7
LC/MS: THEORY,
INSTRUMENTATION, AND
APPLICATIONS TO SMALL
MOLECULES
Guodong Chen, Li-Kang Zhang, and Birendra N. Pramanik
7.1 INTRODUCTION
The discovery of a new drug is a challenging task that includes (a) identifica-
tion of a biochemical target for certain diseases and (b) screening of a large
number of compounds from libraries of compounds arising from synthetic
chemistry, combinatorial chemistry, and natural product isolation for lead gen-
eration. The lead compound is then optimized based on biological activity,
selectivity, pharmacokinetic property, and metabolism. This process produces
a large volume of samples requiring rapid and accurate analysis, with the speed
of analysis contributing directly to the drug discovery cycle time.
As one of primer analytical techniques, mass spectrometry (MS) developed
from nineteenth-century physics, starting with the pioneering work of J. J.
Thomson on the electrical discharges in evacuated tubes. In 1913, Thomson
wrote “I feel sure that there are many problems in Chemistry which could be
tackled with far greater ease by this than any other method. The method is
surprisingly sensitive—more so than that of Spectrum Analysis—requires
infinitesimal amount of material, and does not require this to be specially puri-
fied. . . .” Indeed, MS offers speed, high sensitivity and isotopic specificity. This
technique separates mixtures of ions on the basis of mass-to-charge ratios,
281
HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto
Copyright © 2007 by John Wiley & Sons, Inc.
providing the molecular weight of a compound and its structural information
from fragment ions. It is widely used for identification and quantification of
known/unknown organic compounds. Rapid development of MS in recent
decades has further expanded its role in the structural characterization of

matographic separation techniques, especially with HPLC. Various ionization
methods have been developed over the years, including electron impact (EI),
chemical ionization (CI), desorption ionization (DI), matrix-assisted laser des-
orption/ionization (MALDI), desorption electrospray ionization (DESI), elec-
trospray ionization (ESI), and atmospheric pressure chemical ionization
(APCI). Note that ESI and APCI are part of LC/MS interfaces that will be
282 APPLICATIONS TO SMALL MOLECULES
discussed in separate sections. Table 7-1 summarizes some characteristics of
different ionization methods.
EI and CI are two early developed ionization methods. They are extremely
useful for ionizing volatile compounds. In EI process, molecules are ionized
by collisions with energetic electrons (typically 70 eV) produced from a heated
filament. It produces highly reproducibly mass spectra with extensive frag-
mentation of molecular ions. Thus, library searching with existing EI mass
spectra is possible for unknown identifications. The nature of fragmentation
in EI often leads to lower abundances or absence of molecular ions. On the
other hand, CI is a soft ionization method that generates mainly molecular
ions by ion/molecule reactions of regent ions with analyte molecules [4].
IONIZATION METHODS AND LC/MS INTERFACES 283
TABLE 7-1. Summary of Ionization Methods
Ionization
Method Ionization Agent Strengths Limitations
EI Electrons (∼70 eV) Extensive Limited to
fragmentation, volatile/
reproducible nonpolar
spectra, searchable molecules
large reference
compound EI
libraries
CI Gaseous ions Abundant molecular Limited to

proton affinities of reagent gas and analyte is largely responsible for the extent
of fragmentation if ionization of the analyte occurs.
EI and CI methods are complementary to each other, providing molecular
weight and structural information. As an illustration, Figure 7-1 shows an EI-
MS spectrum of mometasone furoate, an anti-inflammatory steroid drug. A
very low abundant molecular ion at m/z 520 is visible. However, the base peak
in the spectrum is the fragment ion at m/z 295 corresponding to the loss of
furoate ring, HCl, and a moiety of [COCH
2
Cl]. Other fragment ions in the
spectrum yield structurally characteristic fragmentations for this molecule. In
contrast, a CI-MS spectrum of the same compound exhibits the protonated
molecular ion as the most abundant peak at m/z 521 along with some frag-
ment ions (Figure 7-2). The appearance of these two spectra clearly demon-
strates the utility of EI-MS and CI-MS methods.
An inherent limitation for EI and CI methods is the requirement that the
sample analyzed must be volatile. Both methods do not produce MS data for
284 APPLICATIONS TO SMALL MOLECULES
Figure 7-1. EI-MS spectrum of mometasone furoate
.
polar compounds. One solution to this limitation is to employ DI methods to
ionize nonvolatile samples with high molecular weights [5]. In the DI process,
energetic particles or photons impact onto samples on a surface and result in
the liberation of intact molecular ions via selvedge region without direct trans-
fer of the energy to the sample molecules. The particle bombardment includes
keV atoms (e.g., Ar, fast atom bombardment [6]), keV ions (e.g., Cs
+
, liquid
secondary ion MS [7]), and MeV ions (e.g., plasma desorption [8]). Both fast
atom bombardment (FAB) and liquid secondary ion MS (LSI) utilize large

