et al. (2001) used fast gradients within
1 min for quantitative screening of drug discovery compounds
in monkey plasma. Using generic methods to avoid method
development is another trend to enhance the throughput for the
analysis of combinatorial libraries. Figure 1 is an example of
rapid chromatography using fast gradients and high flow without
compromising chromatographic resolution. Although the run
time is significantly decreased from 20 to 1 min, the overall peak
capacity for all the separations is still acceptable to determine
sample purity. The significant developments in separation speed
have shifted the efforts for further optimization and time savings
toward the autosampler and injector components (Zweigenbaum
et al., 1999).
As previously discussed, the MUX interface has been used
successfully with FIA–MS to increase the overall throughput.
Davis and Griffith (2004) described a high-throughput parallel
HPLC–MS–ELSD method for the analysis of combinatorial
libraries with an eight-channel TOF system. This configuration
could analyze approximately 300,000 compounds per year. Fang
et al. (2003) investigated parallel high-throughput accurate mass
measurement using a nine-channel multiplexed HPLC–UV–
TOF–MS system. Accurate mass measurements were simulta-neously
obtained from compounds eluting from eight parallel
columns. Mass accuracy better than 10 ppm was achieved for
80% of the samples investigated.
In other examples of higher throughput, Cremin and Zeng
(2002) applied an eight-way fully automated parallel LC–MS–
ELSD system to enhance the throughput for the analysis of a
natural product library. This method has been successfully
applied to analyze a library of 36,000 partially purified fractions
derived from plants.
The quest for additional separation speed and higher
efficiency, which in turn will increase the overall analysis
throughput, continues with the development of new stationary
phase materials. Much of the early development in this area has
been focused on decreasing the particle size of the packing
material. However, the higher operating pressure associated with
particle size reduction has limited this advancement. Monolithic
columns are a single solid piece of stationary material with pores
through the main skeleton. These structures can increase column
performance by increasing the column permeability and
efficiency (Tanaka et al., 2002). There have been a number of
applications in related research areas using monolithic columns
that exemplify the beneficial performance of these materials
(Asperger et al., 2002; Borges et al., 2004).
C. CE–MS
Capillary electrophoresis (CE) is a powerful separation techni-que
that has been used to separate ionic compounds (Foulon et al.,
2004; Liu, Jo¨nsson, Jiang, 2005) or chiral mixtures (Millot,
2003; Erny Cifuentes, 2006; Souverain et al., 2006). The
different mode of separation of CE results in a unique selectivity
compared to reverse phase HPLC. Moreover, high efficiency
associated with CE promise enhanced separation resolution,
which will lead to even faster analyses. This technique can be
used as an orthogonal complimentary approach to HPLCor as the
primary analysis. However, the difficulties in interfacing CE to
the mass spectrometer and the limited amount of material that can
be loaded on the column have hindered wider use of CE–MS.
Dunayevskiy et al. (1996) was one of the first to apply CE–
MS to combinatorial chemistry. They successfully identified the
171 individual components of a combinatorial library mixture
using CE–MS. Visky et al. (2005) discussed the application of
CE–MS and its role in impurity profiling of pharmaceutical
products. More recently, Smyth reviewed the broader applica-tions
of CE–MS in drug analysis (Smyth, 2005). Generally, CE–
MScan be a valuable technique for rapid compound separation. It
is also useful for analyzing extremely small sample volumes with
high separation efficiency and has been applied to the analysis of
complex biological matrixes (Moini, 2002; Wan Thompson,
2005).
Goetzinger and Cai (2005) described a newway of preparing
a micellar buffer system for the separation of neutral compounds
based on micellar electrokinetic chromatography (MEKC). The
substitution of the traditionally used alkyl sulfates, such as SDS,
with carboxylates together with the substitution of inorganic
cations with organic bases provided a different separation system
with interesting properties. It was shown that these novel buffer
systems could be used as micellar separation systems with lower
conductivity compared to SDS buffers and thus provided more
flexibility for separation conditions. Although this article was
conducted using UV detection, the buffers used are volatile and
amenable to interfacing with MS. The inevitable coupling of this
novel MEKC system with MS could be used to increase
separation speed or alternatively to improve the detection limit
by using larger diameter capillaries.
