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SUPPLEMENT TO
Advances in
Pharmaceutical Analysis
October 2016
Volume 29 Number s10
www.chromatographyonline.com
Joanna Simpson
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4 Advances in Pharmaceutical Analysis October 2016
Advances in
Pharmaceutical Analysis
8 Advances in Pharmaceutical Analysis: Introduction
Pat Sandra and Davy Guillarme
An introduction from the guest editors of this special supplement
9 Advances in Sample Preparation for Biological Fluids
Lucie Nováková
This article explores microextraction techniques, selective approaches, on-line sample preparation, and dried
matrix spots that aim to provide solutions to sample preparation problems in bioanalysis.
16 Quality by Design: A Tool for Separation Method Development in Pharmaceutical Laboratories
Karen Gaudin and Ludivine Ferey
The trends observed when applying QbD to the development of separation methods in pharmaceutical
analysis are discussed.
26 High-Throughput Analysis of Drugs and Metabolites in Biological Fluids Using Quan–Qual Approaches
Ronald de Vries, Rob J. Vreeken, and Filip Cuyckens
Recent high-resolution MS instruments can be used for both quantitative and qualitative measurements, making
these a worthwhile alternative to triple quadrupole MS systems for quantitation, as well as a powerful tool for
identification of unknowns within the same analysis.
31 Contemporary Analysis of Chiral Molecules
Eric Francotte
The state-of-the-art in chiral recognition separation methods with an emphasis on LC and SFC is presented.
38 Characterization of Counterfeit and Substandard Medicines Using Capillary Electrophoresis
Julie Schappler and Serge Rudaz
As a simple, reliable, and cost-efficient technique, CE can offer significant benefits in counterfeit drug analysis.
Ph
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PHARMACEUTICAL Q� HEALTH SCIENCES Q� FOOD Q� ENVIRONMENTAL Q� CHEMICAL MATERIALS
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In the LCGC Europe October
2015 supplement “Advances in
Biopharmaceutical Analysis”, the
analytical challenges in the analysis
of large and heterogeneous protein
biopharmaceuticals were addressed.
Biopharmaceuticals are indeed
the fastest growing area within the
pharmaceutical industry but innovation
in small-molecule drugs is still of great
importance in the sector. It is estimated
that research in both fields, small and
large, will recognize an even distribution.
This supplement therefore focuses on
recent advances in small-molecule drug
analysis, complementing the October
2015 supplement.
Giving a complete and detailed
overview of all innovations in
contemporary analytical methods
for pharmaceutical analysis in a
supplement is unrealistic and this
issue is therefore based on selecting
some important keywords within this
research domain: sample preparation
for biological fluids, quality by
design (QbD), the power of mass
spectrometry (MS), chiral recognition,
and considerations on the analysis of
counterfeits medicines.
Sample preparation is the first
step in an analytical procedure for
biofluids and its importance is often
underestimated. The first contribution,
authored by Lucie Nováková, provides
an overview of conventional sample
preparation techniques and presents
the most recent developments for high
throughput bioanalysis. Microextraction
techniques, on-line sample preparation,
and selective methods, as well as
dried matrix (blood) spot analyses, are
presented in detail.
Analytical procedures are critical
processes in drug development and
quality control. Quality by design (QbD)
is gaining widespread acceptance
in the pharmaceutical industry
and is also more and more widely
implemented in analytical work. Karen
Gaudin and Ludivine Ferey describe
the present trends in applying QbD
to separation methods (particularly
in liquid chromatography [LC]) in
pharmaceutical analysis. A detailed
description of tools involved in QbD
methods are presented, and the
benefits of implementing QbD in the
laboratory are provided.
Developments in MS have gained
momentum in recent years and
are very rapidly implemented into
pharmaceutical laboratories. Ronald
de Vries, Rob J. Vreeken, and
Filip Cuyckens show how recent
high-resolution MS instruments can
be used for both quantitative and
qualitative measurements, making
these a worthwhile alternative to “the
golden standard” triple quadrupole
MS systems for quantitation, as well
as a powerful tool for identification of
unknowns within the same analysis.
A significant number of
small-molecule drugs are single
enantiomers and chiral resolution
methods are required during the
synthetic routes of these drugs and
for the final active pharmaceutical
ingredient (API). Eric Francotte is an
expert in chiral separation methods
and he describes the state-of-the-art in
chiral recognition separation methods
with emphasis on LC and supercritical
fluid chromatography (SFC). This
contribution is a follow-up of the work
described in LCGC Europe April 2016
entitled: “Practical Advances in SFC
for the Purification of Pharmaceutical
Molecules” (1).
Counterfeits or fake medicines
are a growing problem, especially in
developing regions. They may contain
the active ingredients in a lower dose
or even not at all, they may contain
another active ingredient, or they may
be contaminated with toxic ingredients.
Because counterfeits are produced
under uncontrolled conditions and
have not been assessed by health
authorities, they may be very harmful to
the health of users. Control of imported
medicines in developing countries is
therefore of the utmost importance,
but the analytical technique must
be sufficiently simple, reliable, and
cost-efficient. Julie Schappler and
Serge Rudaz describe how capillary
electrophoresis (CE) can fulfil these
requirements. As CE is now recognized
as a valuable technique by numerous
Pharmacopoeia, it is indeed a good
candidate in counterfeit analysis.
We hope that the contributions in this
supplement are of interest to the LCGC
Europe readers. Editing and reviewing
the contributions was a pleasure for
us and we thank our colleagues for an
excellent job.
Reference(1) E. Francotte, LCGC Europe 29(4),
194–204 (2016).
Advances in Pharmaceutical AnalysisPat Sandra1 and Davy Guillarme2, 1Research Institute for Chromatography, Kortrijk, Belgium, 2University of Geneva,
University of Lausanne, Geneva, Switzerland
An introduction from the guest editors of this special supplement from LCGC Europe revealing recent
developments in small-molecule drug analysis.
An additional paper titled
“On-Line Two-Dimensional
Liquid Chromatography
(2D-LC) in the
Analysis of Pharmaceuticals”
by Pat Sandra,
Gerd Vanhoenacker,
Mieke Steenbeke, Frank David,
Koen Sandra, Claudio Brunelli,
and Roman Szucs will be
featured in an upcoming
issue of LCGC Europe.
Advances in Pharmaceutical Analysis October 20168
Pat Sandra Davy Guillarme
The determination of drug
concentrations in biological matrices
is an important aspect of the drug
development process. Bioanalytical
methods are needed to obtain
information on drug profiles from
animal toxicokinetic studies and from
clinical trials, including bioequivalence
studies. These results are used to
make critical decisions supporting
the safety and efficacy of a drug
substance or drug product. Biological
fluids are not convenient for direct
analysis using instrumental methods
because of high sample complexity
and the high content of many
interfering compounds. Generally,
very complex liquid samples including
whole blood, plasma, serum, urine,
or saliva are handled in bioanalysis.
The target analytes are often
present at very low concentrations,
while the interfering compounds
are abundant. Among the most
important, salts and phospholipids are
responsible for matrix effects in liquid
chromatography–mass spectrometry
(LC–MS) analysis. Proteins are
damaging for the analytical
instrumentation because they can
irreversibly adsorb onto the stationary
phase, resulting in a substantial loss
of column efficiency, an increase in
back pressure, or system clogging.
To prevent these issues, adequate
sample preparation is mandatory in
bioanalytical methods (1,2,3).
With a well-designed sample
preparation technique, isolation,
cleanup, and preconcentration of the
analytes of interest from the complex
biological matrices can be achieved,
while the interfering compounds can
be removed. Sample preparation
is an integral part of a bioanalytical
method influencing all further steps
of the analysis, with a crucial impact
on the accuracy and precision of
the final results. Unfortunately, it is
still the most labour-intensive and
time-consuming step of the analytical
procedure, representing 60–80% of
the total analysis time. This is in strong
contrast to modern, fast LC methods
and detection approaches and makes
sample preparation a limiting step to
fast bioanalysis (1,4).
The selection of an appropriate
sample preparation technique is
made with regards to the analyte type,
sample type and amount available,
requested selectivity and sensitivity of
the procedure, extraction time, solvent
consumption, and the possibility of
automation. A well-designed sample
preparation technique should involve a
small amount of sample and a simple
method, which is “just adequate” prior
to analysis. This feature is important
because complicated multistep
procedures can introduce errors.
Sample preparation techniques used
for the treatment of biological fluids
can be classified into two main groups
based on the history and frequency
of their use: conventional methods
and modern approaches to sample
preparation.
Conventional Sample
Preparation Techniques
Conventional sample preparation
approaches involve well-established
and well-optimized techniques,
which are commercially available
and widespread across analytical
laboratories. Straightforward,
reliable, and high-throughput
sample preparation techniques
are a guarantee for achieving
accurate results and meeting
rigorous requirements for method
validation in bioanalysis (5,6) within
a reasonable time frame. These
conventional approaches involve
protein precipitation (PP), liquid–liquid
extraction (LLE), and solid-phase
extraction (SPE) (1,7,8).
Protein Precipitation (PP): Protein
precipitation (PP) followed by
centrifugation (or another precipitate
separation step) belongs among
the leading sample preparation
techniques in modern bioanalysis,
despite its very low selectivity and
clean-up efficiency. Important
benefits, such as very fast sample
treatment, easy and fast method
optimization, minimum number of
steps, and no requirement for special
equipment has brought this method to
the attention of bioanalytical scientists.
However, method sensitivity might
be compromised and matrix effects
may be serious. Another important
drawback of PP is the difficulty of
automating the centrifugation step. To
facilitate the separation of precipitated
phase and supernatant, new solutions
have recently become available in the
form of well-plates to enable filtration
of the precipitated samples (1,7,8).
Liquid–Liquid Extraction (LLE):
Liquid–liquid extraction is based
on a transfer of the analyte from the
aqueous sample to a water-immiscible
solvent. Because of the high
consumption of organic solvents
and thus the production of a large
Advances in Sample Preparation for Biological FluidsLucie Nováková, Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Hradec Králové,
Czech Republic
Sample preparation techniques in bioanalysis are multistep, time-consuming, and labour-intensive
procedures that can take up 60–80% of the total analysis time. Sample preparation is often the limiting
step of fast bioanalysis and the most error-prone part of the analytical method. There is currently a
focus on improving the sample preparation process by shortening sample preparation time, cutting the
cost of analysis, decreasing sample volume and solvent consumption, reducing the number of sample
preparation steps, and adapting the whole process for automation. This article explores microextraction
techniques, selective approaches, on-line sample preparation, and dried matrix spots that aim to provide
solutions to sample preparation problems in bioanalysis.
9www.chromatographyonline.com
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volume of environmental pollutants in
a conventional LLE setup, miniaturized
versions of LLE are currently preferred.
Such extraction is performed in glass
vials or small test tubes (9) using
typically 50–100 μL of sample and
about 600–2000 μL of organic solvent
(10,11). The amount of solvents and
sample can be further decreased in
modern microextraction approaches
(see “Microextraction Techniques
Based on LLE”).
LLE is a simple and straightforward
extraction technique. Advantages
include no requirement for special
equipment or skills, lower cost
compared to SPE, and potential to
remove matrix effects in
LC–MS because ionized compounds
such as salts do not partition
into the organic layer (3). Some
disadvantages arise when large-scale
LLE is performed, such as bubble
and emulsion formation. The high
consumption of toxic organic solvents
and production of a large amount of
organic waste make LLE expensive
and environmentally harmful.
