Tài liệu HPLC for Pharmaceutical Scientists 2007 (Part 14) docx

14
ROLE OF HPLC IN PROCESS
DEVELOPMENT
Richard
Thompson and Rosario LoBrutto
14.1 RESPONSIBILITIES OF THE ANALYTICAL CHEMIST
DURING PROCESS DEVELOPMENT
In the drug discovery area, a compound with desired therapeutic properties
is identified, and its structure may be modified by synthetic alterations to
enhance potency and specificity or to decrease toxicity and undesired side
effects. The lead drug candidate is then transitioned into the drug develop-
ment area. Only small amounts of drug (typically less than a gram) are
required to support the required studies in the Drug Discovery area. However
larger amounts are required to support the studies conducted in the Drug
Development area. The amount required in the preclinical stage typically
ranges from 20 to 2000g. This material is required to support studies includ-
ing subchronic toxicity, genotoxicity, ancillary pharmacology, early animal
pharmacokinetics (PK), salt/form selection, and formulation development.
As the drug candidate progresses through the various clinical stages, the
drug requirements typically range from 1kg to 200kg. This material supports
the various clinical studies as well as chronic toxicity, carcinogenicity, develop-
ment and reproductive toxicity, and formulation development. Finally, tons
of drug may be required upon successful approval and commercialization
(Figure 14-1).
The synthetic pathway to the drug substance is likely to evolve during the
various stages of development. It is highly unlikely that the synthetic process
641
HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto
Copyright © 2007 by John Wiley & Sons, Inc.
utilized in drug discovery will be the same as that used to provide commercial
scale quantities

.The discovery chemist may utilize a large number of synthetic
steps, use a number of reagents that are expensive or not practical at scale-up,
use a number of chromatographic steps for purification, and experience very
low yields.For scale up, the process development chemist must factor in safety,
economical, and ecological considerations while producing a robust and repro-
ducible synthesis. He must consider operating limitations such as heat and
mass transfer. Economic factors will dictate minimization of the synthe-
tic steps, maximization of yield, and choice of raw materials. In addition,
the process must meet environmental, occupational health, and safety
requirements.
Furthermore, the process development chemist must follow guidelines from
the Food and Drug Administration (FDA) in relation to the control and iden-
tification of impurities in drugs that will be used in humans. Regulatory bodies
require that the maximum possible human exposure to an impurity in a drug
substance be supported by toxicological studies in animals that indicate no sig-
nificant adverse effects. Consequently, impurities that exceed a 0.1% tolerance
limit in clinical material must first be qualified in animal toxicological studies.
Scale up of a synthesis,however,may generate a different impurity profile than
observed for the smaller quantities prepared to support the toxicological
studies. Kinetic factors, changes in raw materials, or changes in reaction con-
ditions may result in the introduction of new or elevated impurities.These new
impurities may be qualified in additional chronic toxicity and genotoxicity
studies, but this strategy is often not economically feasible and is undertaken
more as a last resort. A better strategy is to identify and then control impuri-
ties that are generated during the continuously evolving stages of process
development.
642 ROLE OF HPLC IN PROCESS DEVELOPMENT
Figure 14-1. Stages of process development in the context of drug development.
As a consequence of process evolution and regulatory requirements, the
analytical chemist supporting process development is faced with a number of

challenges
. He must evaluate the purity and stability of raw materials, inter-
mediates, and drug substance. He must evaluate yield and impurity generation
across the various synthetic steps. Impurities in drug substance, intermediates,
and raw materials may require identification.Analytical methods may have to
be adapted to accommodate process changes. Finally he must set specifica-
tions, validate analytical methods, provide regulatory documentation, and
perform a technology transfer prior to drug approval and commercialization.
To this end, HPLC is a critical tool to perform many of the above tasks. Most
pharmaceutical compounds are amenable to analysis by HPLC. HPLC is a
powerful technology that is capable of separating complex mixtures into indi-
vidual components that can then be quantified. A well-developed HPLC
method resolves and quantifies impurities from an analyte of interest in a
reproducible, rugged, precise, and accurate fashion.
14.2 HPLC SEPARATION MODES
The multitude of available separation modes, mobile phases, and columns
provide a plethora of parameters that can be manipulated to meet the crite-
ria for a well developed HPLC method. Conversely, it also creates a dilemma
in choosing the optimal parameters from the myriad of possibilities.The com-
monly utilized modes of HPLC in pharmaceutical development are reversed
phase (RP) and normal phase (NP) for small organic molecules (<1000Da).
Sub-/supercritical chromatography (SFC) is utilized as an alternative to
normal phase chromatography. Ion chromatography (IC) is utilized for ionic
species. Other modes utilized include hydrophilic interaction and chiral sepa-
rations. In-depth discussion of the theory and method development of RPLC,
ion exchange, and NPLC are highlighted in the first section of the book. How
each of these chromatographic modes could be applied for the analysis of com-
pounds that the analytical chemist encounters within the framework of process
development will be discussed in more detail.
14.2.1 Reversed-Phase Liquid Chromatography

Reversed-phase chromatography is the preferred HPLC mode in the phar-
maceutical industry. Its popularity is in part derived from its mobile-phase
compatibility with the typical polar drug substance, the higher efficiencies
associated with this mode, shorter re-equilibration times, and the ability to run
gradient methods covering a large range in polarity. This allows for its use in
reaction monitoring, qualifying synthetic intermediates, and for stability and
release testing of the drug substance. Several case studies of reversed-phase
method development of drug substances and drug substance intermediates are
given in Chapter 8 sections 8.2 and 8.5.
HPLC SEPARATION MODES 643
14.2.2 Normal-Phase Chromatography
Normal-phase or adsorption HPLC utilizes a polar stationary phase and a
less polar mobile phase
. Retention occurs through polar interactions, such as
hydrogen bonding and dipole interactions, between the solute and the sta-
tionary phase. Retention is more predictable than for RP chromatography.
Carboxylic acids tend to show the strongest retention followed by amines,
ketones, ethers, aromatic hydrocarbons, and saturated hydrocarbons in
decreasing order of retention. Good selectivity is often observed for positional
and stereoisomers.The mobile phase is usually a mixture of a nonpolar solvent
such as heptane or hexane(s) and a more polar solvent. The polar organic
solvent can be chosen based on it physicochemical properties (hydrogen
bonding capabilities,lipophilicity, and polarizability).There are a large number
of options for eluent components, and extensive selectivity changes are
observed with the use of various mobile-phase components. In addition, small
changes of the polar organic solvent can cause large changes in retention,
and this should be investigated during method development. Common
solvents include ethanol, isopropanol, tetrahydrofuran, ethyl acetate, and
dichloromethane. The level of water in the solvents needs to be controlled as
well, since differences in retention may be observed. Note that heptane and