D. SFC–MS
Supercritical fluid chromatography (SFC) has been used for
chiral separations (Mukherjee, 2007). Recently there has been a
growing trend in the application of SFC–MS to the analysis of
NCEs (Berger et al., 2000). SFC offers the potential benefit of
high-speed separations that can be complementary to reverse
phase HPLC–MS (Berger, 1997; Coe, Rathe, Lee, 2006). Due
to the lower viscosity of supercritical fluids, SFC can be run at
higher flow rates compared to HPLC. Bolan˜os, Ventura, and
Greig (2003) demonstrated the advantage of SFC–TOF MS
for ultrafast qualitative applications. High-speed MS detection
allows the superior resolving power of SFC to be exploited
in high-throughput analytical settings. Bolanos et al. (2004)
extended their work and summarized the applications of SFC–
MS in drug discovery at Pfizer, La Jolla, CA. They rely heavily
on SFC–MS for analysis, as well as, purification of diverse
compound sets.
SFC for purification applications is especially appealing due
to rapid evaporation of the carbon dioxide mobile phase (Toribio
et al., 2006; Zhang et al., 2006). During separation, the carbon
dioxide mobile phase is under high pressure and behaves like a
fluid. However, once the pressure is reduced after the detector, the
fluid rapidly expands into a gas that quickly evaporates leaving
behind the analyte and any organic modifier such as methanol.
The rapid evaporation of the solvent after purification signifi-cantly
increases the overall throughput of preparative SFC
compared to HPLC.
MASS SPECTROMETRY ANALYSIS

Although there are many theoretical and practical advan-tages
to SFC, most laboratories have not embraced the technique
for characterization nor purification. Much of the resistance is
probably due to the perception that SFC is more complicated than
HPLC. In addition, there are significantly fewer vendor options
for instrumentation and application support. Finally, SFC more
closely resembles the separation of normal phase chromatog-raphy
and many scientists may assume that SFC will not be
applicable to many of the polar and ionic compounds typically
associated with pharmaceutical research and development.
Pinkston et al. (2006) have recently shown that approximately
75% of a 2266 compound subset, representing their corporate
collection, could be adequately assessed by SFC–MS. This
compares to approximate 80% success of the same subset by
HPLC–MS. These results further reinforce the value of SFC as an
important technique for high throughput analysis in support of
modern drug discovery and development.
E. Multiple Hyphenated Detection Techniques
One of the benefits of HPLC–MS is the ability to couple a variety
of detectors to generate a range of qualitative, as well as
quantitative information. It is quite common for one or more
auxiliary detection methods to be combined with HPLC–MS to
provide a better overall assessment of sample purity. A typical
approach has been to couple UV and ELSD. Although the
different detection methods often vary regarding detection
capabilities, selectivity, sensitivity range, and linear response,
collectively they provide complementary information.
Due to variations of quantitative data from different
detection techniques, Letot et al. (2005) investigated HPLC–
CLND and HPLC–NMR for quantification of combinatorial
library compounds. In general, he found that purity values
obtained by NMR were more accurate than those obtained by
CLND. However, the differences in purity are not always of the
same order for the two methods. For low concentration
quantification, NMR is less accurate than typically expected
with higher sample concentrations.
Yurek, Branch, and Kuo (2002) developed a new assay
system for the simultaneous determination of identity, purity and
concentration of sample components from combinatorial libra-ries
produced by parallel synthesis. The system employed HPLC
with photodiode array (PDA), ELSD, CLND, and TOF–MS
detectors. The use of TOF–MS with CLND provides a
synergistic combination, enabling target and side-product
structures to be identified and quantified. This approach allows
characterization, purity analysis and quantification in a single
experiment from a synthesis solution containing microgram
levels of materials. Recently, Peake et al. (2005) described a high-throughput
screening method using ELSD, CLND and accurate
mass HPLC–MS–MS to obtain both quantitative data and
structural information.