Another principal drawback of LLE
is its unsuitability for hydrophilic
compounds, which might be a
significant issue in bioanalysis
because some drugs and many
metabolites possess polar structures.
Last, but not least, most of the LLE
procedures require the evaporation
of nonpolar organic solvent and its
reconstitution in the mobile phase
to reach sample preconcentration
and to obtain a sample compatible
for injection into the LC system.
This step substantially prolongs the
extraction time and may be critical
for sample recovery because of
solubility issues (1,7,8,9). In order
to improve the extraction efficiency,
salting-out (SALLE) (12), sugaring-out
(SULLE) (13), or in-vial derivatization
approaches may be applied in LLE (9).
Although LLE is widely and
commonly used in bioanalysis, its
on-line coupling and full automation is
difficult and requires substantial effort.
An important approach to facilitate
and automate LLE is supported liquid
extraction (SLE). SLE columns contain
specially processed wide-pore
diatomaceous earth, a chemically inert
matrix, which only acts as a holder for
the aqueous sample. A 48- or 96-well
plate format enables automation
and high sample throughput using
a positive-pressure manifold. The
adoption of this approach has
initially been very slow because of
the well-established conventional
LLE procedures in routine practice.
However, there has been an increased
interest in this arrangement as a result
of this miniaturization and automation
(14,15).
Solid-Phase Extraction (SPE):
SPE is probably the most widely
accepted sample preparation
technique, enabling sample cleanup
and preconcentration of the target
analytes. The extraction of these
analytes is based on partitioning
between a solid phase (SPE cartridge)
and a liquid phase (sample). The
selection of SPE sorbent is the key
factor, defining important parameters
of selectivity, affinity, and capacity.
SPE protocols are more selective
compared to those of LLE because
of the selectivity of these sorbents.
Moreover, cleanup is also enabled
during individual washing steps.
Further advantages include lower
consumption of organic solvents
(compared to conventional LLE),
high recovery, removal of nonvolatile
salts, ease of operation, and ease of
automation (1,16,17).
Currently, SPE is most widely
performed in a conventional way using
SPE cartridges. Other formats involve
SPE discs and well plates. An SPE
extraction protocol involves several
steps, including sorbent activation
and conditioning, sample load, a
washing step, and, finally, elution of
the analytes. It is performed using
an SPE vacuum manifold, and so the
requirements on equipment are slightly
higher than in LLE or PP. It is important
to point out that manual SPE is quite a
time-consuming, multistep procedure,
especially when the evaporation of
SPE eluate and its reconstitution in
mobile phase are needed. SPE is
also relatively expensive because the
cartridges are manufactured
for single use only and require
relatively large amounts of organic
solvents. Therefore, modern
development in SPE is focused on
miniaturization (see “Microextraction
Techniques Based on SPE”),
automation (see “On-Line Sample
Preparation Techniques”), enhanced
selectivity (see “Sample Preparation
with High Selectivity”), and new
materials (see “New Materials in
Sample Preparation”) (1,7).
Because phospholipids are an
important source of matrix effects,
the use of hybrid precipitation and
SPE plates for the simultaneous
removal of precipitated proteins and
phospholipids has recently become
popular (18).
Modern Advances
in Sample Preparation
The research and development
of modern sample preparation
techniques is focused on
miniaturization and facilitation of the
sample preparation step. One of
the main goals is to decrease both
sample and solvent volumes, to be in
agreement with the green principles in
chemistry (19). Further requirements
include simpler equipment, a
reduction of handling steps, and a
shortening of sample preparation time,
with the final objective of reducing
sample preparation cost and the
susceptibility to errors (1,7,8). As
ultrahigh-performance LC (UHPLC)
and other fast-LC approaches are
now replacing conventional high
performance LC (HPLC) methods,
Advances in Pharmaceutical Analysis October 201610
Nováková
Figure 1: (a) Schematic illustration of the extraction well using a supported liquid membrane. (b) PALME setup: (A) 96-well donor plate, (B) acceptor plate with supported liquid membranes, and (C) and lid. Photo provided by Astrid Gjelstad, University of Oslo, Norway.
there is a need for fast and high
throughput sample preparation
techniques.
Microextraction Techniques Based
on LLE: Microextraction approaches
based on liquid-phase extraction are
a very extensive group of techniques.
They may use very simple devices,
quite a complex setup, or membrane
assistance to achieve better phase
separation (20). These techniques
provide important benefits such
as almost solventless extraction
and very high preconcentration
factors (21). Basic classification of
the liquid-phase microextraction
techniques based on the extraction
principle involves single drop
microextraction (SDME), dispersive
liquid–liquid microextraction (DLLME),
and membrane-supported–LLE.
The membrane techniques include
various hollow-fibre liquid-phase
microextraction (HF-LPME)
configurations (22) and its advanced
variant electromembrane extraction
(EME) (21). The last development
of membrane supported-LLE is
represented by parallel artificial
liquid membrane extraction
(PALME). Despite a growing number
of applications of liquid-phase
microextraction approaches in the
scientific literature, the adoption in
routine bioanalytical laboratories
is much slower. Indeed, their use
may be limited in routine bioanalysis
because of the following drawbacks:
manipulation with the devices may
require skilful operation; method
development may be time-consuming
in more complex setups; longer
extraction times compared to
other techniques to accomplish
the same task; and only a small
preconcentration factor obtained
from a low amount of biological
sample compared to other matrices,
where the enrichment factors
can be much higher. Among the
discussed LLE-based microextraction
approaches, PALME (23,24), EME (25)
— both in 96-well plate format — and
DLLME show the greatest potential
in bioanalysis. The latter has been
more widely applied in many fields
and also in bioanalysis, particularly
because of its speed and ease of use,
which is unique among the LLE-based
microextraction approaches (20).
To perform DLLME, an immiscible
solvent and a miscible disperser
solvent are injected into an aqueous
sample, leading to the formation of
a cloudy solution of fine droplets of
an extraction solvent. As a result of
the high surface contact between
extraction solvent droplets and
aqueous samples, high recovery and
enrichment factors are obtained even
when using very small volumes of the
extraction solvent (20).
PALME has been recently
developed to take advantage of
clean extracts obtained when using
back-extraction and to facilitate the
automation of the LLE procedure
(23,24). Similar to HF-LPME, the
analytes are extracted with the help of
a supported liquid membrane (SLM).
The extraction setup is composed of
a 96-well donor and acceptor plates
(Figure 1). The acceptor plate contains
96 artificial membranes that are
impregnated with an organic solvent
to create the SLMs. The pH gradient
across the SLM serves as the driving
force for mass transfer. The aqueous
acceptor solution can be directly
analyzed with LC–MS/MS (23).
EME is another technique using
a similar setup to the one used in
HF-LPME (SLM) with the addition of a
power supply and two electrodes. A
power supply provides d.c. potential
to enhance the extraction rate of
ionizable analytes from the donor
solution (sample) to the acceptor
phase, which is contained in the
lumen of a hollow fibre (25).
Microextraction Techniques Based
on SPE: Microextraction approaches
based on SPE involve three different
groups of extraction techniques
using diverse extraction principles. A
miniaturized version of SPE is applied
in microextraction by packed syringe
(MEPS) and μ-SPE in pipette tips
(μ-SPE-PT). In MEPS, the extraction
is made using a specially adapted
syringe handled manually or with an
automated analytical syringe (26,27).
The solid packing material (1–4 mg)
is inserted into the barrel of a syringe
as a plug or between the needle and
the barrel as a cartridge. In μ-SPE-PT,
the sorbent is firmly placed into
the pipette tip. Both commercially
available or laboratory-made sorbents
can be used for extraction. A great
advantage of the latter is the option
to tailor-make sorbents using a
combination of different chemistries
(Figure 2). This approach originates
from proteomics and is sometimes
designated as STop And Go
11www.chromatographyonline.com
Nováková
(a) (b)
(c)
Figure 2: Microextraction using μ-SPE-PT with laboratory-made pipette tips. (a) Preparation of the pipette tips. (b) Resulting μ-SPE-PT tip containing three combined sorbent layers: C18-polymer-CAX. (c) Extraction procedure using centrifuge as a driving force. Photos and figure provided by Ota Blahoušek and Ondrej Novák, Palacky University Olomouc.
Extraction tips (StageTips) (28,29,30).
In μ-SPE-PT extraction procedures,
pipetting or centrifugation may be
used as a driving force to perform
the individual steps of SPE. The latter
enables simultaneous treatment of a
large number of samples (Figure 2[c]).
In both μ-SPE-PT and MEPS
techniques, the same protocol used
for conventional SPE is applied with
much lower sample and solvent
volumes. Straightforward method
development (a result of the similarity
with conventional SPE) is another
very important benefit. It is easy
and straightforward to automate the
procedure in μ-SPE-PT, but somewhat
more challenging for MEPS, requiring
a dedicated autosampler.
The second approach, which is
called dispersive SPE (d-SPE), is
used in μ-dSPE-PT or in QuEChERS
(quick, easy, cheap, effective, rugged,
and safe) extractions. Compared
to the previous approach, it differs
in the sorbent placement and the
extraction process. In μ-dSPE-PT,
the extraction also takes place in the
standard pipette tips. The sorbent
is loosely placed between the two
frits. This variant is also designated
as disposable pipette tips extraction
(DPX). However, sorbent price is
higher and stationary phase choice is
more limited compared to traditional
SPE (31). Individual extraction steps
are performed using a standard
pipette, which allows the sorbent
to be dynamically mixed with the
extracted material and leads to a rapid
adsorption equilibrium between solid
phase and the analyte. In this way,
every sorbent particle actually faces
analyte several times. This is contrary
to conventional SPE, where a greater
amount of sorbent is needed because
the analyte comes into contact with
the sorbent particles only once. This
extraction procedure leads to fast and
efficient extractions (8).
The third solid-phase–based
extraction principle is different from
conventional SPE. Here, adsorption,
absorption, and desorption processes
take place on the solid sorbent,
allowing the need for solvents to be
completely eliminated. The sorbent
is placed on the fused-silica fibre in
the modified syringe in solid-phase
microextraction (SPME) (32,33), in
a piece of capillary in in-tube SPME
(32), or on the stir bar in stir-bar
sorptive extraction (SBSE) (34).
In SPME, the coated fibre can be
moved in and out using a plunger.
Using such simple equipment, all
steps — extraction, preconcentration,
derivatization, and transfer to
the chromatographic system —
are integrated into one device.
Various materials such as PDMS
(polydimethylsiloxane) or many other
materials may be used for the fibre
coating. New types of SPME fibres
use biocompatible coatings prepared
by immobilizing various sorbents with
polyacrylonitrile (35). Since 1990,
when SPME was first introduced,
a series of modifications and
improvements have been proposed.
Initially, either direct immersion
(DI-SPME) or head-space fibre
(HS-SPME) were applied; however,
in vivo SPME sampling devices
and fully automated 96-well format
multifibre SPME are now preferred
(33). The main benefits of SPME
include no need for solvents, the ease
of automation, minimal equipment
requirements, good linearity, and
relatively high sensitivity. Some of
the drawbacks include a longer time
needed for extraction, limited capacity
and fragility of SPME fibre, generally
lower recoveries than those using
LLE and SPE, and the incidence of
carryover effects. The use of modern
SPME in routine laboratories is still
limited.
On-Line Sample Preparation
Techniques: The use of on-line
sample preparation techniques
reduces sample manipulation and
provides high preconcentration
factors, recoveries, high speed, and
throughput. SPE using 96- or 384-well
plate formats in fully automated robotic
workstations, semi-automated, or in
an off-line configuration is therefore
the most well-established technique in
bioanalysis (36,37).