methanol have limited miscibility and only a maximum of 5% methanol should
be used. Mobile-phase miscibility should be checked prior to pumping a par-
ticular composition on the HPLC. A simple mixture of the solvents in the
beaker should allow the chromatographer to discern if the two components
are miscible. Additives such as triethylamine and trifluoroacetic acid are rec-
ommended to reduce retention and improve peak shape for the analysis of
bases and acids, respectively, by reducing interactions of the solute with the
highly active sites of silica. Commonly utilized stationary phases include cyano,
diol, amino, and silica. However, unmodified silica possesses greater surface
heterogeneity and is more retentive than the other three phases. Very little
selectivity differences are observed as a function of the type of stationary
phase.
When using NP the chromatographer must remember to convert their
system to NP mode if RP mode was used previously. Any aqueous/buffer left
in the system could precipitate out when the normal-phase solvents are
pumped than the system. Water contamination in the mobile phase lines can
also lead to water absorption on the column and change the chromatography
significantly. It is generally recommended, that if the system was previously in
RP mode, to flush the system with pure water for about 15 minutes at 2
mL/min. Then use IPA to flush the system for an additional 10 minutes at 2
mL/min.The system should then be flushed with the desired NP mobile phase
for 5 minute at 2mL/min. Then the NP column can be installed and equili-
brated with the NP mobile phase.
Despite the popularity of RP chromatography, NP has its usefulness in the
analysis of compounds during drug development. It can be used for polar
644 ROLE OF HPLC IN PROCESS DEVELOPMENT
solutes that are poorly retained in RP, nonpolar solutes that are strongly
retained in RP
, positional and stereoisomers, or solutes that are labile or
possess poor solubility in RP mobile phases.An example of an RP-incompat-

ible method involves reaction monitoring of a mesylation step:
RCOOH + MeSO
2
Cl → RCOOSO
2
Me + RCOOOCR + HCl
The anhydride was formed as a side product in this reaction impacting yield.
However, in an RP mobile phase, both the mesylate and anhydride would
revert back to the carboxylic acid. Derivatization would produce the same
product for both the mesylate and the anhydride. The reaction components
were separated and quantified under NP conditions using a diol column with
a 0.1v/v% TFA in heptane/THF mobile phase (Figure 14-2). This method
was used monitor the reaction such that the level of the carboxylic acid inter-
mediate was less than 0.5% in the reaction mixture.
14.2.3 Sub-/Supercritical Chromatography
Sub-/supercritical fluid chromatography is essentially NP chromatography
with the added advantage that the lower viscosity and higher diffusivity of the
mobile phase results in higher column efficiencies allowing for rapid resolu-
tions. The columns employed are the same as those utilized in conventional
NP chromatography. Carbon dioxide is the most commonly used nonpolar
eluent but requires a more polar modifier such as an alcohol for the elution
of polar solutes. The modifier increases the polarity of the mobile phase and
HPLC SEPARATION MODES 645
Figure 14-2. Normal-phase separation of a mesylate from corresponding acid.
Chro-
matographic conditions:YMC Pack Diol 150 × 4.6mm, 90% 0.1% TFA in heptane/10%
0.1% TFA in THF.
occupies active sites on the stationary phase, leading to reduced retention of
solutes
. As with conventional NP chromatography, the use of triethylamine

and trifluoroacetic acid as additives is recommended for the analysis of amines
and acids, respectively. The polar nature of most drug substance requires the
use of high levels of organic modifier, and thus the mobile phase is most often
in the subcritical state. Retention characteristics are the same as in conven-
tional NP chromatography.
Subcritical fluid chromatography was applied for the resolution of a
bromosulfone drug intermediate from various process-related compounds
(Figure 14-3). Initial steps toward method development were performed in RP
mode. However, significant fronting of the bromosulfone peak was observed,
indicating on-column degradation that was later determined to occur through
(a) nucleophilic substitution of the bromo group with a hydroxyl group to form
646 ROLE OF HPLC IN PROCESS DEVELOPMENT
Figure 14-3. Structures of bromosulfone and process-related impurities
. (Reprinted
from reference 1, with permission.)
an alcohol and (b) addition to the ketone group to form a gem diol. Separa-
tion of bromosulfone from seven process-related compounds was achieved
under subcritical conditions using a silica column and a mobile phase of carbon
dioxide and 50% modifier (80/20 methylene chloride/acetonitrile) within three
minutes (F
igure 14-4) [1].
14.2.4 Hydrophilic Interaction Chromatography
Another option to conventional NP chromatography is hydrophilic interac-
tion chromatography (HILIC). This mode utilizes a polar stationary phase
with aqueous/organic modifier but with very high percentages of organic mod-
ifier. A simple acetonitrile/aqueous buffer mobile phase is commonly utilized
in conjunction with a silica or amino stationary phase. Ammonium acetate is
often used as a buffer salt in the mobile phase because it possesses good sol-
ubility at high organic content. At lower pHs, phosphoric acid can be utilized.
An adsorbed water layer on the silica substrate is formed under these chro-

matographic conditions. Polar solutes partition from the highly organic bulk
mobile phase into the adsorbed water layer where they can undergo polar
interactions. In addition, positively charged solutes, such as amines, can
undergo ionic interactions with charged silanol groups. As a consequence,
retention of solutes increases with their increasing polarity. This mode is par-
ticularly useful for the separation of very polar solutes drug substance inter-
mediates and/or raw materials that show minimal or no retention under RP
conditions and are very strongly retained under NP conditions. Figures 14-5
and 14-6 depict the separation of nine very polar pyridine derivatives [2]. In
process research environment, for example, one of these pyridine derivatives
HPLC SEPARATION MODES 647
Figure 14-4. Separation of bromosulfone from process-related impurities by
SFC. Chromatographic conditions: 50% Carbon dioxide/50% (80/20 methylene
chloride/acetonitrile), Zorbax silica 250 × 4.6 mm. (Reprinted from reference 1, with
permission.)
could be a key raw material in a synthetic process.The possible isomeric forms
of the key raw material should be well-resolved from the key raw material and
needs to be controlled (sometimes a certain set of acceptance criteria are set
for both the overall purity of the key raw material and maximum amount
of undesired impurity) to avoid undesired reactions in the downstream
processing
.
648 ROLE OF HPLC IN PROCESS DEVELOPMENT
Figure 14-5. Structures of pyridine-related compounds.
Figure 14-6. Separation of pyridine-related compounds by HILIC. Chromatographic
conditions:Atlantis HILIC silica 3µm, 150 × 4.6mm. Mobile phase A: 0.1% phosphoric
acid in D.I water. Mobile phase B:Acetonitrile. Gradient at 95% B to 60% B in 7 min
and then hold 8min.
14.2.5 Ion-Exchange Chromatography
Ion-exchange chromatography is useful for the separation of ionic or ioniz-