Although the complementary nature of the different
detection methods is often a benefit, it can also be a challenge.
In particular, sensitivity and detector response times may vary
significantly among the various detectors. Therefore, it is very
important to assure that all operating parameters are appropri-ately
optimized. This is especially important for rapid separation
methods, which produce chromatographic peaks with 1–2 sec
widths. These systems are very susceptible to operating vari-ations
and must be diligently optimized for peak performance.
Another consideration when using multiple detectors to
assess purity is developing protocols to deal with major purity
inconsistencies between multiple detectors. Of course the
definition of purity depends on the application and can vary for
different experiments. In general, however, purity values of
around 80–85% for both UV and ELSD have been found
acceptable for many early discovery applications. It is important
to note that for hyphenated detection, all purity values have to be
above some predefined threshold. One failed value can indicate
the presence of major impurities that have not been identified by
the other methods. In our experience, we found that ELSD tends
to overestimate the larger components in a mixture at the expense
of the smaller ones. UVon the other hand, even monitoring low
wavelengths, may exhibit more variability depending on
structural differences of the mixture components. This is the
advantage of the complementary detector approach, which
generates higher confidence data (Letot et al., 2005).
F. Open-Access HPLC–MS
Although not a true high-throughput technique in the typical
sense, the open-access or walk-up HPLC–MS system does
provide quick single sample HPLC–MS analysis. This system
has presently become the standard setup in many laboratories.
The open-access system can be used at any time by the medicinal
chemist in need of rapid confirmation of synthetic products or to
monitor the extent of reactions. The quick single sample analysis
can provide information about the completeness of a reaction,
byproducts or unexpected reaction failure. These data add
another layer of information to that obtained from NMR analysis
or other traditional low throughput approaches. Once imple-mented,
open-access systems accept individual samples in an
ongoing queue and can be run without much support from the
analytical department.
The open-access approach was pioneered by Hayward,
Snodgrass, and Thomson (1993) and optimized by Pullen and
coworkers at Pfizer Central Research (Pullen et al., 1995a,b).
Initially, an automated column-bypass thermospray MS system
was used but later this was reconfigured into an ESI–MS system.
The instrument is usually set up to email the results as completely
processed data to the submitting chemists (Mallis et al., 2002).
Because of its success, open-access software modules are
currently offered by most instrument manufacturers to be used
with single-quadrupole and TOF instruments. The use of an
open-access facility can enhance sample throughput and allow
the analytical chemists to spend more time on nonroutine work.
Spreen and Schaffter (1996) have extensively reviewed applica-tions
of open-access MS.
It has been shown that open-access MS works extremely
well for fast characterization of synthesis samples. Most small
molecules that are of interest for pharmaceutical research ionize
well using a standard set of instrument conditions and time
consuming adjustments are typically not needed. Although
the typical setup for a walk-up mass spectrometer includes a
low resolution instrument, high resolution instrumentation has
also been used effectively (Dykes et al., 2003). There have also
been attempts to extend the walk-up approach to purification
FANG ET AL.

systems (Blom et al., 2004). However to date, purification
systems have not been adopted as readily as the characteriza-tion
systems.
V. MIXTURE ANALYSIS
Most high throughput synthesis approaches being used generate
a single discrete compound per reaction vessel. Solid phase
mix-and-split combinatorial synthesis was one of the earliest
synthesis techniques that generate mixtures of closely related
analogs in each reaction vessel. The emphasis for this approach is
to synthesize as many compounds as possible in a small number
of reaction pools and then screen these mixtures to identify hits.
Once a hit is identified in a mixture pool, that mixture pool is
deconvoluted to identify the active component(s). Alternatively,
the active mixture can be resynthesized in a series of smaller
mixture pools and these pools screened for activity. By iteratively
synthesizing and screening ever smaller mixture pools, the active
compound(s) can ultimately be identified. This approach does not
require complete characterization of the individual members
of a mixture pool. The emphasis for analysis here is a quick
assessment of how well the reaction has proceeded in general.