On-line SPE technology in
combination with LC–MS/MS analysis
has experienced many developments
in recent years and enables faster
and precise determination in both
conventional and miniaturized
arrangements (see “Microextraction
Techniques Based on SPE”). On-line
hyphenation is accomplished using
switching valves and an additional
pump. The experimental parameters
to be optimized in on-line SPE are the
type of sorbent, the solvents used
in the different SPE steps, and their
flow-rates (extraction time). In addition,
further pretreatment, such as filtration,
centrifugation, or protein precipitation,
might sometimes be required prior to
on-line SPE. Other constraints may
include reduced sorbent capacity,
too strong retention, slow kinetic of
the sorption process, and possible
absorption of the analyte on the system
tubes (9,36,38). Specific approaches
to remove macromolecules from the
biological fluids include molecularly
imprinted polymers (MIPs),
restricted-access media (RAM), and
turbulent flow chromatography (TFC).
RAM sorbents are used for the
direct injection of biological fluids
into a chromatographic system to
enable the fractionation of a biological
Advances in Pharmaceutical Analysis October 201612
Nováková
Complex
formation
Cross-linker
Polymerization
Monomer
+
+Template
Template
Template
Template
removal
Figure 3: Schematic procedure of the synthesis of a molecularly imprinted polymer (MIP).
sample into a protein matrix and analyte fraction. This will
lead to the extraction and enrichment of low molecular
compounds into the interior phase via partition. The
exclusion of macromolecules can be accomplished using
the outer surface of the RAM particle as a physical or a
chemical barrier. The use of RAM sorbents for the direct
and repeated analyses of biological fluids is already well
established. However, the complete elution of the analytes
from RAM on the analytical column might sometimes be
quite challenging (36,39).
TFC enables the direct injection of biological fluids and the
separation of small analyte molecules from the macromolecular
matrix based on the low-diffusion coefficients of proteins
using turbulent flow. The generation of turbulent-flow requires
short, narrow-bore columns, packed with large-size particles
(typically 50 mm × 1.0 mm, 20–60 μm) and flow-rates in the
range of 4–5 mL/min. High-molecular-weight compounds are
quickly eluted using a pure water or buffer mobile phase and
are usually directed to the waste. The retained small molecular
weight analytes are subsequently eluted onto an analytical
column for the chromatographic separation using an organic
mobile phase (36).
To increase the limited capacity and facilitate its
automation, on-line coupling of SPME with separation
techniques has been established and termed in-tube
SPME. In this setup, a capillary column is placed as the
injection loop in a standard autosampler. While the typical
advantages attributed to the on-line setup are obtained
with this technique, there are also several drawbacks,
such as the need for more complex instrumentation with
commercially unavailable adjustments; a requirement
for very clean samples, because the capillary can be
easily clogged; low extraction efficiency; selectivity;
and mechanical stability. To overcome these issues,
development is focused on coupling in-tube SPME with
miniaturized LC techniques and the preparation of new
extraction phases (36,40,41).
Sample Preparation with High Selectivity: The selectivity
of SPE can be further improved using specific sorbents,
such as MIPs, immunoaffinity sorbents, or aptamers.
MIPs are stable synthetic polymers possessing
tailor-made recognition sites, which can specifically retain
target analyte molecules. The preparation of MIPs involves
the copolymerization of a complex formed by the template
molecule and a functional monomer with a cross-linking
agent in the presence of a suitable porogenic solvent
(Figure 3). After removing the template, the resulting cavity
is complementary to the target analytes in terms of size,
shape, and functionality. An SPE cartridge is the most
popular form of MIP used in sample preparation. However,
growing interest in miniaturization has led to the use of MIPs
in SPME, SBSE, MEPS, μ-SPE, membranes, and magnetic
beads. High extraction selectivity and capability to
eliminate the matrix effects are definitely the most important
benefits of MIPs. Other advantages include reusability,
ease of use, and low cost of preparation. Some features
that need to be improved in future research include the
need for higher yields of specific binding sites, the need for
some rules for MIP design, and the application of MIPs to
aqueous samples (42,43).
Immunoaffinity-based SPE (IA-SPE) uses sorbents
with attached antibodies to obtain high selectivity from
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antigen–antibody interaction.
For small molecules, the target
analyte is typically coupled to the
carrier protein (hapten) to achieve
an immune response. While the
cross-reactivity of antibodies with
structural analogues is considered as
a negative feature in immunoassay,
it is exploited in immunoaffinity
extraction. An important limitation
of IA-SPE is the difficult, expensive,
and time-consuming synthesis
of antibodies with no certainty of
success, which probably prevents
wider use of IA-SPE in routine
bioanalytical laboratories (44).
Aptamer-functionalized
materials (AFMs) are promising,
specific-recognition materials in
sample preparation, providing
many advantages such as high
specificity and binding affinity,
good stability, low cost, nontoxicity,
ease of synthesis, and easy and
controllable modification. Aptamers
are the artificial single-stranded
oligonucleotides generated by an in
vitro selection process called SELEX
(systematic evolution of ligands by
exponential enrichment). By folding
into distinct secondary or tertiary
structures, aptamers can bind to
certain targets with extremely high
specificity. So far, they have been
widely used in biosensors or in
immunoassays, while their use in
sample preparation in bioanalysis is
still at the research stage (45,46).
New Materials in Sample
Preparation: New types of materials
have been introduced in line with the
green analytical chemistry concept.
Among them are sol-gel-based
materials, which allow inorganic and
organic-inorganic hybrid polymers
with typically higher thermal and
chemical stability, controlled
morphology, surface properties, and
pore structures to be obtained due to
the controlled synthesis conditions.
These sorbents are successfully used
in SPE, SBSE, SPME, and MEPS (47).
Ionic liquids (ILs) are used to
enhance extraction efficiency and
selectivity in LLE-based procedures
and as sorbents in SPE-based
techniques. They are liquid salts at
room temperature, with a melting point
lower than 100 ºC. Their structure
consists of organic cations derived
from Lewis bases and polyatomic
anions. The addition of different
structures enables their hydrophobic or
hydrophilic abilities to be tailored (47).
Along with the development of
nanotechnology, various nanomaterials
have been introduced into the sample
preparation domain. Nanomaterials
refer to a special kind of materials
with nanometric scales. Compared
to conventional materials, some
exceptional properties, such as ultrahigh
specific area and increased surface
activity, are facilitating the application
of nanomaterials in sample preparation.
Further advantages include tunable
compositions, various morphologies,
and flexible functionalization.
Nanomaterials used in sample
preparation include nanoparticles and
nanoporous materials (48,49).
Magnetic separation techniques
(Figure 4) (50) are an interesting
approach used in sample preparation
and provide several advantages, such
as efficient, gentle, and nondestructive
separation, especially for large
molecules. Magnetic separation
techniques are therefore able to
facilitate or accelerate many separation
and purification procedures because
of rapid isolation of analytes using an
external magnetic field (51,52).
Dried Matrix Spot Analysis:
Dried matrix spot (DMS) analysis
has recently gained attention in
bioanalytical routine laboratories.
Although dried blood spot (DBS)
analysis is the most widely used
(53,54), other matrices, such as dried
saliva spots (DSS) (55), dried plasma
spots (DPS) (56), dried urine spots
(DUS), and even cerebrospinal fluid
(CSF) sample spots (57), have been
successfully analyzed.
The DBS and other matrix spot
sampling is minimally invasive and
uses substantially lower sample
volumes (<100 μL for four spots)
compared to other procedures. DBS
uses capillary blood from a finger
prick with a lancet. After collection,
the samples are dried on the special
sample collection cards. As the
samples are collected, transported,
and stored in dry form, the handling
and storage of such biological
material becomes easier and may
lead to increased stability of some
unstable compounds. For subsequent
analysis, the disk is punched out from
the spot and this disk is extracted for
the isolation of target analytes, which
is typically followed by LC–MS/MS.
Despite some challenges,
such as proper DBS calibration,
analyte stability, the influence of
hematocrit on blood spot size,
and the need for clinical validation
because of the differences between
venous, whole blood, and plasma, its
wider use in bioanalysis is expected
in the future, as it is a fast, relatively
simple, and straightforward sample
preparation technique (53,54).
The importance of microsampling
in bioanalysis is confirmed by the
development of a new microsampling
Advances in Pharmaceutical Analysis October 201614
Nováková
Vortex Magnetic separation
Elution
Discard liquid
supernatant
HPLC
analysis
MNPs ADR Impurities Magnet
Figure 4: A schematic procedure of magnetic SPE. Extraction of Adriamycin hydrochloride from human plasma. Adapted with permission from reference 50.
approach termed volumetric
absorptive microsampling (VAMS).
This approach should compensate
for the area bias and homogeneity
issues associated with conventional
DBS (58,59).
Conclusions
Modern method development in
bioanalysis is focused on speed
of analysis, efficiency, selectivity,
sensitivity, low cost, miniaturization,
and automation to obtain high sample
throughput and high quality data.
While significant improvements have
been made in instrumental methods
— both in chromatographic separation
and MS/MS detection — the sample
preparation step still remains the most
time-consuming and labour-intensive
aspect of a bioanalytical method,
particularly when compared to
ultra-fast chromatography. The efforts
to improve this situation have resulted
in the development of many new
sample preparation techniques, which
have helped to overcome some of the
drawbacks of the conventional sample
preparation techniques.
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Rogerg-Larsen, C. Aass, and S.R. Wilson,
J. Pharm. Biomed. Anal. 87, 120 (2014).
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J. Chromatogr. B 801, 141 (2004).
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P. López-Mahía, S. Muniategui-Lorenzo,
and D. Prada-Rodríguez, Anal. Chim. Acta
906, 41 (2016).
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J. Verdú-Andrés, and C. Molins-Legua,
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Tan, Trends Anal. Chem. 39, 72 (2012).
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J. Li, and G. Li, Trends Anal. Chem. 67, 134
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Domingues, and F.M. Lanças, Trends Anal.
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Pharm. Biomed. Anal. in press (2016).
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(2015).
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Lucie Nováková is an associate
professor in the Department of
Analytical Chemistry, Charles
University, Faculty of Pharmacy in
Hradec Králové, Czech Republic.
She has a Masters degree in
pharmacy and obtained her Ph.D.
in pharmaceutical analysis from
Charles University, Faculty of
Pharmacy. Her research is focused
on fast LC and SFC techniques,
especially UHPLC, UHPSFC, and
their coupling to MS. She is involved
in a wide variety of research projects
focused on pharmaceutical analysis,
plant analysis, doping control, and
bioanalytical methods. An important
part of her research therefore lies in
the sample preparation step, where
the focus is put on the current trends
enabling facilitation, miniaturization,
and reduction of time and sample
requirements. She has published over
75 scientific articles with about 1650
citations and is widely involved in
teaching and education activities, such
as HPLC and SFC training courses,
seminars, and congresses.
15www.chromatographyonline.com
Nováková
The concept of quality by design
(QbD) was first introduced in 2004 by
the US Food and Drug Administration
(FDA), as an essential pharmaceutical
quality model to be considered in
the development of new products
and processes (1). Later, QbD
was supported by the International
Conference on Harmonization (ICH).