able solutes and resolves solutes based on the strength of their ionic interac-
tions with ionic functional groups on the stationary phase
. The mobile phase
is aqueous.The solute and the functional group on the stationary phase possess
opposite charges, and the mobile phase contains a counterion with the same
type of charge as the solute and thus effectively competes with the solute ion
for ion pair interactions with the stationary phase. The retention of the solute
is dependent upon the ionic size, charge magnitude, and polarizability of the
solute and mobile-phase counterion as well as the ionic strength of the mobile-
phase counterion. Gradients of counterion concentration can be employed.
Retention is also dependent upon the mobile-phase pH and the dissociation
constants of protolytic solute and mobile-phase species.
The stationary phase can be categorized as strong or weak ion exchangers.
The capacity of strong ion exchangers is independent of pH, while the capac-
ity of weak ion exchangers varies as a function of their protonated state.Strong
ion exchangers include sulfonate functionalities for the analysis of cationic
species and quaternary ammonium functionalities for the analysis of anio-
nic species. Weak ion exchangers include carboxylate functionalities for the
analysis of cationic species and amines for the analysis of anionic species.
The functionalities are commonly attached to a polymeric matrix such as
poly(styrene-divinylbenzene), polyacrylate, or polymethylacrylate.
Ion chromatography can be applied for the quantitation of inorganic impu-
rities, drug substance counterions, and ionic synthetic impurities and degrada-
tion products. The most common forms of detection are by conductivity
detection and indirect photometric detection (IPD), which allows for the use
of conventional UV detectors. With IPD the mobile-phase anion possesses
a significant chromophore. When a solute molecule, with a weaker chro-
mophore, is eluted and passes through the detector cell, it is manifested as a
negative peak. This form of detection can be used for analysis of ionic impu-
rities in API [3–5]. Alendronate is a highly ionic bisphosphonate species that

also possesses a primary amine functionality that can be derivatized with
9-fluorenylmethyl chloroformate (FMOC) and analyzed by conventional
RPLC. However, alendronate does not possess a significant chromophore, and
process-related impurities may also have low chromophores and may also not
have an amine functionality that can be derivatized by FMOC. Such impuri-
ties would not be detected in the conventional RPLC method. To address
this issue, an ion exchange method was developed to separate alendronate
from similar bisphosphonates, synthetic impurities, and inorganic impurities
(Figure 14-7) [4].
The addition of a compatible organic solvent may also influence selectivity,
particularly when the stationary phase has a polymeric substrate. With these
types of phases, the solute can undergo both hydrophobic and ion-exchange
interactions. The addition of an organic solvent will result in increased
HPLC SEPARATION MODES 649
retention for solutes such as inorganic ions that only undergo ion interactions.
Ions such as acetate and alendronate
, which can undergo both types of inter-
actions, may be more or less strongly retained depending on the ratio of
hydrophobic to ion exchange interactions [4, 5].
14.2.6 Chiral Chromatography
Chiral separations can be considered as a special subset of HPLC. The FDA
suggests that for drugs developed as a single enantiomer, the stereoisomeric
composition should be evaluated in terms of identity and purity [6].The unde-
sired enantiomer should be treated as a structurally related impurity, and its
level should be assessed by an enantioselective means. The interpretation is
that methods should be in place that resolve the drug substance from its enan-
tiomer and should have the ability to quantitate the enantiomer at the 0.1%
level. Chiral separations can be performed in reversed phase, normal phase,
and polar organic phase modes. Chiral stationary phases (CSP) range from
small bonded synthetic selectors to large biopolymers.The classes of CSP that

are most commonly utilized in the pharmaceutical industry include Pirkle
type, crown ether, protein, polysaccharide, and antibiotic phases [7].
Pirkle-type phases are amino acid derivatives possessing an aromatic
entity which can undergo π–π interactions with the solute.The aromatic entity
can be either a π donor or π acceptor. The CSP and the solute form a π
donor/acceptor pair.This complex is then stabilized by additional interactions
such as hydrogen bonding, dipole interactions, or steric repulsion [8]. The
Pirkle-type phases are most commonly used in normal-phase mode in order
to enhance the π–π and hydrogen bond interactions. Hexane with an alcoholic
modifier, such as isopropanol,is the mobile phase of choice.These phases have
650 ROLE OF HPLC IN PROCESS DEVELOPMENT
Figure 14-7. Separation of organophosphonates and process-related impurities. Chro-
matographic conditions: Hamilton PRP-X100, 250 × 4.6 mm, 1 mM trimesic acid
(pH 5.5). 1, Phosphonopyrrolidine; 2, alendronate; 3, phosphite; 4, chloride; 5, methane-
sulfonate; 6, alendronate dimer; 7, etidronate; 8, clodronate. (Reprinted from reference
4, with permission.)
also been utilized in the reversed-phase mode but with poorer enantioselec-
tivity and in some cases different elution orders indicating a change in the
chiral recognition mechanism.
These phases can also be utilized in super-/
subcritical mode.
Crown ethers are heteroatomic macrocycles possessing a hydrophobic exte-
rior and a hydrophilic cavity. Crown ethers show a strong affinity for primary
amines through a hydrogen bonding interaction. The introduction of bulky
groups, such as binaphthyl or carboxylate groups, onto the exterior of the
crown ethers provides steric barriers and induces enantioselective interactions
with solute molecules. Separations are performed in reversed-phase mode.
Retention and selectivity is controlled by the concentration and type of coun-
teranion in the mobile phase and the percent of organic modifier. One com-
mercially available stationary phase contains a crown ether phase, with

binaphthyl appendages, that is dynamically coated onto a silica substrate. An
aqueous mobile phase is recommended when using this column. Retention
increases with the chaotropicity and concentration of the counteranion [9]. A
second commercially available phase utilizes a crown ether with carboxylate
appendages and is covalently bonded to a silica substrate. Organic solvents
can be used in the eluent. In an in-house study for a series of amines (drug
substance intermediates), retention increased with organic content opposite
for what is expected from a reversed-phase system. This behavior can be
explained due to the fact that the primary interactions are hydrogen bonding
and ion pairing, both of which would increase in strength with decreasing
polarity of the mobile phase. Retention also increases with increasing depro-
tonation of the CSP’s carboxylate groups as a consequence of increased sites
for ion pair interactions [10].
The antibiotic glycopeptides—vancomycin, teicoplanin, and ristocetin A—
have been extensively utilized as chiral selectors [11]. These macrocyclic
antibiotics possess several characteristics that enable them to stereoselectively
interact with solutes. They contain an aglycon bucket consisting of three or
four macrocyclic rings. They also possess multiple stereogenic centers and a
number of functional groups including sugars, aromatic rings, phenol groups,
amide linkages, amine, moieties, and acid/esters moieties. As a consequence,
they can interact with a solute through hydrogen bonding, dipole interactions,
π–π interactions, hydrophobic interactions,electrostatic interactions,and steric
hindrance. The phases can be used in normal-phase, reversed-phase, polar
organic, and sub-/supercritical modes. These columns show very good selec-
tivity to amino acids and other carboxylic acids but also resolve many neutral
and basic solutes.
A number of proteins are commercially available as CSPs including α-acid
glycoproteins (AGP,the major plasma binding protein for basic drugs), human
serum albumin (HSA, the major plasma binding protein for weakly acidic
drugs), bovine serum albumin (BSA), ovomucoid (OVM), and cellobiohydro-