The intent is often to eliminate complete synthesis failures rather
than identifying and characterizing an individual compounds.
Although HPLC–MS has been used in characterizing these types
of complex mixtures, it is quite difficult and labor intensive. For
such a general evaluation of a synthetic procedure, direct analysis
of the mixture using mass spectrometry has been used quite
effectively. By knowing the compounds expected in a synthesis
pool and assuming that each compound is equally represented in
the mixture, theoretical mass distributions can be generated.
These theoretical mass spectra can then be compared to the actual
spectra and conclusions about the success of the synthesis can be
drawn. These types of analyses have been successfully conducted
using both lowand high resolution instrumentation (Carrell et al.,
1995; Dunayevskiy et al., 1995; Nawrocki et al., 1996).
Fourier transform ion cyclotron resonance (FTICR) instru-mentation,
also known simply as Fourier transform mass
spectrometry (FTMS), are high field magnetic instruments that
are usually installed in a laboratory to handle nonroutine
applications. The high resolution capabilities of FTMS and
its more complex operation make it somewhat of a specialty
analytical tool. Although FTMS can be used as an HPLC detec-tor,
as described earlier in this review, the high costs and the more
complex operation associated with it have limited this use. The
primary advantages of FTMS are its high sensitivity (sub fmol),
high mass accuracy (sub ppm levels), high resolution for complex
mixtures (600,000 FWHM) and sequencing capabilities (exact
MS(n) capability).
The high resolution capabilities of FTMS are particularly
appealing for the characterization of mixture analysis since
accurate mass measurements can be used to correctly identify
potential isobaric compounds without the use of chromato-graphic
separation. Furthermore, the ability to conduct sequential
MS–MS and obtain accurate mass assignment for the fragments
helps to accurately identify the molecular structure of specific
compounds in a complex synthesis mixture. Although HPLC–
MS–MS may be able to adequately separate and characterize
individual structures in a complex matrix, direct analysis of
the synthesis mixture using high resolution accurate mass FTMS
in some cases may be easier and faster. On the other hand,
conducting sequential MS–MS for each component of a large
complex mixture is quite tedious, time consuming and not
particularly amenable to high throughput environment. Several
publications have focused on FTMS as a technique to character-ize
(Winger Campana, 1996; Fang et al., 1998; Schmid et al.,
2001) or screen (Poulsen, 2006) combinatorial library mixtures
of discrete small molecules (Bristow Webb, 2003; Herniman
et al., 2004, 2005; Zhang et al., 2005).
VI. PURIFICATION OF NCES
The number of new chemical entities undergoing purification
before any biological assays are undertaken has increased over
the last few years.(Ripka, Barker, Krakover, 2001). This is
mostly due to the recognition that valuable resources are often
wasted when impure compounds are submitted for screening
only to end up generating false positives. This issue has led many
companies to institute specific purity thresholds for compounds
supporting medicinal chemistry projects. An industry standard of
at least 80–85% purity by UVat 210–214 nm has become fairly
typical (Letot et al., 2005).
A. Preparative HPLC–MS
Preparative HPLC using small efficient columns that allow for
rapid separations have become the mainstay of the industry.
Many laboratories use traditional UV detection to collect all
fractions above a certain threshold. The collected fractions are
then rapidly analyzed by FIA–MS to identify the component(s)
of interest. Due to collecting multiple fractions from one sample,
the overall throughput of this approach is severely constrained.
The collection decks can accommodate only a limited number of
fraction collection tubes and once those tubes are used, the
operation stops. In an effort to alleviate this limitation, most of the
current high throughput purification setups use some variation of
HPLC–MS instrumentation as the method of fraction collection.
The specificity of the mass spectrometer is used to trigger
collection only when the expected mass of the product is seen in
the sample. This approach significantly reduces the total number
of fractions collected per sample and allows more samples to be
purified per run.We have pushed the throughput to the maximum
by allowing only one fraction to be collected for each sample.
This type of setup is becoming more common and can purify
approximately 3,000 compounds each month in a completely
automated fashion using a single instrument. Setups for this type
of analysis include simple single channel HPLC–MS systems as
well as MUX systems, as described above.