Since the adoption of the ICH Q8
(R2) guideline encouraging the
implementation of a QbD approach
in pharmaceutical development, QbD
has become a key methodology in the
pharmaceutical industry (2). Indeed,
the number of publications discussing
QbD has exponentially increased over
the last 10 years, as shown in Figure 1.
In the ICH Q8 (R2) guideline, QbD
is defined as “a systematic approach
to development that begins with
predefined objectives and emphasizes
product and process understanding
based on sound science and quality
risk management” (2). The main
purpose is to improve the quality of
pharmaceutical products and ultimately
patient safety by designing quality
directly into pharmaceutical processes.
Processes are better understood
compared to the quality-by-testing
(QbT) approach, which is traditionally
implemented in the pharmaceutical
industry. In fact, QbT does not ensure
product quality since processes and
products cannot be sufficiently known
and controlled.
A QbD approach is mainly
applied to the development of
new pharmaceutical formulations,
as encouraged by the specific
guideline (2). However, in the ICH
Q8 (R2) guideline, the need to
gain a greater understanding of
all processes involved in product
development to achieve a proper
control strategy is outlined. By
supporting drug development
and quality control (QC) activities,
analytical procedures form an integral
part of pharmaceutical processes.
Analytical methods play a key role
throughout a drug product life cycle
because they ensure efficacy and
safety of a pharmaceutical product
by including scientific and regulatory
knowledge, as well as QC needs.
As a result, QbD is being adopted
more and more by pharmaceutical
and biopharmaceutical analysts to
achieve a higher quality of analytical
methods and thus a higher quality of
the pharmaceutical product. The ICH
Q8 (R2) guideline is completed by the
ICH Q9 (3) and Q10 (4) guidelines,
which provide a comprehensive
overview of the basic tools that can be
used to effectively build quality into
products.
This article presents examples
of applications of QbD in the
development of separation methods
for pharmaceutical analysis. The
global methodology of QbD is
summarized, followed by a focus on
some essential tools and concepts
related to QbD strategy. Finally,
this article concludes on how QbD
stands out as a powerful tool for the
pharmaceutical analyst.
Applications
Table 1 classifies the use of QbD
for the development of separation
methods dedicated to pharmaceutical
analysis in four main applications: the
simultaneous analysis of an active
pharmaceutical ingredient (API) and
its related substances; multimolecule
separation; enantioselective
separation; and natural product
analysis. All of these applications are
encountered in the pharmaceutical
industry and are challenging because
of the simultaneous analysis of an
important number of molecules and
the difficulty of separating molecules
with closely related properties.
Related substance analysis
represents one of the most
challenging analytical applications
required in QC and research
and development activities. As
these methods are included in
the Common Technical Document
(CTD) of a pharmaceutical product,
the robustness of the method is
an added value. Samples to be
analyzed can be highly complex
because of the combination of
unreacted starting materials,
impurities originating from starting
materials, unreacted intermediates,
reaction byproducts, and degradation
products. Compounds with closely
related structures are present at
very different concentrations in the
Quality by Design: A Tool for Separation Method Development in Pharmaceutical Laboratories
Karen Gaudin and Ludivine Ferey, School of Pharmaceutical Sciences, Bordeaux University, Bordeaux, France
Quality by design (QbD) has gained in importance in the pharmaceutical industry and is supported
by several regulatory documents (ICH, FDA). The aim is to ensure product quality through a better
understanding of products and processes during pharmaceutical development. As analytical procedures
are critical processes of pharmaceutical product development and quality control (QC), QbD has become
a key tool in the development of analytical methods. This article outlines the general trends observed
when applying QbD to the development of separation methods in pharmaceutical analysis. The main
pharmaceutical applications are reviewed along with a detailed description of tools involved in QbD
methodology. A focus on QbD benefits for the pharmaceutical industry is provided.
Ph
oto
Cre
dit: R
alf H
iem
isc
h/G
ett
y Im
ag
es
Advances in Pharmaceutical Analysis October 201616
samples, making the development of
separation methods difficult. However,
these impurities usually provide no
benefit to patients and can actually
constitute risks to patient safety or
drug efficacy. There is a great need
therefore to be able to control them
during the manufacturing process
and in the final product. To deal with
such expectation, a QbD strategy
has proved to be appropriate. For
the study of related compounds
in a QC context, the most current
techniques coupled to QbD are high
performance liquid chromatography
(HPLC) and ultrahigh-pressure
liquid chromatography (UHPLC)
in reversed-phase mode (5–13).
However, as QbD provides a better
understanding of the development
process, more complex and recent
techniques that suffer from robustness
problems (such as, hydrophilic
interaction liquid chromatography
[HILIC] and capillary zone
electrophoresis [CZE]) could take
advantage of this approach (14–18).
The second important
pharmaceutical application using QbD
is the separation of several APIs for
the detection of counterfeit medicines
(19–23) or for homologous molecule
analysis in QC (24). When a high
number of compounds (above 10)
needs to be simultaneously separated,
the method must be specific and
robust to distinguish one API from
another. QbD is the most appropriate
tool to speed up the development of
such a method, while also improving
knowledge acquired on the analytical
method.
Enantioselective separations
represent a fundamental field of
research in drug analysis because
the potential biological activity of
chiral compounds is mostly down to
one enantiomer only. Optical purity,
that is, enantiomeric excess (ee),
determination represents a major
challenge for the analyst. Indeed,
the main enantiomer is the active
one and is present in high amounts
in the sample, whereas the impurity
enantiomer is expected to be under
0.1% of the main enantiomer. To
quantify the chiral impurity, the
complete separation of the two
enantiomers is required with a low
limit of quantitation for the impurity
enantiomer. However, enantiomers
have closely related physico-chemical
properties, making the separation
challenging. CZE is a powerful
technique for chiral analysis because
of its high efficiency, fast analysis,
and the low amounts of chiral selector
required. Cyclodextrins are the most
commonly used chiral selectors in
CZE. The association of QbD with
CZE proves that robust methods for
enantioseparations can be achieved
(25,26).
The final application combining
QbD and separation method
development in pharmaceutical
analysis is the characterization of
natural products. Traditional medicine
still holds an important place in curing
disease in developing countries. This
also includes occidental countries
with a greater use of phytotherapy
and an increased consumption
of natural medicines and food
supplements. Efficient analytical
methods are required for structural
identification, classification of
varieties or geographical origin, and
purification purposes. The safety and
quality aspects of finished herbal
medicinal products depend on source
materials of high complexity and
variability because they can include
hundreds of constituents. Fast,
selective, and accurate analytical
methods are therefore needed. QbD
can manage this natural heterogeneity
to provide the best controlled
separations (27–29). In addition, the
optimization offered by QbD can
combine separation parameters
with those of the extraction that may
be intercorrelated (27). Therefore,
the optimum condition and the
design space are designed taking
into account the influence of both
separation and extraction methods.
The main use of QbD in the
optimization of separation method
development lies in the separation
of samples composed of a large
number of molecules. This approach
17www.chromatographyonline.com
Gaudin and Ferey
ICH Q8 (R2)
Pharmaceutical development
2009
1990
250
200
150
100
50
0
1995 2000 2005 2010 2015
Final concept paper
ICH Q8 Pharmaceutical development
2003
Year
Nu
mb
er
of
pu
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tio
nFigure 1: Evolution of the number of publications based on the QbD approach following its acceptance by regulatory authorities. Source: Scopus search of the chemical abstracts database from 1990–2015.
QbDstrategy
Qualitycontrol
Method goals
Method selection
Risk assessment
Knowledge space
Design space
Method validation
and control
strategy
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Figure 2: QbD approach for analytical method development.
takes advantage of the design of
experiments to find the optimum
method for the separation and to
provide method robustness. QbD
is a powerful tool that can offer a
guarantee of separation quality.
QbD Methodology
Traditional univariate methods vary
only one factor at a time (OFAT)
between consecutive experiments.
Such method development strategies
can lead to nonoptimized methods
and offer no warranty of robustness.
On the contrary, by conducting a
systematic development, QbD allows
optimal method performance to be
obtained.
An implementation of the QbD
approach during analytical method
development involves several steps,
as described in Figure 2.
QbD requires the determination of
predefined objectives of the analytical
method, called the analytical target
profile (ATP). The intended purpose
of the method can be stability testing,
quantitative determination, or a limit
test. This means that the goals of the
method — including its performance
—must be precisely described before
the development. For separation
methods, the objectives could be
described as the separation of
compounds with an identification
or quantification purpose, with
no specification of the separation
technique at this step.
The method is selected based on its
ability to fulfill the objectives specified
in the ATP. A significant amount of
knowledge of analytical technologies
and the drug helps with the selection
of an appropriate method. Through
method scouting experiments and
prior knowledge, the most suitable
analytical technique is chosen: HPLC,
UHPLC, or CZE. At the same time,
Critical Quality Attributes (CQAs)
are selected. They are defined in
ICH Q8 (R2) as “physical, chemical,
biological, or microbiological property
or characteristic that should be
within an appropriate limit, range,
or distribution to ensure the desired
product quality” (2). These elements
convert the intended purpose
of the method into performance
criteria. Therefore, the CQAs are a
measure of method performance in
accordance with the ATP — in other
words, the method specifications.
In chromatographic methods,
resolution of the critical pairs
(5,7,8,17,18,21,25), time differences
between chromatographic peaks
(19,20,22), analysis time (24–26),
and even robustness as allowed by a
QbD approach (5,21) are examples of
CQAs to evaluate separation quality.
Once the CQAs are selected, the
method under development follows a
Advances in Pharmaceutical Analysis October 201618
Gaudin and Ferey
1
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Figure 3: Response-surface designs for three factors: (a) central composite design (at least 15 experiments), (b) Box-Behnken design (at least 13 experiments), and (c) Doehlert design (at least 13 experiments).
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“ChromasterUltra Rs
with ChromSwordAuto 5
method development
platform exclusively
available through
VWR.”
Solvent switching
valves
Easy to maintain
6 column switching valve
Ideal for use with
QbD method
development software
ChromSwordAuto 5.0
Continuing the Hitachi tradition of
manufacturing highly robust HPLC
systems but now with additional
novel new technologies. The
ChromasterUltra Rs effortlessly
delivers a top class performance
and the fexibility needed for
today’s laboratory.
quality risk assessment to investigate
Critical Process Parameters (CPPs).
CPPs are “parameters whose
variability has an impact on CQAs
and therefore should be monitored
or controlled to ensure the process
produces the desired quality” (2).
Risk assessment tools can be used
to identify and rank parameters with
a potential impact on method quality,
based on prior knowledge and
initial experimental data. A fishbone
diagram, also known as Ishikawa
diagram, identifies potential factors
and classifies risks associated with
these factors into groups related
to instrumentation, materials,
methods, chemicals and reagents,
operators, and laboratory environment
(5,9,30,31). Risk factors can be
further ranked and prioritized through
a Failure Mode and Effect Analysis
(FMEA) approach (32). This approach
guarantees that all parameters of the
analytical method were listed and their
influence evaluated.
High-risk parameters, known as
Critical Process Parameters (CPPs),
are then assessed experimentally
through screening designs, which are
first-degree Design of Experiments
(DOEs). For separation methods,
mobile phase or buffer pH often
appears as a CPP when ionizable
compounds have to be separated,
since pH has an impact on resolution
between compounds (5,8–20,25,26).
Others factors identified as CPPs are
flow rate (6,9,27); gradient slope (5,
7–13,19–23,28,29) in reversed-phase
HPLC; percentage organic solvent
in the initial and final mobile phases
(6,10,14,15,28,29) in reversed-phase
HPLC and HILIC–HPLC; buffer
concentration (14–18) in HILIC–HPLC
and CZE; and column or capillary
temperature in HPLC and CZE. During
this stage, the knowledge space is
fixed, which corresponds to the range
of CPP variation.