lase (CBH) [12]. The proteins are bonded to silica and utilized in reversed-
phase mode with an aqueous buffer/organic modifier eluent. Mobile-phase
HPLC SEPARATION MODES 651
optimization is performed through variation of the pH, ionic strength, tem-
perature
, and organic modifier [13]. It is believed that chiral recognition occurs
predominantly through hydrophobic interactions in an apolar calyx that is
buried in the interior of the structure. In the calyx, additional interactions such
as electrostatic interactions, hydrogen bonding, dipole interactions, and steric
hindrance occur.The protein CSPs are very broad-based in the types of drugs
that they can enantioseparate.
Several variations of the triphenylesters and triphenylcarbamates of
amylose and cellulose are commercially available from Diacel. These poly-
saccharide phases show the broadest applicability of all of the commercially
available CSP and are capable of resolving a large and diverse selection of
chiral solutes [14, 15]. The more popular phases are the 3,5-dimethylphenyl-
carbamates of amylose and cellulose (Chiracel OD and Chiralpak AD, respec-
tively).For most of these phases,the polysaccharide is dynamically coated onto
a silica substrate. A 3,5-dimethylphenylcarbamate derivative of amylose that
is covalently bonded to silica was recently introduced (Chiralpak IA). The
polysaccharide phases are very flexible in that they can be used in normal-
phase, reversed-phase, polar organic, and sub-/supercritical mode. Chiral
recognition on polysaccharide phases are attributed to shape-selective inclu-
sion into the chiral grooves enhanced by additional interactions such as hydro-
gen bonding, dipole interactions, π interactions, and van der Waal forces,
depending upon the chromatographic mode [16, 17]. Enantioselectivity can
vary as a function of amylose versus cellulose, ester derivative versus carba-
mate derivative, mobile-phase components, temperature, and chromato-
graphic mode.
A more detailed discussion of the stationary phase types and mechanism

of interaction and separation theory in relation to chiral compounds is given
in Chapter 22. A large number of chiral stationary phases are currently avail-
able to meet the needs of the pharmaceutical industry for determination of
the enantiomeric purity of active pharmaceutical ingredients, raw materials,
and metabolites. As a consequence, there are a multitude of options in terms
of columns, separation mode, and separation conditions to explore in achiev-
ing an enantioseparation.
For chiral liquid chromatography method development, the first choice to
be made is the separation mode. The popular options are reversed-phase and
normal/subcritical mode.The reversed-phase mode generally offers the advan-
tage of sensitivity. Peak efficiency tends to be greater in reversed-phase mode
relative to the normal-phase mode because of faster mass transfer. Combined
with the ability to use low-UV-cutoff mobile-phase solvents, one can gener-
ally detect 0.1% of the enantiomeric impurity. Moreover, premixed solvents
may be used to increase the detection limits as this will lead to a flatter base-
line (no pulsation due to the pump mixing will be observed). Subcritical mode
also offers the same level of sensitivity but is hampered somewhat by instru-
mental limitations with respect to ruggedness and robustness. Normal-phase
652 ROLE OF HPLC IN PROCESS DEVELOPMENT
and the subcritical modes allow the analysts to take advantage of interactions
such as hydrogen bonding and dipole interactions that are strongest in
apolar media.
T
he polysaccharide phases are known to separate a large range of phar-
maceutical compounds. Chiral screening should include at least the Chiralpak
OD and AD columns. Other popular columns that can be utilized include
protein and antibiotic columns in reversed-phase mode and crown ether sta-
tionary phases for the separation of primary amines. Chirbase (http://chirbase.
u-3mrs.fr/chirbase), a database specializing in chiral chromatographic sepa-
rations, offers comprehensive structural, experimental, and bibliographic

information on both successful and unsuccessful separations. It lists over
100,000 separations. This database indicates that polysaccharide-based sta-
tionary phases are the most frequently utilized phases accounting for ∼40%
of the separations.This database can be utilized as a starting point for method
development.
14.3 SAMPLE PREPARATION
Sample preparation is required for the removal of potential interferents, to
increase or decrease the concentration of an analyte and to convert the analyte
into a suitable form for separation and detection. Sample preparation can be
performed manually or through automation. In the process support area,
sample preparation is seldom more complex than a simple “dilute and shoot.”
In some cases it may be required to dilute the sample in a solution that
quenches an ongoing reaction for in-process samples. In other cases, solid-
phase extraction (SPE) may be required for analysis of certain species in the
presence of an interfering component. The SPE sorbent is chosen either to
retain the analytes of interest while the interfering component is unretained,
or the interfering component is retained while the analytes of interest are
unretained. As an example, a method for determination of azide in the pres-
ence of a triazole derivative utilized a cation-exchange SPE step prior to analy-
sis on an anion-exchange column [18]. The triazole derivative was strongly
retained on the cation-exchange cartridge.The sorbent for SPE can be normal-
phase, reversed-phase, or ion-exchange packings. SPE can also be used for
enrichment of low-level analytes.
Derivatization is another form of sample preparation. It is utilized for the
analysis of labile analytes or to enhance retention or detection with a pre-
ferred type of detector. Derivatization can be performed to enhance detection
by UV/Vis, fluorescence, or electrochemical detection. Consideration must be
given to the stability of the derivatize to solvolysis and thermal degradation.
In our labs alendronate, a bisphosphonate with a primary amine functionality,
was derivatized with FMOC to enhance detection by UV/Vis as well as to

increase retention in RPLC mode [19]. An acylchloride was derivatized with
SAMPLE PREPARATION 653
aniline to form a stable anilide derivative prior to analysis in the RPLC mode
to quantitate the content of the corresponding carboxylic acid and other impu-
rities [20].
The triflation of a drug intermediate alcohol formed an active
trifluoromethanesulfonyl ester. This active ester was derivatized with tetra-
butylammonium bromide to form the bromo analog prior to analysis by
reversed-phase LC [21].
14.4 HPLC DETECTORS
The detectors utilized for HPLC are designed to respond to the solute being
eluted. HPLC detectors can be classified into two broad categories: universal
and selective. Selective detectors respond to some physicochemical property
of the solute, while universal detectors respond to all solutes independent of
their physicochemical properties. The ideal detector would be highly univer-
sal and highly sensitive,have a wide linear range,and not be affected by change
in temperature or mobile phase composition. Commercially available detec-
tors possess some of these characteristics but not all.
The most commonly utilized detectors used in process development are the
UV/Vis detectors that can be fixed-wavelength, variable-wavelength, or diode
array.These detectors are sensitive,have a wide linear range, and are relatively
unaffected by temperature or mobile-phase composition. They respond to
solutes containing double bonds,and compounds with unpaired electrons such
as bromine, iodine, and sulfur. Their response, however, is not equivalent. A
variable-wavelength detector uses a deuterium or xenon lamp source, and the
desired wavelength is isolated by a monochromator. A diode array detector
performs a simultaneous measurement of absorption as a function of analysis
time and over a chosen wavelength range.Thus a UV spectrum is obtained for
each eluted peak.The main advantage of a diode array detector is for method
development where wavelength maxima of the drug substance and its impu-