For purification of crude synthesis products, the most critical
step in the process is the mass directed fraction collection. After
separation by the HPLC column and detection by UV, the eluant
is connected to a tee that splits the chromatographic flow. The
majority of the HPLC eluant is directed to a switching valve
connected to the fraction collector. This valve, which is
controlled by the MS output signal, toggles between the
collection tubes and the waste line. A smaller portion of the
eluant is coupled to the MS system via a split flow interface.

The mass spectrometer monitors the expected m/z trace of
the component of interest. When the expected m/z rises above a
predetermined threshold value, the collector valve is switched
‘‘on’’ to collect the fraction of interest. When the monitored m/z
trace drops below a certain threshold, flow through the switching
valve is directed to waste. In our approach, the waste line is
connected to a second UV detector and the eluant is monitored
prior to discharging it in the waste system. The overall efficiency
of the collection process can be monitored for each sample in
real time by comparing the trace of the UVimmediately after the
HPLC column with the chromatographic trace from the waste
UV (Goetzinger et al., 2004).
The amount of sample being split and infused into the mass
spectrometer must be small. Since mass spectrometry consumes
the sample during detection, only the absolute minimum of the
sample should be sacrificed. Moreover, the sensitivity of MS is
quite high, therefore, the amount necessary for good signal-to-noise
detection is relatively small. In fact, given the high sample
concentration eluting off the preparative HPLC column and
the minimal sample necessary for good MS detection, a 1:10,000
split ratio is fairly typical. To effectively and reproducibly
accommodate this split, small lengths of capillary tubing ranging
from 5 to 25 mm in diameter are typically used. The high split
ratio necessary to limit the amount of material entering the mass
spectrometer requires additional make up flow provided by a
separate pump.
It has been suggested that a major drawback of mass directed
fraction collection is the lack of ruggedness. This apparent
limitation is more of a perception issue because of the more
complex nature of MS and the experience necessary to set up and
use split flow systems. Our experience has been that the system
will be fairly stable and perform well as long as performance is
continually monitored and regular preventive maintenance is
conducted. Our protocols include running daily standards,
checking system performance and recovery every month and
regularly replacing in-line filters to prevent clogging of the flow
split capillary. It has been shown by us and others that using the
setup described above, more than 200 compounds can be purified
in unattended overnight runs (Isbell et al., 2002, Kyranos et al.,
2001b). In addition to implementing diligent preventive main-tenance
procedures, custom monitoring systems can be
employed that trigger an alarm and/or action when a variety of
potential failures are detected. Potential system failures include
high back pressure, injector clogs, collector failures, no anti-cipated
masses to trigger collection are found or data acquisition
errors. By properly programming these system monitoring
devises, responses to the aforementioned failures can be
customized, ranging from system shutdown to selectively
rebooting an unresponsive computer to allow for continued
operation. This type of custom sensor can be highly effective for
unattended overnight runs. It can prevent samples from being
injected into a failing system or will reboot a nonresponsive
acquisition system and get it back into operation without wasting
much time during the purification run (Paulson, 2005).
B. Connecting Characterization and Purification
The focus of the initial characterization analysis for synthesis
samples is on developing high performance methods that can be
used as general universal methods for all samples. Speed with
maximum resolution of all the components in the mixture across
the entire run time are important considerations. Once the pre-purification
analysis is completed, the emphasis shifts to
optimizing the separation conditions exclusively for the peak of
interest. It is irrelevant if several other peaks are overlapping or if
the majority of the peaks are closely eluting either early or later in
the chromatogram. As long as the product peak is well separated
from other components and can be collected with ease,
purification will be successful. Ideally, in a high throughput
environment where compound diversity is the rule, each
synthesis mixture needs to be treated differently. This challenge
contributed to developing automation routines that use the
information obtained from initial characterization data to adjust
the separation gradients used for purification.