additional peak broadening), high transfer efficiency from LC to MS, and no
degradation in mass spectrometric performance. Historically, a main challenge
in LC/MS interfaces was that high liquid flows from HPLC make it very dif-
ficult to maintain the high vacuum required for the function of a mass spec-
trometer. A number of different LC/MS interfaces have been developed over
the years to address this issue and overcome the difficulty [3].
7.2.2.1 Direct Liquid Introduction. One of the first attempted experiments
to introduce liquids into a mass spectrometer was to minimize the amount of
liquid into an MS, removing solvent by the vacuum system and ionizing the
analyte in the gas phase. The pioneering work carried out by Tal’roze et al.
[11] described the simplest direct liquid introduction interface. In their
experiments, solvent was introduced into the mass spectrometer through a
capillary at a flow rate below 1 µL/min. The ionization of analytes occurred by
EI. The low flow rate used in the experiments was a limitation and would not
give good sensitivities for analytes. In 1970s, McLafferty’s group employed
a direct liquid introduction (DLI) interface to directly introduce a small
fraction (<1%) of the liquid from HPLC into the ion chamber of a CI mass
spectrometer [12]. The solvent acted as the ionizing reagent. The maintenance
of the vacuum was assisted by using large pump systems and differential
pumping. Micro-and nanobore chromatography (<1-mm-i.d. column) were
suitable for DLI. A detection sensitivity of picogram level was achieved for
full-scan analysis.
7.2.2.2 Moving Belt System. Initially developed by McFadden et al. [13],
the moving belt system was based on the physical method of evaporation of
the mobile phase through heat and vacuum that leave analytes as a thin
coating on a continuously cycling polyimide belt. The analytes were trans-
ported from atmospheric pressure region to the vacuum of the ion source
through differentially pumped vacuum locks. Ionization methods used
286 APPLICATIONS TO SMALL MOLECULES
included EI and CI for volatile analytes. The system has excellent enrichments

added FAB matrix (usually 5% aqueous glycerol) is continuously transported
through a fused-silica capillary to the tip of a FAB probe residing inside of the
ion source. The HPLC liquid with matrix deposited on the tip of the FAB
probe is subjected to atom bombardment for ionization of analytes.The matrix
addition can be done either pre-column or post-column, although post-column
addition is preferred. The acceptable liquid flow rate in continuous-flow FAB
is less than 10 µL/min. Flow splitting or the use of capillary chromatography
is often required in the experiments. A major advantage in this method is the
reduced chemical noise since much less matrix is used in continuous-flow FAB
than in standard FAB experiments. This has led to improved detection limits
to subpicomole range. Significantly, this interface allows the LC/MS analysis
of biomolecules that are traditionally analyzed by DI methods.
IONIZATION METHODS AND LC/MS INTERFACES 287
7.2.3 Common Interfaces
The early developed LC/MS interfaces as described above have played impor-
tant roles in the evolution of LC/MS interfaces. However, their applicability,
sensitivity, and robustness are very limited. The overwhelming popularity of
LC/MS today is largely due to the development of atmospheric pressure ion-
ization (API) interfaces, including ESI and APCI.
7.2.3.1 Electrospray. The first description of ESI was made by Zeleny in
1917 [17]. He described how a high electrical potential applied to a capillary
caused the solvent to break into small droplets. In late 1960s and early 1970s,
Dole and co-workers attempted to generate gas phase ions from macromole-
cules in solution using an atmospheric pressure electrostatic sprayer by ion
mobility spectrometry [18, 19]. In the late 1970s, Thomson and Iribarne suc-
cessfully demonstrated the production of macro-ions from electrically charged
droplets using MS [20, 21]. The very first applications of ESI were reported
independently by Yamashita and Fenn [22] and Aleksandrov et al. [23] in
the mid-1980s. Now ESI has become one of the most successful ionization
methods / interfaces used in mass spectrometry [24].