DOEs based on higher-order
polynomial models are then
performed to optimize CPPs through
a response-surface methodology, that
is, the modelling of the CQAs as a
function of the CPPs.
The main goal of a QbD approach
is, in fine, to define the Method
Operable Design Region (MODR)
or the Design Space (DS). This is
the multidimensional subspace of
the experimental domain where
assurance of analytical method
quality is provided. This domain
is determined using a desirability
function. Further information about
the concept of DS for analytical
methods is provided later in the
article. A detailed review on DS
for analytical methods is also available
in the literature (33). The final step
of the QbD approach is the
validation of the DS to prove
experimentally that the ATP is
achieved inside the DS. The range of
each CPP is thus validated and the
robustness domain of the achieved
method is described.
To use the optimized method
routinely in QC laboratories, a formal
validation is necessary (34–36).
The final step is the implementation
of a control strategy to ensure
that the method will perform as
expected during its routine use
(4). Some responses measuring
method performance, known as
system-suitability criteria, are selected
and monitored at each analytical run
during routine applications. Therefore,
risk assessment and DS definition
involved in QbD approach can help
to identify an appropriate control
strategy (36).
Advances in Pharmaceutical Analysis October 201620
Gaudin and Ferey
4 5
55
50
45
40
35
30
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40
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Imp. A efficiency
Imp. A efficiency
Imp. A efficiency
Imp. A efficiency
Imp. A efficiency
EtOH volume
EtOH volume
EtOH volume
EtOH volume
EtOH volume
EtOH volume
Mobile phase volume
Mobile phase volume
Mobile phase volume
Mobile phase volume
Mobile phase volume
Mobile phase volume
Imp. C efficiency
Imp. C efficiency
Imp. C efficiency
Imp. C efficiency
Imp. C efficiency
Rs DXM/Imp.A
Rs DXM/Imp.A
Rs DXM/Imp.A
Rs DXM/Imp.A
Rs DXM/Imp.A
Rs DXM/Imp.C
Rs DXM/Imp.C Rs DXM/Imp.C
Rs Imp. A/Imp. C
0.40.25 0.35
Flow rate Flow rate
0.2 0.3 0.40.25 0.35
0.2 0.3 0.40.25 0.35
Flow rate
2 3 42.5 3.5
Gradient slope
2 3 42.5 3.5
Gradient slope
Temperature
Tem
pera
ture
Tem
pera
ture
30 40 5035 45
2 4
44
Figure 4: Contour plots of the seven CQAs of interest (Rs DXM/Imp.A, Rs DXM/Imp.C, Rs Imp.A/Imp.C, Imp.A efficiency, Imp.C efficiency, mobile phase volume, and EtOH volume) as a function of the four studied CPPs. In the white zone, all CQAs fulfill the requirements (Rs >2.5, Imp.A >3000 theoretical plates, Imp.C >30,000 theoretical plates, mobile phase volume <1.2 mL, and EtOH volume <0.35 mL). Adapted with permission from reference 9.
21www.chromatographyonline.com
Gaudin and Ferey
Table 1: Applications of QbD to the development of separation methods in pharmaceutical analysis
API + Related Substances Analysis
Technique Analyte Matrix Modelling ApproachDesign Space
Determination/ValidationReference
Reversed-
phase
HPLC–DAD
Abacavir, Lamivudine, and Dolutegravir + 11 related substances + excipients
In-house manufactured tablets
Central composite designFactors: pH, column temperature, gradient slope3 levels
Monte Carlo simulationsDetermination of DS through process capability index calculation
(5)
Reversed-
phase
HPLC–DAD
Pitavastatin calcium + 15 related substances
Forced degradationLivalo tablets
Central composite designFactors: % solvent, column temperature, flow rate5 levels
Desirability plotsValidation of DS through 2 verification trials, one fixing all the factors at the higher ranges and another fixing all the factors at the lower ranges
(6)
Reversed-
phase
UHPLC–DAD
Ebastine + related substances
Oro-dispersible tablets
Full factorial designFactors: gradient time, 2 levels, column temperature, 2 levels, ternary composition of the organic eluent, 3 levels, 12 experiments (2 × 2 × 3)
3D resolution mapsValidation of DS through 6 experiments visually selectedSimulated robustness testing (729 experiments) by varying 6 factors
(7)
Reversed-
phase UHPLC–
DAD
Omeprazole + related substances
Standard
Full factorial designFactors: gradient time, 2 levels, column temperature, 2 levels, pH, 3 levels12 experiments (2 × 2 × 3) in two organic solvents
3D resolution mapsValidation of DS through 6 experiments visually selected Simulated robustness testing (729 experiments) by varying 6 factors
(8)
Reversed-
phase
UHPLC–DAD
Dextromethorphan + impurities + excipient
Syrup
Central composite designFactors: flow rate, gradient slope, pH, column temperature5 levels
Response surface overlayValidation of DS through a PB design
(9)
Reversed-
phase
UHPLC–UV
Mixture of benzalkonium chloride
Preserved drug formulation
Stepwise optimization using commercial softwareFactors: pH, gradient slope, % solvent in initial and final mobile phases
Response surface overlayRobustness validation of optimum during validation
(10)
Reversed-
phase
UHPLC–DAD
Amlodipine besylateBisoprolol fumarate+ related substances
Standards
Full factorial designFactors: gradient time, 2 levels, column temperature, 2 levels, pH, 3 levels12 experiments (2 × 2 × 3)
3D resolution mapsSimulated robustness testing (729 experiments) by varying 6 factorsand 3 experiments fixing worst case conditions
(11)
Reversed-
phase
UHPLC–DAD
2 active pharmaceutical ingredients + 9 impurities
Eye drop solution
Full factorial designFactors: gradient time, 2 levels, column temperature, 2 levels, pH, 3 levels12 experiments (2 × 2 × 3)
3D resolution mapsDS validation through 12 experiments by varying the 3 factors at the edge of the DS Simulated robustness testing by varying 6 factors
(12)
Reversed-
phase UHPLC–
UV–TOF-ESI-
MS/MS
Imatinib mesylate + related substances
Forced degradation
Box–Behnken designFactors: gradient slope, column temperature, pH3 levels
Response surface overlayRobustness validation of optimum during validation
(13)
HILIC–
HPLC–UV
Iohexol + related substances
Omnipaque Solution for parenteral use
Box-Behnken designFactors: % solvent, pH, buffer concentration3 levels
Monte Carlo simulationsValidation of DS through a fractional factorial design 2 3-1 Robustness validation of optimum through a PB design
(14)
HILIC–
HPLC–UV
Bilastine + related substances
Nixar tablets
Box-Behnken designFactors: % solvent, pH, buffer concentration3 levels
Monte Carlo simulationsRobustness validation of optimum through a fractional factorial design 2 5-2
(15)
CZE–DADZolmitriptan + 5 related substances
Zomigtablets
Box-Behnken design Factors: buffer concentration, pH, temperature3 levels
Monte Carlo simulationsValidation of DS through a PB design Robustness validation of optimum through a PB design by expanding the number of factors
(16)
CD–CZE–DAD
Metformin hydrochloride + 5 related substances
Metforalcoated tablets
Doehlert design Factors: injection time, 3 levels, anionic CD concentration, 7 levels, buffer concentration, 5 levels, pH, 7 levels
Monte Carlo simulationsValidation of DS through a PB design Robustness validation of optimum through a PB design by expanding the number of factors
(17)
CZE–DADGlibenclamide + 2 related substances
Eugucontablets
Box-Behnken designFactors: voltage, injection time, buffer concentration, pH, 3 levels
Monte Carlo simulationsValidation of DS and robustness of optimum through a 23 full factorial design
(18)
Advances in Pharmaceutical Analysis October 201622
Gaudin and Ferey
Table 1: Contd....
Multimolecule Separation
Technique Analyte Matrix Modelling Approach Design Space Determination Reference
Reversed-
phase
HPLC–DAD
19 antimalarial drugsdivided in two groups
Arteplus pharmaceutical formulation
Full factorial designFactors: pH, 5 levels, gradient time, 3 levels, column temperature, 3 levels
Monte Carlo simulationsOne method for each groupNo robustness validation
(19)
Reversed-
phase
HPLC–DAD
18 non-steroidal anti-inflammatory drugs, 5 pharmaceutical conservatives, paracetamol, chlorzoxazone, caffeine, salicylic aciddivided into five groups
5 capsules from the Democratic Republic of Congo
Augmented central composite designFactors: pH, 7 levels, gradient slope, 5 levels, column temperature, 5 levels
Monte Carlo simulationsOne method for each groupRepeatability at the optimal conditions
(20)
Reversed-
phase
UHPLC–UV
15 antipsychotic drugs
Standards
Full factorial design Factors: column temperature and gradient time3 levels
Monte Carlo simulationsDetermination of DS through robustness criterion Cp calculation Experimental validation of robustness
(21)
Reversed-
phase
UHPLC–DAD
19 antibioticsdivided into 3 groups
Powder for injection
D-optimal experimentsFactors: pH, 6 levels, gradient time, 3 levels, column temperature, 3 levels
Monte Carlo simulationsRepeatability at the optimal conditions
(22)
Reversed-
phase
HPLC–UV
8 antidiabetic drugsGlimepiride plustablets
Full factorial design on 3 columnsFactors: pH, 3 levels, ternary solvent ratio, 3 levels, gradient time, 2 levels
DS through modelling error propagationIn-silico robustness testing
(23)
MEKC–DAD 7 triptans
AlmogranAuradolZomigtablets
Central composite designFactors: SDS concentration, n-butanol and ethanol percentages5 levels
Monte Carlo simulationsValidation of DS through a PB design Robustness validation of optimum through a PB design by expanding the number of factors
(24)
Enantioselective Separation
Technique Analyte Matrix Modelling Approach Design Space Determination Reference
CD–MEKC–
DAD
Ambrisentan + related substances
VolibrisCoated tablets
Central composite face centred designFactors: voltage, CD concentration, pH, 3 levels
Monte Carlo simulationsValidation of DS through a PB designRobustness validation of optimum through a PB design by expanding the number of factors
(25)
CD–CZE–DAD LevosulpirideLevobrenAmpoules
Doehlert design Factors: anionic CD concentration, 5 levels, neutral CD concentration, 7 levels, pH, 7 levels, voltage, 3 levels
Monte Carlo simulationsValidation of DS through a PB design Robustness validation of optimum through a PB design by expanding the number of factors
(26)
Natural Product Analysis
Technique Analyte Matrix Modelling Approach Design Space Determination Reference
SPE/reversed-
phase UHPLC–
UV/ELSD
Isoflavonoids, triterpenoid saponins
Pharmaceutical liquid herbal preparation of Shenqi Fuzheng
Box–Behnken designFactors: flow rate, column temperature, ELSD evaporation temperature, and ELSD gas flow rate3 levels
Monte Carlo simulationsRobustness validation of optimum during validation
(27)
Reversed-
phase
HPLC–UV
13 aporphine alkaloids
Leave extras of Spirospermum penduliflorum Thouars
Full factorial designFactors: pH, 3 levels, gradient slope, 3 levels, methanol proportion at the beginning of the gradient, 4 levels
Monte Carlo simulationsNo experimental validation of DS or robustness of optimum
(28)
Reversed-
phase
HPLC–DAD
7 tertiary alkaloidsLeave extras of Strychnos usambarensis
Full factorial designFactors: proportions of organic solvents in the initial phase, gradient slope, % acetonitrile, 3 levels
Monte Carlo simulationsNo experimental validation of DS or robustness of optimum
(29)
CD: cyclodextrin, DS: design space, ELSD: evaporative light scattering detector, PB: Plackett-Burman
DOE and Response Modelling
DOEs are key components of a
QbD methodology. Compared to the
OFAT approach, DOEs minimize the
number of experiments, while gaining
knowledge about the analytical
method. Such multivariate strategies
allow the evaluation of the effects of
the factors and their interactions, the
modelling and the prediction of the
responses as a function of the factors,
and, in fine, the determination of the
DS. The factors are the CPPs and the
responses the CQAs.