For example, if the compound of interest was found co-eluting
with impurities late in the gradient, then the purification
gradient is automatically adjusted to start with a high percentage
of organic solvent followed by a shallow gradient until the
desired product is collected. Using this approach, co-eluting
impurities are adequately separated from the desired peak more
effectively and a cleaner fraction is collected. This process is
illustrated with the example in Figure 2. Figure 2a represents the
pre-purification chromatogram, identifying the peak of interest
(shaded). Based on this separation, the gradient was modified in
order to further separate the product from the side products
during the preparative run (Figure 2b).
In reality, not all characterization data will identify perfect
gradient assignments for all samples. There can be challenges
regarding continued co-elution or target peaks that are broadened
by a shallow gradient. But compared to running a routine
universal gradient of 5–95% acetonitrile for all compounds, this
method of adjusting the gradients based on characterization data
routinely outperforms the rigid traditional approach. Thus it
provides an effective way to optimize purification conditions for
the large majority of compounds without significant operator
effort.
C. Post-Purification Challenges
Other recent advances have been in the combination of analytical
and preparative scale instrumentation that yields an analytical-to-prep
combination. This instrument has the ability to perform an
initial characterization analysis and under preset guidelines,
utilizes the processed data from the first run to automate a
purification analysis. In some cases this has been extended to
subsequent characterization of the collected fraction(s).
Since many high-throughput environments now need to
handle large amounts of liquids from collected fractions, some
efforts have been made to simplify these tasks. One such effort is
the design of a fraction pooling station as described by Koppitz,
Brailsford, andWenz (2005). In their approach, product fractions
are automatically identified from their corresponding mass
spectra, even accounting for sodium adducts or dimers. The
key elements of this approach are automated identification of the
target compounds. This information is then used to optimize
preparative gradients for purification of the target compounds
followed by automated purity assessment of fractions with
FANG ET AL.

FIGURE 2. a: Pre-purification QC analysis using a gradient of 5–95% acetonitrile. The product peak
(4) partially co-elutes with an impurity at 1.43 min. b: Purification run with conditions adjusted to a gradient
of 30–95% acetonitrile based on pre-purification results. The product peak is completely resolved from the
co-eluting impurity that was detected in pre-purification QC run.
subsequent pooling of validated product fractions. Purity
assessment of product fractions allows cherry-picking of pure
fraction tubes on the basis of an algorithm that calculates MS
purities and considers fragmentation. Using this type of system,
one person can easily process 100–200 compounds per day.
D. Microscale Separations and Collection
Besides the potential for sample path blockages, the sample loss
associated with the use of small diameter capillaries for flow
splitting is not a major factor for mass directed fraction collection
with semi-preparative HPLC flows. Flow splitting can indeed
become a problem once the scale and the flow rate become
significantly smaller than traditional semi-preparative or ana-lytical
applications. This has been reported byWachs and Henion
(2001) and is part of the driver for the development of an
integrated nano scale fraction collection system that is being
marketed commercially as an ultra-low-volume fraction collec-tor
and analyzer. This type of system monitors mass traces using
an automated chip-based nanospray and collects fractions that
can subsequently be reanalyzed automatically.
VII. CONCLUSION AND FUTURE DIRECTIONS
In the early 1990s, there was an explosive surge of new
technology development primarily focused on characterization
of NCE’s in support of early pharmaceutical discovery. During
this period, mass spectrometry emerged as the central analytical
technique in the development of modern high throughput

analysis. The MS technologies covered in this review have made
a leap from primarily research purposes to becoming an integral
part of modern medicinal chemistry. However, over the last
5 years there has been greater emphasis on fine tuning the existing
characterization techniques and making them more robust and
reliable rather than pioneering new technologies. The emphasis
on future developments in MS characterization is anticipated to
be continuing to streamline and further increase throughput.
There is still opportunity for significant improvements. Data
handling and analysis can be automated further. Instrument and
method troubleshooting can be automated. Development of
additional methods that can accommodate more complex
samples and new MALDI matrices that are more amenable to
low molecular weight compounds are also needed.
In contrast toMScharacterization, there has been significant
development of high throughput purification using mass directed
fractionation. Areas of future development are anticipated in
automatic gradient or column assignments specific to the need
of each sample, as well as improved automated fraction
handling.