100 kDa [27]. Another important characteristic of ESI is the softness of the
ionization. It is a very mild process and can generate mainly molecular ions
with little fragmentation. For small molecules, the singly charged molecular
ions usually dominate the mass spectrum. The third characteristic of ESI is the
simplicity of the source design and its operation at atmospheric pressure,
allowing ESI to be readily coupled to HPLC. It is important to note that a low
flow rate (~200 µL/min) of the sample solution is required in order to main-
tain a stable spray in ESI. Thus, flow splitters are often utilized in ESI-LC/MS
applications. This does not reduce the concentration sensitivity of ESI since
ESI responses are directly related to the concentration of the analyte enter-
ing the ion source. However, the mass sensitivity can be substantially increased
with a lower flow rate if the same concentration sensitivity is maintained
(c = m/v). This has led to the wide use of nano-spray (~nL/min) LC/MS for
analysis of proteins and peptides, achieving femtomole sensitivity [24].
7.2.3.2 Atmospheric Pressure Chemical Ionization. APCI is closely related
to ESI. It was developed by Horning et al. [28] in the early 1970s. Figure 7-4
illustrates a typical APCI source. The sample solution is introduced into a
nozzle spray device similar to that used in ESI, but without the high electrical
potential applied to the nozzle. The nebulizing gas (usually N
2
) is often added
to assist the desolvation/ionization process. Although a heater at a tempera-
ture of 400–500°C is used to vaporize solvents, minimal degradation of the
sample occurs.A corona discharge needle at a high voltage (3–5 kV) is respon-
sible for producing a discharge current and inducing solvent ionizations. The
generated solvent reagent ions react with analyte molecules via gas-phase
ion/molecule reactions and produce analyte ions. Clearly, the ion formation
process is separated from solvent evaporation process in APCI (in contrast to
ESI), allowing the use of solvents unfavorable for ion formation. For example,
IONIZATION METHODS AND LC/MS INTERFACES 289

tion of ions involves the absorption of a photon by the molecule and ejection
of an electron from the molecule to form the radical cation.The necessary con-
dition for the ionization to occur is that the photon energy has to exceed the
ionization potential of the molecule of interest. Like ESI and APCI, APPI uses
nebulizer and vaporizer for desolvation. The ionization occurs at atmospheric
pressure with UV light source [30].The standard UV lamp has a photon energy
of about 10 eV that is sufficiently high to ionize most organic molecules.
Common HPLC solvents and permanent gases usually have higher ionization
potentials that will not be ionized. This results in relatively noise-free mass
spectra, as opposed to ESI or APCI. In some cases, analyte molecules may
exhibit higher ionization potentials (>10 eV) and the direct photoionization
may not produce ions. Then, addition of a large excess of a dopant such as
toluene and acetone is necessary to yield charge carriers for ionizing analytes
of interest. The undesired consequence of this dopant-assisted APPI is the
increase of background ions.
The potential application of APPI includes analysis of compounds (non-
polar and neutral analytes) that are not effectively ionized by ESI or APCI.
APPI appears to be less influenced by matrix suppression as seen in ESI or
APCI. It can serve as a complementary ionization source to ESI/APCI.
7.3 MASS ANALYZERS
The basic function of a mass analyzer is to measure the mass-to-charge ratios
of ions (charged particles) and provide a means of separating the ions. The
operating principles of mass analyzers depend on interactions of charged par-
ticles with electrical or magnetic fields. Commonly used mass analyzers include
magnetic sector, quadrupole, ion trap, time-of-flight (TOF), and Fourier trans-
form ion cyclotron resonance (FT-ICR). The combination of different mass
analyzers can provide additional capabilities of performing mass spectrome-
try/mass spectrometry (MS/MS) or tandem MS experiments for structural
characterization. Table 7-2 lists some characteristics of various mass analyzers.
7.3.1 Magnetic Sector