Method optimization often begins
with a screening phase followed by
an optimization step using screening
and response-surface designs,
respectively.
Screening Designs: After a risk
prioritization, where a high number
of factors have been revealed
as potentially critical on method
performance, screening designs are
used to identify which parameters
are effectively critical. The aim is
the selection of CPPs. Such designs
allow the evaluation of a relatively
high number of factors (qualitative
or quantitative) — mostly at two
levels in a relatively low number of
experiments. The relation between
the responses and the factors is
described by a first-order polynomial
model. An estimation of the main
effects of factors on the considered
responses, mainly the CQAs, can
be assessed by calculating model
coefficients for the linear terms.
Factors with the most significant
affect on the analytical method are
retained as CPPs. Interaction effects
between factors are rarely evaluated,
while higher order terms, for example,
quadratic effects, cannot be studied.
Indeed, two-level screening designs
don’t allow modelling of curvature.
Such designs are therefore not
sufficient to investigate a process
involving interactions and higher-order
effect terms. The most often applied
screening designs are two-level
fractional factorial or two-level
Plackett-Burman designs (9,27,37).
Response-Surface Designs: Once
identified, CPPs are further studied
through a response-surface method to
predict CQAs. For a given number of
parameters, many more experiments
are required compared to first degree
screening designs. The analysis of
the results is focused on building
a mathematical model linking the
responses (CQAs) to the factors
(CPPs). More than two levels for each
factor are needed to fit quadratic
or higher-order-term polynomial
functions and, thus, to model the
curvature in the function responses.
Interactions between factors and
higher-order effect terms can then be
evaluated.
Full factorial designs
(7,8,11,12,19,21,23,28,29), central
composite designs (5,6,9,20,24,25),
Box-Behnken (13–16,18,27), and
Doehlert designs (17,26) are
commonly used. Figure 3 shows the
experimental domains resulting from
the study of three factors for central
composite designs, Box-Behnken
designs, and Doehlert designs.
D-optimal designs can also be
performed in particular cases, for
example, when experimental space
is constrained or standard factorial
designs require too many runs (22).
Such DOEs are essential tools
to define the DS of an analytical
method (33). Indeed, the model
enables response values (CQAs)
within the investigated experimental
domain to be predicted. Moreover,
model-parameter uncertainty and
the prediction uncertainty of the
model can be estimated, providing
information on the probability of
meeting the specifications imposed on
CQAs. The resulting DS corresponds
to a subspace of the experimental
domain where a level of quality is
ensured, that is, the CQAs meet the
specifications with a given probability.
Response Modelling: Responses
can be modelled in different ways.
The first approach uses optimization
software for an automated method
development. Such software is mostly
dedicated to reversed-phase HPLC
and based on the well-known linear
solvent-strength theoretical model.
Several software systems are available
commercially (7,8,11,12,38). However,
such theoretical models do not usually
take into account all the parameters
that may impact the analytical method.
Moreover, the variability or the method
quality (the robustness) is not included
in the method development.
A second approach is the use of
empirical models based on DOEs
and multiple linear regressions (MLR).
Such models link the responses to
the factors investigated by fitting an
appropriate polynomial model on the
data obtained from DOEs. Software
used for such empirical approaches
is that classically dedicated to
experimental designs. However, there
is software that is appropriate for QbD
developments by combining DOE,
MLR, and some specific CQAs to
evaluate method robustness (21,39).
Design Space: As mentioned
previously, the main aim in
applying QbD to analytical method
development is to establish a DS.
In the ICH Q8 (R2) guideline, DS is
defined as “the multidimensional
combination and interaction of input
variables (such as material attributes)
and process parameters that have
been demonstrated to provide
assurance of quality” (2).
Most of the time, DS is represented
graphically through response surface
overlay. A graph displaying multiple
CQA objectives is plotted using
software (Figure 4). This example
deals with the development of a
UHPLC method for the simultaneous
analysis of dextromethorphan (DXM)
and its main degradation products
(9). Contour plots of the seven studied
CQAs as a function of the four CPPs
are overlaid and DS is represented by
the white zone where all CQAs meet
predefined criteria.
Up to now, the DS of an analytical
method has been mainly defined
through mean-response surfaces.
However, mean-response surfaces
do not provide any guarantee about
method quality, and so it is impossible
to know with which probability the
CQAs will reach the performance
criteria. As “assurance of quality”
is clearly required by ICH Q8 (R2)
guideline (2), the level of assurance
that the analytical method will meet
the specifications must be known.
This is why a group of researchers
highlighted the need to consider
the prediction uncertainty by taking
into account the model-coefficient
uncertainty, that is, the model
uncertainty (40). The Monte Carlo
study is the most implemented
strategy (5,14–26). By including
model coefficient uncertainty, a
model’s predictive errors can be
calculated. This provides access
to the distributions of the modelled
responses for each operating
condition of the DOE. Consequently,
when establishing the DS, a level
23www.chromatographyonline.com
Gaudin and Ferey
of quality assurance can be set by
imposing a probability threshold
above which the CQAs meet the
requirements. As an example,
Debrus et al. performed Monte Carlo
simulations to compute and model
a “robustness criteria”, called Cp.
As such criterion was a quantitative
measure of the variation of the
predicted responses, it was modelled
as an additional CQA to guarantee
the quality of the method (21). Quality
should be assured inside the DS and
as specified by ICH Q8 guideline
“working within the design space is
not considered as a change” (2), DS
should represent a zone of robustness
of the analytical method.
The final step of the QbD method
is to experimentally validate the DS.
To limit the number of experiments,
DOEs are usually required. Screening
designs, such as two-level fractional
factorial or two-level Plackett-Burman
designs, are sufficient to achieve
this validation. To validate the whole
DS, CPPs are usually varied from the
lowest to the highest limits of the DS
borders (6,9,14,16–18 24–26). DS
represents the robustness zone of the
method, and so a classical robustness
study verifying the influence of
small changes of the CPPs around
the working point on the CQAs
performance is not required. However,
after DS determination, it may be
preferred to perform a robustness
study before or juxtaposed to a
complete method validation (6,14,
16–18,24–26). Additional parameters
may be added, but the range of
variation of each parameter is often
reduced in comparison to the values
defined by the DS.
Another approach has been
proposed by Molnár et al. Using
chromatography modelling software,
a robustness testing is simulated over
more than 700 conditions by enlarging
the number of tested factors compared
to the number of CPPs retained for
the optimization step. Some selected
points are then experimentally
checked (7,8,11,12).
Some authors limit the DS validation
to a traditional robustness validation of
the optimum point, leading to methods
with a smaller robustness domain
because the method operable region
is also reduced (10,13,27). Finally,
in several papers DS validation and
robustness validation have been
replaced by a single repeatability
study at optimal conditions (20,22)
without verifying if the DS is correctly
defined. This latter approach cannot
provide a DS for the analytical method
under development, while robustness
inside is not experimentally checked.
This conflicts with the objectives of
QbD to develop a robust method.
Conclusion
A QbD approach for analytical method
development aims at determining a
multivariate domain, called the design
space (DS), where method critical
parameters have been demonstrated
to provide assurance of quality.
In fact, the analytical method is
no longer restricted to one single
combination of parameters, but is
now identified by a set of operating
conditions defining the DS. Inside
the DS, the method achieves the
predefined performance with a high
level of probability.
Many benefits are intrinsic to QbD
for the pharmaceutical industry.
Robust methods are developed so that
fewer method failures may
appear during routine use in QC
laboratories. Moreover, higher flexibility
is achieved because moving inside the
DS is not considered a change and
does not require any further regulatory
approval. QbD offers a deeper
understanding of the analytical method
through the evaluation of the main
and interaction effects of the method
parameters on its performance.
This additional knowledge gained
during development is crucial for
pharmaceutical analysts. Method
validation becomes less prone to
unpredictable errors, analyst reactivity
is improved to face future operating
problems, and strong scientific
justification may influence future
regulatory frameworks. Finally, method
transfer from one laboratory to another
is simpler because the adaptation of
the transferred method will be eased
by the knowledge of its DS at the
receiving site.
Throughout the product life cycle,
companies have the opportunity to
evaluate innovative approaches to
improve product quality. QbD, through
a more systematic approach, plays a
major role in assessing the potential of
new techniques and thus contributes
to continual improvement within the
pharmaceutical industry.
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N. Kalenda, E. Rozet, Ph. Hubert, and
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G.M. Hadad, J. Chromatogr. A 1399,
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Bubba, E. Bertol, and S. Furlanetto,
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91, 4–16 (2008).
Karen Gaudin completed a Ph.D.
in analytical chemistry in 1999 at
the School of Pharmacy of Paris XI
University in France. She is a professor
and director of a research team focusing
on analytical and pharmaceutical
developments at the University of
Bordeaux. These main developments
concern strategies to analyze APIs or
lipids in new pharmaceutical products
using various separation techniques
(reversed phase, HILIC) and detectors
(CAD) eventually in green chemistry and
QbD. She has published more than 50
papers in reputed journals and serves
as an editorial board member of repute.
Ludivine Ferey has completed a
Ph.D. in analytical chemistry at the
Industrial Physics and Chemistry
Higher Educational Institution (ESPCI)
in Paris, France. She is an associate
professor in the ChemBioPharm
team (ARNA, INSERM U1212) at the
University of Bordeaux in France. Her
research is focused on the use of
chemometric tools, mostly QbD, for
the development and optimization of
separation analytical methods in the
field of pharmaceutical analysis.
25www.chromatographyonline.com
Gaudin and Ferey
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Liquid chromatography coupled to
tandem mass spectroscopy (LC–MS/
MS) using triple quadrupole mass
spectrometers (TQ-MS) has evolved
into a mainstream approach for
quantitative bioanalysis of drugs and
metabolites over the past 20 years.
The high selectivity of triple quadrupole
instruments in the selected reaction
monitoring (SRM) mode makes the
technique very suitable for targeted
analysis in a high-throughput mode with
short run times. The disadvantage of this
approach is that only information on the
analytes for which SRM mass transitions
are put into the method are obtained.
Qualitative information on other analytes
will not be obtained using this approach.
The sensitivity and dynamic range
of the latest generation of HRMS
instruments now approaches that
of TQ-MS, but at a higher and still
increasing resolution. When HRMS
is used, no SRM transitions for the
analytes of interest need to be entered
into the method. The system is run
in full-scan MS mode and selectivity
is achieved by the high resolution of
the system, instead of by selecting
a specific precursor–product ion
combination in SRM. As a result, a much
richer dataset is obtained, containing
not only quantitative information on the
analytes of interest, but also qualitative
information on the other analytes present
in the sample.