rities may be unknown or where the UV spectra can be used to track peaks
as operating conditions are changed. If the solute of interest does not possess
a significant chromophore, then indirect photometric detection can be utilized.
In this mode it is the mobile phase that possesses a chromophore and absorbs
light. The detector still measures the difference in absorption between the
mobile phase and the solute. When an analyte without a significant chro-
mophore passes through the detector cell, the absorption of the mobile phase
is decreased and is recorded as a negative peak. An ion-exchange method for
the resolution of alendronate from other bisphosphonates, ionic synthetic
impurities, and inorganic impurities utilized indirect photometric detection
(Figure 14-7) [4].
Fluorescence occurs when a compound absorbs radiation then emits it at
a longer wavelength. It is highly selective. Fluorescence is exhibited by rigid
molecules possessing a large number of delocalized π electrons. Electron-
654 ROLE OF HPLC IN PROCESS DEVELOPMENT
donating groups enhance while electron-withdrawing groups decrease fluo-
rescence
. Few drugs possess natural fluorescence but for those that do, fluo-
rescence detection is an option that offers increased specificity and sensitivity
over UV/Vis detectors. Fluorescence is more sensitive than UV/Vis detection,
particularly for laser-induced fluorescence. Care must be taken in choosing a
compatible mobile phase because fluorescence can be quenched by highly
polar solvents or halide ions. Fluorescence efficiency is also dependent upon
pH of the mobile phase.
Some solutes may not have a significant chromophore, and alternate detec-
tors must be utilized. These detectors include refractive index, evaporative
light-scattering, element-specific, electrochemical, and mass spectrometric
detectors. Refractive index (RI) detectors monitor changes in the refractive
index of the mobile phase that occur due to the presence of solute molecules.
Detection is universal but less sensitive than UV detectors. It is suitable for

solutes without significant chromophores. The refractive index of the mobile
phase must be constant, and thus this mode of detection is not amenable to
gradient elution. Slight variations in temperature will also change the refrac-
tive index of the mobile phase. Therefore, very good temperature control is
required.
Evaporative light-scattering detectors (ELSD) require nebulization of the
eluent after which the aerosol is transported through a heated tube allowing
the mobile phase to be evaporated. The residual particles pass through a light
beam, and scattered light is then detected at a fixed angle from the incident
light. Volatile mobile-phase components such as trifluoroacetic acid, formic
acid, acetic acid, and ammonium hydroxide must be used. The ELSD is a uni-
versal detector as long as the solute is less volatile than the mobile phase. The
linear range is not wide. It is intermediate between UV and RI detectors in
terms of sensitivity and can be utilized with gradient elution. ELSD is useful
for detection of solutes that do not possess a significant chromophore [22] but
should not be used for thermolabile solutes.A recently commercialized alter-
native to ELSD is a corona discharge detector.The HPLC effluent is similarly
converted to an aerosol, the aerosol particles are then charged by a positive
corona discharge, and the current from the charged particle flux is then mea-
sured. This detector is generally regarded as more sensitive than ELSD [23].
Chemiluminescent detectors (CLND) are very selective and sensitive. If a
solute contains at least one nitrogen atom, it can be detected. The effluent is
nebulized, and then it is oxidized by combustion in a high-temperature
furnace. Nitrogen-containing solutes are converted into nitric oxide, which is
then passed into a chamber where it reacts with ozone to produce excited-
state nitrogen dioxide that emits a photon upon relaxation. The photon flux is
then measured by a photomultiplier tube [24]. The signal generated is pro-
portional to the number of nitrogen atoms in the solute molecule. This detec-
tion mode requires volatile mobile phases that are free of nitrogen-containing
molecules (no acetonitrile). CLND have been determined to have a wider

linear range and greater sensitivity than ELSD [24].
HPLC DETECTORS 655
Electrochemical detection can be utilized for compounds that are ionic or
readily oxidizable or reducible
.Thus, this form of detection can be used for the
analysis of inorganic ions, protolytic organic compounds such as amines and
carboxylic acids, and other compounds such as phenols, thiols, and alcohols.
Conductivity detectors measure differences in the equivalent conductance of
the solute and ions in the mobile phase. The conductivity response is maxi-
mized through the use of ion suppressors that effectively eliminate the con-
ductivity of the mobile-phase ions through chemical removal or electronic
subtraction.The linearity range is wide,and detection is highly sensitive. In our
labs, conductivity with ion suppression was utilized to detect residual levels
(∼0.1%) of choline (quaternary saturated amine) in drug substance [25].Amper-
ometric detection is less commonly utilized and is suitable for compounds that
can be electrolytically oxidized such as phenols. This mode is not generally
applied in the reductive mode due to interference from dissolved oxygen in the
mobile phase. Amperometric detection is highly sensitive and selective.
Mass spectrometric detection is close to being a universal detector. Ioniza-
tion techniques such as atmospheric pressure chemical ionization (APCI) and
electrospray ionization (ESI) are routinely employed. These techniques allow
the transfer of the LC effluent into the gas phase. With APCI, the eluent is
converted to an aerosol by a sheath gas. The aerosol is then subjected to a
chemical ionization plasma created by a corona discharge, leading to forma-
tion of solute ions. These ions are then transferred into the mass spectrome-
ter.With ESI, the eluent is converted to charged droplets. ESI is preferred for
compounds that are ionized in solution. APCI is better for compounds of
medium polarity. Both techniques can be used in positive or negative ion
mode. Positive ion mode is commonly used. Negative ion detection is useful
for negatively charged ions such as acids. Nonpolar compounds are difficult to