Successful chip-based MS analytical techniques for both,
characterization and purification have been reported. This area of
research is promising to be the most significant development in
NCE characterization and purification. The small volumes that
are typically used with chip based techniques have the potential
for rapid and ultra-high throughput while maintaining opera-tional
and consumable costs low. However, the rate of future
development of these techniques and how fast they may be
adopted in mainstream applications is yet to be determined.
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Annette S. Fang, Ph.D. Dr. Fang received her undergraduate education in Germany and her
Ph.D. degree in Analytical Chemistry from Northeastern University. She was a Staff
Investigator in the Analytical Department at ArQule, primarily focused on purification of
new chemical entities synthesized for early drug discovery. Her prior work experiences
include positions at Research Biochemicals, Inc., Natick, MA and Bruker Daltonics,
Billerica, MA.

Xiusheng Miao, Ph.D., Senior Investigator, Preclinical Development and Clinical
Pharmacology, ArQule, Inc., Woburn, MA. Dr. Miao is currently responsible for in vitro
and in vivo drug metabolite identification and bioanalytical method development. He
obtained his Ph.D. from the Chinese Academy of Sciences, China and has co-authored over
40 journal articles and book sections.
Peter W. Tidswell, Ph.D., Associate Research Fellow, Pfizer, Inc. Dr. Tidswell leads the
purification laboratory at the St. Louis research site, which supports the medicinal chemistry
department and therapeutic programs. Prior to joining Pfizer, he was a Group Leader in the
Chemical Technologies’ analytical department for ArQule, where he led the Character-ization
group. His other experience encompasses positions at the Bristol-Myers Squibb
Pharmaceutical Research Institute, EvotecOAI and ChiroScience. Dr. Tidswell received his
Ph.D. under the direction of Dr. Mel Kilner in Organometallic Chemistry from the
University of Durham, UK.
Marc H. Towle, Scientist, Discovery Analytical,Vertex Pharmaceuticals, Inc., Cambridge,
MA. Mr. Towle provides analytical development support to assess the purity, potency and
stability of drug substances and drug products for late research, pre-clinical, and phase I
development. Prior to joining Vertex, he was a Senior Investigator at ArQule, where he
managed the analytical purification group responsible for supporting high throughput
production of chemical libraries for early drug discovery. His prior experience includes
research positions with BioDevelopment Laboratories, Arthur D. Little and Groundwater
Technology. Mr. Towle received his BS in Biochemistry from the University of New
Hampshire.
Wolfgang K. Goetzinger, Ph.D., Director Research, Molecular Structure, Amgen, Inc.,
Cambridge, MA. Dr. Goetzinger is responsible for the Discovery Analytical group at
Amgen, Inc. where he leads a team supporting all analytical aspects of small molecule
pharmaceutical discovery. Prior to joining Amgen, he was Director of Analytical Chemistry
at ArQule, Inc., where he managed a group focused on high throughput analytical
approaches to support parallel synthesis efforts. Dr. Goetzinger worked as a Post Doctoral
Fellowunder Professor Barry L. Karger at Northeastern University, where he focused on life
science applications of capillary electrophoresis. He received his Ph.D. from the University
Saarland under Professor Heinz Engelhardt working on the synthesis and characterization
of chiral stationary phases for the separation of enantiomers.
James N. Kyranos, Ph.D., Vice President, Preclinical Development, Wolfe Laboratories,
Watertown, MA. Dr. Kyranos leads the technical and business operations of WLI’s
preclinical development services focused on bioanalytical and pharmaceutical sciences
support. Prior to joining WLI, he was Vice President of Chemical Technologies for ArQule,
where he managed the chemistry services business focused on providing early discovery
services to the pharmaceutical industry. His other experience includes leadership positions
at BioDevelopment Laboratories and Arthur D. Little. Dr.Kyranos received his Ph.D. under
the direction of Professor Paul Vouros in Analytical Chemistry from Northeastern
University.
FANG ET AL.

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