An example of the different data types
obtained by HRMS and TQ-MS is shown
in Figure 1. For TQ-MS, only information
on the parent drug is obtained, whereas
with HRMS, information on adducts,
in-source fragments, metabolites, and
endogenous compounds, including
biomarkers and background ions, is
obtained.
It can be very useful to obtain both
quantitative information on the drug and
qualitative information on other analytes
that are present in the sample, and not
be limited by only the analytes for which
SRM mass transitions were entered. In
the literature, this approach is referred
to as a quan–qual approach, and the
terminology quan–qual is mainly used
in the context of obtaining quantitative
information on the drug and qualitative
information on drug metabolites (1–3).
For example, in metabolic stability
studies, quantitative data on the drug,
as well as qualitative data of the in vitro
metabolites, can be relevant (4). In this
way, not only the metabolic stability
of the drug is assessed, but also
information is obtained on the metabolic
hotspot(s) of the molecule, which is
relevant for compound optimization to
improve metabolic stability.
Our group, however, prefers to use
a broader definition for quan–qual,
because there is much more potential in
the use of the qualitative data besides
looking for drug metabolites. Looking
at the presence and regulation of
endogenous metabolites can be of
interest, although the number and type
of biomarkers that can be followed will
be limited by the more generic sample
preparation and LC conditions applied
for quan–qual analyses. The availability
of all ion information can be beneficial
for speeding up method development,
or for the resolution of analytical or
other issues. A few examples of this are
given below. Certain compounds, like in
formulations (for example, PEG 400 and
polysorbate [tween]) or endogenous
compounds, such as phospholipids,
can cause ion suppression and
influence the quantitative result if
they are coeluted with the analyte.
Therefore, it is useful to know what is
coeluted with the analyte(s) of interest,
so this information can be used for
potential troubleshooting. If an in vitro
experiment does not seem to provide
the expected result, it might be useful to
check whether the right cofactors were
present and in which form (for example,
to determine the ratio of glutathione
[GSH] and oxidized GSH in a reactive
metabolite screening), or to check if
cells were present during the in vitro
experiments by looking for the presence
of phospholipids via their marker ions at
m/z 104 and 184.
Quan–qual approaches have clear
advantages compared to doing “quan”
alone but there are some caveats to
take into account. First, for quantitative
targeted analysis, very short analytical
run times (typically 0.5–1.5 min) are
preferred to enable the analysis of
many samples in a short timeframe. For
qualitative analysis, significantly longer
analytical runtimes are more beneficial.
For drug metabolite identification, longer
analytical runtimes (typically 10–20 min)
High-Throughput Analysis of Drugs and Metabolites in Biological Fluids Using Quan–Qual Approaches
Ronald de Vries, Rob J. Vreeken, and Filip Cuyckens, Pharmacokinetics, Dynamics & Metabolism, Janssen R&D,
Beerse, Belgium
The new generation of high-resolution mass spectrometry (HRMS) systems offers high sensitivity,
dynamic range, resolution, accuracy, and scan-to-scan reproducibility, enabling high-throughput
quantitative analyses in combination with information-rich qualitative data. The most recently released
HRMS systems offer an alternative to triple quadrupole (TQ)-MS systems. This provides a huge opportunity
to obtain quantitative and qualitative information from one analysis, but also requires a different mindset
and expertise to make the right choices and compromises to get the most information from your sample.
Ph
oto
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Ge
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Advances in Pharmaceutical Analysis October 201626
are important to reduce the risk of
coelution of isobaric metabolites and, in
this way, to correctly identify metabolites.
Therefore, we see quan–qual more
as a quantitative approach providing
additional qualitative information “for
free”, rather than using the approach as
a replacement for separate metabolite
identification studies.
When metabolite identification is (one
of) the main goal(s) of the study, it could
be better to reanalyze a small selection
of the samples using a longer analytical
runtime and use these data for metabolite
identification, rather than analyzing
all samples with a longer analytical
runtime (5). Also, if obtaining biomarker
information is one of the main goals, it
may be better to rerun samples using a
method where sample preparation and
chromatography have been optimized for
the analysis of the required biomarkers.
Furthermore, the large amount of
data produced by quan–qual analysis
increases the risk of confusion. It is
important to ensure that the qualitative
data produced are used with a focus
on answering specific key questions,
rather than spending time trying to
extract all available information on
a routine basis simply because it
is available. The availability of the
qualitative data should be used to avoid
repetition of experiments and analyses
if specific questions come up later in
the development programme. Data
can be reevaluated at the moment that
the specific questions arise, rather
than directly after the data has been
acquired. For example, if a critical in
vivo experiment is performed, where
only quantitative data on the drug
are requested, it might be useful to
perform the analysis in quan–qual mode
but initially only use the quantitative
data. If, later on, questions arise (for
example, the potential involvement of a
metabolite explaining a pharmacokinetic
or pharmacodynamic [PK or PD]
disconnect, or the presence of a human
metabolite in animal species), it is easy
to go back to the qualitative data months
or years after analyses to find the answer.
In this paper, sample preparation
methods and mass spectrometry
approaches suitable for high throughput
quan–qual analysis are presented. High
performance liquid chromatography
(HPLC) optimization for high throughput
quan–qual analysis is also discussed.
Furthermore, potential uses, challenges,
and future perspectives for quan–qual
analysis will be discussed.
Sample Preparation
Since the qualitative part of a quan–
qual analysis is an untargeted analysis
with a potential interest in molecules
27www.chromatographyonline.com
Cuyckens et al.
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Figure 2: Separation of buspirone (1) and its major metabolites (2: +O; 3: -2H; 4: +O-2H; 5: +O+gluc) in rat hepatocytes with: (a) a traditional metabolite profiling chromatographic method using a 100-mm length porous 1.7-μm particle packed column at 0.4 mL/min and (b) a shorter 50-mm solid core 1.6-μm particle packed column at a higher 0.9 mL/min flow rate. Adapted with permission from reference 6.
that are unidentified at the start of
the analysis, the sample preparation
should be kept to a minimum to avoid
any potential degradation or recovery
issues of the compounds of interest.
This is in line with what is done in
metabolite profiling studies and might
be in contrast with quantitative assays
that often use liquid–liquid extraction
(LLE) and solid-phase extraction (SPE)
to improve the specificity and sensitivity
of the assay. For quan–qual analyses,
typically one or multiple volumes of
an organic solvent (usually 3 or more
volumes of acetonitrile) are added to the
sample to stop any remaining enzymatic
activity, break the analyte–protein
binding, and precipitate the majority
of the proteins. The samples are then
centrifuged and the supernatant
injected onto the LC–MS system.
Liquid Chromatography
Optimization
Metabolites are often structurally
related molecules. Therefore, longer
LC gradients are typically used in
metabolite profiling and metabolomics
studies to reduce the risk of missing
coeluting isobaric compounds.
Quantitative LC–MS runs are usually
very short, allowing a higher throughput
of the analysis of large(r) batches of
samples. A compromise needs to be
made providing sufficient throughput
with decent separation power. This
is also a function of the batch size
because there is no use in running
short gradients if the instrument sits
idle for a couple of hours because the
run finishes in the middle of the night.
Nevertheless, longer runs also result in
larger file sizes and processing times,
and a higher potential for MS drift.
Longer LC gradients do not
necessarily give rise to the separation
of more compounds in one run —
expressed as peak capacity for
gradient elution. The effect of a longer
run time on separation power is often
overestimated because an increase
in gradient time gives a less than
proportional increase in peak capacity.
When chromatographic conditions are
optimized using shorter LC columns
packed with sub-2 μm particles in
combination with higher flow rates
and the extracolumn dead volume
is reduced, it is perfectly feasible to
achieve peak capacities similar to
those obtained in traditional metabolite
profiling studies in 2–3-fold shorter
run times. This was demonstrated
on a mix of pharmaceutical
standards and an in vitro incubation
of buspirone containing multiple
isobaric metabolites (6). The latter is
illustrated in Figure 2, which shows a
slightly better separation between the
major metabolites of buspirone in rat
hepatocytes using a 2.5-fold shorter
generic method, which applies a
short (5 cm) ultrahigh-pressure liquid
chromatography (UHPLC) column
packed with solid core 1.6-μm particles
in combination with a higher flow rate.
While the peak capacity of the LC
separation is worthwhile optimizing
because metabolites are often isobaric
compounds, the selectivity provided by
mass spectrometry — high-resolution
MS in particular — has a much bigger
impact on the total peak capacity of the
overall analytical assay.
Mass Spectrometry Methods
To take full benefit of the optimized
chromatographic systems boosted for
peak capacity, the mass spectrometry
methods should provide high-quality
quantification and a maximum of
qualitative information in accordance
with the short timeframe of high
resolution chromatographic peaks.
To obtain high-quality quantitative
information with good reproducibility,
linearity, and sensitivity, an adequate
description of the chromatographic
peak is essential. This requires typically
>12 data points per chromatographic
peak. Current attainable peak widths
when using UHPLC are around 2–3 s
and are distinctively smaller than
traditional LC peak widths. Current
HRMS systems are well equipped
with respect to scan speed to obtain
at least 12 data points across the
peak in full scan mode, even with
these narrow peak widths. From full
scan HRMS data, relevant qualitative
information can be obtained. Analytes
can already be identified to a certain
extent because information on the
likely elemental composition can be
obtained based on the accurate mass
measurement and isotope ratios, or
by using very high-resolution systems
(Fourier-transform MS) one can even
look at the isotopic fine structure. Scan
routines to obtain MS/MS spectra can
be added to the MS method to obtain
additional qualitative information to aid
in structure identification, for example,
when coeluting isobaric metabolites
show differences in fragmentation.
However, when additional scan modes
are added to the mass spectroscopy
method, in addition to the full scan
mode, challenges can arise with
regards to the available acquisition
time for these experiments. Optimizing
both the “quan” and “qual” world will
result in a “sweet spot” of settings
where, on the one hand, an acceptable
number of data points allows reliable
quantitation and, on the other, MS/MS
spectra of sufficient quality further aids
identification.
The MS/MS scan routines can be
divided into targeted (data-dependent
[7,8]) and untargeted (data-independent
[8,9]) strategies (see Figure 3). Targeted
approaches relate to acquisition of
product ion scans of preselected
m/z values. A targeted approach
can be (but does not need to be)
part of a data-dependent strategy.
A data-independent approach is by
definition untargeted.
Data-dependent strategies, such
as Data Directed Analysis (DDA) or
Information Dependent Analysis (IDA),
Advances in Pharmaceutical Analysis October 201628
Cuyckens et al.
Ion source
DDA/IDA
SWATH
MSe/MSall
Targeted
Un-Targeted
Collision cell
TOF
TOF
TOF
Q1
Figure 3: An overiew of different quan–qual MS acquisition strategies. Information or Data Dependent Acquisition (IDA or DDA), Sequential Window Acquisition of all THeoretical fragment ion spectra (SWATH), MS/MS of all ions (MSall or MSe). Adapted with permission from reference 8.
have been known for more than a
decade and were first used on triple
quadrupole instruments. The system
acquires data in full-scan mode and
upon passing a threshold (either
intensity [untargeted] or intensity at
predefined m/z values [targeted]), the
system automatically switches to one
or more product-ion scans of that m/z
value to obtain product ion spectra
that can be used for identification
purposes. Instead of using a predefined
list of m/z values (targeted approach),
MS/MS spectra can also be acquired
through different parameters, such as
the 10 most intense ions. This can be
combined with dynamic inclusion and
exclusion lists, isotope pattern filtering
(if the analytes of interest contain
chlorine, the isotope pattern filtering
can be used to obtain MS/MS spectra
of only chlorine-containing analytes),
dynamic background subtraction,
and on-the-fly mass defect filtering to
maximize the number of MS/MS spectra
obtained for relevant analytes, while
reducing the acquisition of MS/MS
spectra of irrelevant background ions.