analyze with these atmospheric ionization techniques due to their soft ioniza-
tion mechanisms. Atmospheric pressure photoionization is an emerging tech-
nology for the analysis of these nonpolar compounds.This technique is similar
to APCI; however, a gas discharge lamp that emits photons in the vacuum UV
region is utilized. Sensitivity can be increased by the use of dopants such as
toluene or acetone added post-column to the eluent.The dopant is first ionized
and then ionizes the analytes through further reactions [26].
Mass spectrometric detection in the process development area is generally
performed with a single-quadropole, triple-quadrupole, or ion trap mass spec-
trometer. Other options include sector and time of flight spectrometers. A
single quadrupole provides information pertaining to the mass to charge ratio
(m/z) of the solute. Ion traps and triple quadrupoles provide additional infor-
mation through tandem MS, allowing for a more definitive structural elucida-
tion of the solute. Volatile buffers such as ammonium acetate or ammonium
formate and low-pH mobile phases such as 0.1% formic or acetic acid are rec-
ommended to prevent blockages of sample cones or capillaries. The relative
sensitivity of MS versus UV/Vis detection may differ by many orders of mag-
nitude in either direction, depending upon the chromophoric properties and
656 ROLE OF HPLC IN PROCESS DEVELOPMENT
the ionizability of the analyte. It can be very selective when used in selected
ion monitoring mode where it is detecting one specific mass/charge ratio
. The
use of MS is extremely valuable in identifying by-products of reactions, impu-
rities in intermediates that may react further in downstream processing, and
impurities that are formed during stability testing.
14.5 METHOD DEVELOPMENT
The approach to method development is dependent upon the physicochemi-
cal properties of the solute and any known potential impurities and the
purpose of the method. The method may be required for an impurity profile,
assay, in-process monitoring, or chiral/isomeric evaluation. Method develop-

ment is usually dynamic.As more knowledge about the properties of the solute
and potential impurities is gained, the method can be further optimized.Ana-
lytical laboratories supporting process development should be stocked with a
variety of columns for RPLC, NPLC, ion-exchange, and chiral separations.
Column switching capability is also an asset for method development. Column
switching allows for analysis of the same sample with as many as six differ-
ent columns in an overnight run to help speed method development. Initial
development can be performed empirically, based on the chromatographer’s
experience, or through the use of simulations with one of the commercially
available method development software packages.The parameters to explore
for method development include separation mode, column selection, mobile-
phase optimization, temperature, detection wavelength, sample diluent and
concentration, injection volume, and sample preparation procedure. The
use of an orthogonal chromatographic method, with the developed method
as a check, is recommended. Having an orthogonal method minimizes the
possibility of peak co-elution, particularly in cases where there is limited
information available regarding the nature of impurities. An orthogonal
method may be employed once the final synthesis is set during the develop-
ment of a drug. The final synthesis is usually set for preparation of the
clinical material used for Phase II clinical studies. When a method has
been developed that is deemed appropriate for the purpose, system suitabil-
ity parameters should be implemented and some degree of validation
should be performed to ensure that the method meets the needs of the
chromatographer.
For developing an impurity profile for raw materials, intermediates, or drug
substance, communication with the process chemists regarding potential reac-
tion by-products is always the best start. This information plus any garnered
knowledge of the physicochemical properties of the solute and potential impu-
rities such as pK
a

, logP (octanol/water partition coefficients), solubility, and
UV spectrum will determine the selection of the appropriate mode, column,
mobile-phase, and other separation parameters. Given the potential for gen-
eration of impurities that are unanticipated by the process chemists, it is
METHOD DEVELOPMENT 657
recommended that for early development a gradient method be employed. A
gradient method will allow for coverage of a wide range of polarity and thus
be able to capture early and late eluting impurities in the same run.
For this
reason, a reversed-phase method is the first choice. Most components can be
eluted in a 10–90% gradient of organic modifier as long as there are no mis-
cibility issues with the aqueous mobile phase.An isocratic hold at 90% organic
should be performed especially in early development to detect the presence
of any extremely hydrophobic impurities. Ideally the peaks of interest should
be eluted with a capacity factor between 1 and 10.
The choice of a column is dictated in large part by the hydrophobicity (eval-
uated as logP when available) of the solute. C18 or C8 columns are commonly
utilized in reversed-phase mode. Retention and selectivity for these phases can
vary, depending upon whether they are conventional, polar end-capped, polar
embedded, or hybrid silica. A high-carbon-load C18 or a graphitic column can
be used to increase retention. A low-carbon-load C8 or a phenyl or cyano
column can be used to decrease retention. Alkyl, phenyl, and cyano phases
may offer different selectivities. Selectivity may also vary as a function of the
substrate: silica versus polymer versus zirconia. The sheer volume of com-
mercially available reversed-phase columns makes selection of the best
column, for a particular separation, anything but a simple task. Much research
has been performed toward the classification of reversed-phase columns.
Approaches include regression of logk versus logP, thermodynamic mea-
surements of retention, and quantitative structure–retention relationships
(QSRR) using experimentally determined or calculated molecular descriptors

[27–35]. For example, classifications in terms of efficiency, hydrophobicity,
silanol activity, and steric selectivity were used in the evaluation by principal
component analysis of 69 columns differing in type of silica, pore size, end-
capped/not end-capped, base deactivated/not base deactivated, and polar
embedded [31]. Based on this classification, one can select four columns, which
fall into separate categories ensuring selectivity differences, for initial method
development. Similarly, classification of 28 columns in terms of selectivity
based on hydrophobicity, steric selectivity, efficiency, and silanol activity using
chemometric approaches led to the selection of eight columns of low, inter-
mediate, and high hydrophobicity that were highly efficient and showed good
steric selectivity [35]. One could then choose one column from each hydropho-
bic class for method development. One should also ensure that the selected
columns are stable within the intended pH and temperature regions that they
will be employed. A good understanding of the chemical stability of the sta-
tionary phases is essential.
An additional variable for varying selectivity is column temperature. Sig-
nificant changes in selectivity may be observed when comparing separations
at 10°C and 50°C. This depends on the nature of the analyte and its interac-
tion with the stationary-phase and mobile-phase components. Elevated tem-
peratures, however, may lead to unwanted compound degradation and should
be avoided for labile components.
658 ROLE OF HPLC IN PROCESS DEVELOPMENT
Mobile-phase composition is another major parameter for affecting selec-
tivity in a separation.
Points to consider include choice of organic solvent,
mobile-phase pH, and use of additives. The three most commonly utilized
organic solvents are acetonitrile, methanol, and THF. Acetonitrile is usually a
good starting organic solvent as a consequence of its lower viscosity and UV
cutoff. Method development can be performed with each of these three
organic solvents or with mixtures of them. The pH of the mobile phase is also

critical. Low pH protonates acids and bases, resulting in neutral acids and
charged bases. Conversely, high pH deprotonates acids and bases, resulting in
charged acids and neutral bases. In general, retention decreases with increas-
ing charge on the solute. Buffers are recommended. Phosphate is a commonly
utilized buffer with pK
a
values of 2.1 and 7.Buffers such as acetate and formate
are useful for detection modes requiring volatilization of the mobile phase.
Care should be taken to avoid working within ±1.5 units of the pK
a
of the
solute, because this may result in poor retention precision. Many pharmaceu-
tical compounds are acidic or basic, and a good starting point for method
development is low pH. A low pH suppresses the ionization of acid solutes
and the silanol sites of the stationary phase. High pH can be used to increase
retention of bases (neutral form) or to take advantage of ion-exchange inter-
actions (with bases in ionized form) to improve selectivity (however, bad
peak shapes sometimes are the result due to strong silanophilic interactions).
Retention can also be enhanced by the use of additives such as chaotropic
anions (perchlorate, hexafluorophosphate) or by ion-pairing agents
(hexanesulfonate) [36].
Knowledge of the UV spectra of the solute of interest can be applied to
choice of wavelength for UV/Vis is detection where amenable.One can choose
a wavelength near or at the UV maxima for detection. This choice suffers the
disadvantage that unknown impurities in the intermediates and/or drug sub-
stance may not exhibit strong extinction coefficients at the chosen wavelength
and may go undetected. An alternative is to work at a low wavelength such as
210 or 220nm where most solutes possessing a chromophore will have signif-
icant absorption (π–π* bands for double bonds and n–σ* bands for amines and
halogens). The choice of wavelength is also dictated by the UV cutoff of the

mobile-phase components. Knowledge of the solubility of the solute as well as
its compatibility directs the choice of diluent. A combination of adequate sol-
ubility and injection volume should be chosen such that ideally 0.05% of the
solute can be detected with a signal-to-noise ratio of greater than 10 to 1.
For development of a weight percent assay, a short isocratic method can be
implemented based on observations from the gradient method used for the
impurity profile. One can use a shorter column such as a 5-cm column and
keep retention of the solute of interest to around a capacity factor of 3 as long
as it is still resolved from impurities observed in the impurity profile. Addi-
tionally, the elution of more hydrophobic species should not co-elute with the
drug substance in later injections. During the method development of an iso-
cratic method, the compound should be injected and then a suitable number
METHOD DEVELOPMENT 659
of blanks injected to ensure that more hydrophobic impurities do not elute at
the same time as the analyte peak in later injections
.
A similar approach using an isocratic method can be applied to in-process
monitoring, where the goal is to monitor the disappearance of the starting
material and appearance of the product. In-process methods will be discussed
in greater detail in Section 14.6.
The use of computer simulations is an alternative approach to method
development. Computer-based expert systems are designed to mimic the
thought processes of an experienced chromatographer.These systems contain
a database that can used to evaluate chromatographic data and provide opti-
mized conditions. Variables such as solute structure, column type, mobile-
phase components, pH, and temperature can be inputted, and proposed
optimized chromatographic conditions are outputted. This approach is gener-
ally faster and cheaper than performing all of the experiments necessary for
method development. Systems with artificial intelligence can plan experi-
ments,collect and evaluate data, and adjust chromatographic conditions in real

time according to predefined decision schemes until a satisfactory separation
is achieved. Further discussion of the different automated method develop-
ment software available is given in Chapter 10.
14.6 IN-PROCESS MONITORING
In-process monitoring is implemented to maximize yield and minimize impu-
rity generation during the various synthetic steps.An ideal in-process method
should quickly evaluate a specific sample and provide results in a timely
fashion such that changes may be triggered to maintain the reaction condi-
tions at the optimal level required to secure production with high purity and
maximum yield. Process analytics using on-line spectroscopic analysis can
provide instantaneous feedback; however, the reaction mixture is often too
complex to provide accurate results.Oftentimes,separation is required to eval-
uate levels of several components. Chromatography can provide the necessary
separation, but the time lag of the analysis must be short enough to monitor
the actual state of the reaction. An emphasis should be placed on providing
near-real-time feedback by using methods with short run times. Ideally, this
would be accomplished by reducing the run time without a concomitant loss
in column efficiency or resolution.
One approach to achieving near-real-time feedback with chromatography
is through the use of short columns with smaller particles.Small particles result
in higher column efficiency,but with increased backpressure limiting the work-
able column length. Short columns (10cm or less) with smaller particle sizes
(1.5 to 3.5µm) can result in comparable separations to longer columns (25cm,
5-µm particles) but with one-half to one-fifth the run time. The efficiency of
these shorter columns is equivalent to, and often superior to, the longer con-
ventional columns. Shorter columns, however, are susceptible to instrumental
660 ROLE OF HPLC IN PROCESS DEVELOPMENT
band-broadening effects,and care must be taken to minimize the extra-column
volume
.

A nonporous silica C18 column was utilized in conjunction with an on-line
HPLC to provide rapid feedback for a deprotecting step for a drug substance
[37]. The conventional method had a run time of 35 minutes, not including
sample preparation time and the time needed to sample the batch and trans-
port the sample from the pilot plant to the analytical lab. The on-line method
had a run time of 10 minutes, and the sample preparation for the subsequent
sample was ongoing during each analysis point. As a consequence, the batch
could be evaluated every 10 minutes as compared to every 60–90 minutes by
the conventional method. On-line sampling was feasible only because the reac-
tion mixture was a homogeneous solution (Figures 14-8 to 14-10).
Another approach is the use of monolithic columns consisting of silica
based rods of bimodal pore structure.They contain macropores (∼1–2µm) and
smaller mesopores (∼10–20nm) [38]. The macropores allow for low backpres-
sure at high flow rates. The mesopores provide the needed surface area for
interactions between the solute and stationary phase. The macropores result
in higher total porosity as compared to porous silica particles. Flow rates of
5mL/min can be tolerated on a 10-cm column without an appreciable loss in
IN-PROCESS MONITORING 661
Figure 14-8. Structures of components in a deprotecting process
. (Reprinted from
reference 37, with permission.)
column efficiency. Monolithic columns show similar selectivity to spherical
particle columns but with shorter retention times due to the ability to use
higher flow rates without compromising efficienc
y. Methods have been devel-
oped, with less than 10-minute run times, using 15-cm Chromolith columns
for a number of in-process samples in our labs. One application was for mon-
itoring a coupling reaction between a biarylpiperazine and an epoxide during
662 ROLE OF HPLC IN PROCESS DEVELOPMENT
Figure 14-9. Schematics of an on-line HPLC system. (Reprinted from reference 37,

with permission.)
Figure 14-10. On-line chromatogram of a typical reaction mixture. (Reprinted from
reference 37, with permission.)
the synthesis of an HIV drug candidate [39]. A second application was for
catalyst screening [39].
14.7
IMPURITY IDENTIFICATION
A critical aspect of drug development is the control and identification of impu-
rities in the active pharmaceutical ingredient. Regulatory requirements dictate
the control of impurities and the identification, where possible, of impurities
that exceed 0.1% [40–42].The identification of these impurities provides infor-
mation toward their potential toxicity. Impurities in the drug substance can
originate from incomplete reactions,over-reactions, and side reactions and can
also originate from impurities in starting materials, reaction solvents, catalysts,
or residual solvents. Impurities may also occur as a result of degradation.
Common degradation pathways are hydrolysis, dehydration, oxidation, dimer-
ization, or a combination thereof. Delineation between process related and
degradation impurities is not always possible. Impurities may be organic or
inorganic in nature. Inorganic impurities may originate from the counterion if
the drug substance is in salt form, from reaction vessels, filters, adsorbents, and
tubing, and also from catalysts and inorganic reagents used in the synthesis.
Control of impurities that appear in the drug substance is not only undertaken
at the final isolation step but also through control of impurities in raw mate-
rials, intermediates, and solvents and at critical synthetic steps.Whenever pos-
sible, the level of impurities originating from the starting materials should be
limited by in-process controls rather than at the final step. Chromatography
as a consequence of its selectivity and precision is an excellent tool for the
monitoring of impurities.
The identification of process-related impurities can ultimately lead to elu-
cidation of their mechanistic pathways. The first step in this process entails

determination of structures for the impurities using tools such as NMR or LC-
MS. With an elucidated structure, one can then attempt to propose a mecha-
nism for its formation and to identify the synthetic steps that lead to its
formation. This information can then be utilized to manipulate synthetic
conditions so as to minimize its formation or develop efficient recrystalliza-
tion procedures to reduce impurity level while still maintaining acceptable
yield.
The retention times of impurities found in a drug substance impurity profile
should be first compared to those of known process-related impurities, inter-
mediates, and starting materials which could have been carried forward to the
final product. Where facile, as a first step, the process chemist can synthesize
some of the potential impurities on a small scale. Potential impurities include
the penultimate intermediates, products of over-, under-, and side reactions,
and degradation products. Analogs may have also been synthesized during
the drug discovery stage for screening that can also be potential impurities.
These isolated impurities can then be correlated to impurities that appear in
IMPURITY IDENTIFICATION 663
the chromatogram. However, a simple matching of retention times is not
satisfactory confirmation of impurity identification.
The use of an orthogonal
chromatographic method is recommended. For example, one can match the
prepared impurity versus an impurity that appears in the reference RP chro-
matographic method by using an orthogonal TLC method or a NPLC method.
If the impurities match up in both chromatographic systems, then there is a
level of confidence that the impurity observed in the reference chromatogram
is the same as the synthesized potential impurity. Further confirmation can be
provided by matching UV spectra using diode array analysis. The definitive
confirmation, which can occur at a later stage in development, is through
matching of MS or NMR spectra.
A logical second option is to evaluate samples where the relative concen-

tration of impurities is higher. These samples include crude product, mother
liquors, and stressed drug substance. Isolation of the impurity and sensitivity
issues for identification can then be overcome. Care must be taken, however,
that the impurity found in these samples are exactly the same as those found
in the drug substance and not just co-eluters. Consequently, the use of orthog-
onal chromatographic methods and UV spectra matching should be strongly
considered.
As a third option, impurities appearing in the reference chromatogra-
phic method may be isolated using analytical (under overloaded conditions)
or semi-prep chromatography and then identified by MS and/or NMR.
Mobile-phase components should be removed prior to analysis. Removal of
mobile-phase components may entail evaporation, liquid–liquid extraction,
freeze-drying, or solid-phase extraction. Identification by MS should be
attempted first because its high sensitivity requires only small sample amounts.
The less sensitive NMR requires the isolation of greater amounts of the impu-
rity. An alternative to this approach is the use of LC-MS or LC-NMR.
LC-MS coupled with UV analysis is now routinely used in the process
development area for the identification of impurities.The UV detection allows
for cross-referencing of impurities because response factors may differ and
changes in retention time may occur as a consequence of differences in instru-
mentation setup or changes in the chromatographic method to make it
MS-compatible (e.g., switch from phosphate to formate buffer). LC-UV-MS
using soft ionization techniques will simply provide a molecular mass of the
impurity. The use of collision-induced dissociation can provide some frag-
mentation information. LC-UV-MS-MS will provide even more structural
information. For example, a triple quadrupole instrument can isolate one ion,
which is then fragmented by gas-phase collisions (argon or xenon) in the
second quadrupole. The produced fragments are then analyzed by the
third quadrupole. The increasing ease of use and the proliferation of LC-MS
instrumentation in the analytical labs have led to its utilization as the first

option for identification of impurities, particularly when small-scale synthesis
of potential impurities is not trivial. For in-depth discussion of LC-MS,
the reader is referred to Chapter 7. However, if the MS data do not provide
664 ROLE OF HPLC IN PROCESS DEVELOPMENT
sufficient structural information, then it may be necessary to isolate and
identify by NMR or LC-NMR.
T
he introduction of NMR probes that can be interfaced with HPLC has
resulted in the emergence of another powerful tool for the structural elucida-
tion of impurities [43].The intense signals from the protons of the solvents can
overwhelm the weak signals of the impurities, rendering solvent signal sup-
pression necessary. However, solvent signal suppression may suppress impu-
rity signals lying under the solvent signal, and thus some information may be
lost. To minimize this effect, simple binary mobile phases such as methanol/
water or acetonitrile/water are used.Another alternative is to use only deuter-
ated solvents, but this is a rather expensive choice given the price of these
solvents. Additionally, there will still be some strong signals from the solvent
as a consequence of contamination.
LC-NMR analysis can be performed in continuous-flow or stop-flow mode
[43]. In continuous-flow mode a series of NMR spectra is collected rapidly as
the HPLC eluent flows through the NMR probe. In stopped-flow mode, the
flow is stopped for short intervals as the peak of interest passes through the
probe, allowing for the collection of sequential spectra through the peak.This
mode can lead to band broadening and affect the resolution of later eluting
peaks. It is best employed for analysis of just one impurity in a chromatogram.
An alternative is to store the peaks of interest in capillary loops. Stopped-flow
allows for 2D NMR experiments such as correlation spectroscopy (COSY) to
be performed. The main disadvantage of HPLC-NMR is sensitivity. Analysis
of peaks at 0.1% to 1% is a challenge. This can be overcome to some extent
by trapping chromatographic peaks on solid-phase extraction cartridges,which

can be subsequently eluted with deuterated solvents [44]. This mode allows
for greater flexibility in mobile-phase choice since the mobile-phase additives
can be separated from the impurity peak on the SPE cartridge. For a detailed
discussion of LC-NMR, the reader is referred to chapter 20.
Finally, impurity analysis can also be utilized to demonstrate illegal use of
patented reaction routes.The impurity profile of a drug substance is influenced
by the synthetic route and the source and quality of the starting materials.
Identification of impurities in drug prepared by two different manufacturers
may provide valuable insight into the manufacturing route and determine if
patent infringement has occurred, because certain impurities may be indica-
tors of a specific synthetic route.
14.8 ESTABLISHMENT OF HPLC SELECTIVITY BY
STRESS STUDIES
According to the ICH guideline on stability testing, the purpose of stress
testing is twofold [45]. First, it can be used to predict the stability of the mol-
ecule and from the degradation products establish degradation pathways.
Second, it can validate the stability-indicating capability of the analytical
ESTABLISHMENT OF HPLC SELECTIVITY BY STRESS STUDIES 665