(Mass-defect filtering is often used in
drug metabolism and filters data on the
basis of the mass-defect of the parent
compound. Unrelated compounds are
then dynamically excluded from MS/
MS acquisition.) The use of HRMS
also allows for “thresholding” based
on accurate mass instead of nominal
mass, further reducing the number
of product ion spectra of irrelevant
background ions. Despite all these
available filtering algorithms, the
disadvantages of data-dependent
strategies remain. Taking individual
MS/MS spectra requires substantial
scanning time and hence some
relevant, often lower abundant, analytes
might be missed. Another disadvantage
of a data-dependent strategy is that
it relies heavily on prior knowledge
of the sample and that it cannot be
used as a generic method, especially
for parameters such as mass defect
filtering, isotope filtering, and using a
predefined list of m/z values for each
drug. Although MS/MS spectral quality
is often superior to that obtained in
data-independent strategies (discussed
below), it is not usually the method of
choice for quan–qual analyses, which
ideally comprise the maximum amount
of information to allow questions that
come up long after the analyses have
finished to be addressed.
In data-independent strategies, no
precursor ions are selected for MS/MS
as it is in data-dependent or targeted
approaches. Instead, MS allows the
passage of all precursor ions at the
same time and no selection of ions on
whatever criteria needs to be made.
Both full scan and MS/MS spectra
of all incoming ions are obtained via
this so-called MSall (also called MSE)
approach by alternately acquiring
spectra at low-collision energy (full
MS) and high-collision energy (MS/
MS). If the UHPLC separation of the
different analytes in the sample is
adequate, good quality MS/MS spectra
can be obtained via this approach.
However, when the complexity of the
sample increases, resulting in coelution
of analytes, mixed MS/MS spectra
are obtained that complicate their
interpretation. One way to obtain cleaner
MS/MS spectra in MSall mode, even for
low-abundant analytes coeluting with
high-abundance interfering analytes,
is to transmit smaller m/z ranges (for
example, 25 amu) instead of the whole
m/z range to pass through the first MS
analyzer and subsequently produce
MS/MS spectra for each separate
window (multiplexed MS/MS, MSX,
SWATH). By consecutively stepping up
the m/z range of ions passing through
the MS system, the complete mass
range of interest is “scanned” over.
Data deconvolution by the vendor
software relates the precursor and
associated product ions. Parameters
like scan speed, width of each window,
number of windows selected, scan
speed or dwell time for each window,
and overlap between windows will
affect the information obtained for
each compound. The disadvantage of
multiplexed MS/MS is that the required
scan time is significantly longer than for
MSall, and inversely proportional to the
width of the m/z windows. Therefore,
the multiplexed MS/MS approach is
usually not compatible with quan–qual
analysis where full MS or MSall is to be
preferred, unless future developments
in HRMS technology result in large
improvements in scan speed. Another
technique resulting in cleaner MS/MS
spectra in MSall mode without affecting
scan times is ion mobility spectrometry
(IMS). In IMS, ions are separated based
on size, shape, and charge rather than
on m/z. The combination of retention
time (LC) and drift time (IMS) alignment
results in cleaner MS/MS spectra
because precursor and product ions
have identical drift times, while coeluting
background ions and their product ions
might be separated in the ion mobility
device preceding the collision cell
(10). In full MS, ion mobility separation
can be beneficial, providing advanced
selectivity and, thus, better detection
limits. The IMS separation also has some
disadvantages: it affects the dynamic
range of the detector (earlier saturation),
the resolving power of current IMS
systems is often not adequate enough,
and it has an impact on scan speed;
however, in general, it is still compatible
with average UHPLC peak widths.
Challenges and Future
Perspectives
There are a lot of advantages to using
HRMS systems for the acquisition
of both quantitative and qualitative
information in the same analyses.
The number of applications is also
gradually growing in different (mainly
non-regulated) fields. Nevertheless, the
majority of LC–MS quantification is still
triple quadrupole-based since quan–
qual approaches and high-resolution
quantification, in general, also come
with some challenges, as described in
previous publications (11,12).
Historically, most laboratories
focusing on LC–MS quantification are
equipped with triple quadrupoles.
The replacement of an MS system
requires substantial investment, which
can hamper a rapid shift to HRMS.
In the last few years HRMS systems
have become available offering
high sensitivity, dynamic range,
resolution, accuracy, and scan-to-scan
reproducibility at a price similar to
modern triple quadrupole systems.
Most of these are still tandem mass
spectrometers (mainly quadrupole
time-of-flight [QTOF]). However, the
selectivity provided by the narrow
extraction windows, based on accurate
mass in full MS, is sufficient. Therefore,
a growing choice in single-stage
high-resolution systems will make the
switch easier because the investment
will be lower than a high-end triple
quadrupole or a tandem HRMS
system. In addition, the ease of use
will be improved over a tandem HRMS
and TQ-MS system. HRMS systems
were historically designed to be used
primarily by more experienced MS
users. The newly released TOF systems
are focused on quantification and
29www.chromatographyonline.com
Cuyckens et al.
ease of use, rather than on flexibility in
scan speed, resolution, and a myriad
in MSn or MSall scanning options. As
is the case in any application, any
good hardware is only useful when
appropriate application software
is available. Many good uni- and
multivariate analysis approaches
and structure identification tools are
available for metabolite profiling,
typically done on HRMS systems.
There is still room for improvement
because identification of molecules
based on mass spectrometry can be
time-consuming and requires expertise.
Another bottleneck in the qualitative
part of the quan–qual analysis is
that appropriate blank samples (for
example, from vehicle dosed animals)
are often lacking. This gives rise to false
positives upon extraction of metabolites
from the background.
Quantification tools based on narrow
extraction windows are also available
in most, if not all, vendor software.
Processing times might be longer
compared to triple quadrupole data
processing because of the exponentially
larger file sizes linked to the richness
of the data, which also require much
larger servers for file storage. However,
a lot more can potentially be done with
the available MS data besides looking
at one accurate mass extracted ion
chromatogram of the analyte(s) of
interest, as is currently the standard for
quantitative processing. Besides the
[M + H]+ ion chromatogram, the ion
chromatograms of the isotopes could be
automatically processed to check their
correspondence with the theoretical
isotope distribution. This could serve as
a peak purity check to highlight potential
coeluting isobaric species or other
coeluting compounds. Isotope peaks
could be processed instead of the
primary isotope in case of interference
or summed to increase signal-to-noise
ratios, especially in cases where the
isotope peaks are equally abundant
(bromine-containing compounds,
multiple charged ions). Isotopes could
also be used in detector saturation
(and no saturation of the ionization)
to increase the dynamic range by
processing a less abundant isotope
peak. The richness of the accurate mass
data provides many more opportunities,
such as automatically highlighting the
presence of adducts and in-source
fragments, automated assignment of
the optimal narrow extraction window,
and alerting the user for coelution with
potentially interfering matrix compounds
(phospholipids, PEG from the
formulation) to name a few. These extra
capabilities and improved ease-of-use
of HRMS systems are key to convincing
the majority of the LC–MS quantification
user community to make the switch
to high-resolution MS quantification
and quan–qual analysis in those
cases where additional information
on metabolites or background ions
might be useful in the future, and
where there is no need for that extra
2–5-fold more sensitivity provided by
the newest high-end triple quadrupole
systems. Furthermore, increasing scan
speed and the resolution of both the
mass spectrometer and ion mobility
separation in newer HRMS instruments
will inevitably lead to more adaptation
to quan–qual workflows. The pace
with which the capabilities of modern
high-resolution systems increase and
the computer power and software
tools improve will dictate the speed
of implementation. However, the split
between “quantitative” and “qualitative”
bioanalytical departments, lack of
experience with high-resolution MS, and
often a more conservative approach
in regulatory environments are some
of the other hurdles to overcome for a
widespread application.
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R. Papp, and L. Taylor, J. Am Soc Mass
Spectrom. 20, 1441 (2009).
(2) W. Korfmacher, Bioanalysis 3(11), 1169
(2011).
(3) L. King, Bioanalysis 6(24), 3337 (2014).
(4) G. Backfish, B. Reder-Hilz, J.
Hoeckels-Messemer, et al., Bioanalysis
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(5) M.F. Grubb, W.G.Humphreys, and
J.L. Josephs, Bioanalysis 4(14), 1747
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G. Gross, T. Hankemeier, and R.J.
Vreeken, J. Chromatogr. A 1374, 122–133
(2014).
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Franklin, and T.A. Baillie, Anal. Chem.
76(3), 823 (2004).
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Anal. Chem. 86(2), 1202–1209 (2014).
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et al., Rapid Commun. Mass Spectrom.
21(9), 1485 (2007).
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Bioanalysis 5(10), 1277 (2013).
(12) L. Dillen and F. Cuyckens, Bioanalysis
5(10), 1145 (2013).
Ronald de Vries graduated in
organic and analytical chemistry at
the Free University of Amsterdam, in
the Netherlands. After working in a
contract laboratory for 7 years, he joined
Janssen R&D in 1998. At Janssen
R&D, he worked in the bioanalytical
department supporting both clinical and
non-clinical bioanalysis. He provided
the bioanalytical support for various
drug development programmes and
led the method establishment group
responsible for development of new
bioanalytical assays using LC–MS/
MS. Since 2014, he has worked in the
drug metabolism group, focusing on
metabolite identification using high
resolution mass spectrometry and
radiodetection. He has (co-)authored
more than 40 peer-reviewed scientific
publications.
Rob J. Vreeken has over 25 years
experience in quantitative and qualitative
analysis by hyphenated MS techniques
in a wide variety of applications. He has
worked in academia, service providers,
and in industry, and is currently
employed at Janssen Pharma R&D. His
team focuses on quantitative assays
in early discovery and exploratory
pharmacokinetics, dynamics, and
metabolism. Next to semi-HT compound
exposure analysis, the team implements
metabolomics strategies to collect
information on efficacy through
semi-quantitative PD-marker analysis.
He is also an associate professor at M4I,
Maastricht University in the Netherlands,
where he focuses on quantitative mass
spectrometry imaging techniques for
pharmaceutical markers. He has (co-)
authored close to 100 peer-reviewed
scientific papers and is a frequent
presenter at international symposia.
Filip Cuyckens is a Scientific Director
& Fellow at Janssen R&D. He is
responsible for analytical sciences
in the pharmacokinetics, dynamics,
and metabolism department,
focusing on metabolite profiling and
identification of drugs in discovery
and development, and quantification
of drug candidates, metabolites, and
biomarkers in biological matrices. He
earned a pharmacist degree in 1998,
a degree in industrial pharmacy in
2002, and a Ph.D. in pharmaceutical
sciences in 2003. He has (co-)authored
more than 50 peer-reviewed scientific
publications, and is a member of the
associate editorial board of Rapid
Communications in Mass Spectrometry
and board member of the Belgian
Society for Mass Spectrometry.
Advances in Pharmaceutical Analysis October 201630
Cuyckens et al.
Analysis of enantiomers is a critical topic
in the life science, food, fragrance, and
environment industries, and is also key
in asymmetric synthesis and catalysis.
Enantioselective chromatography
has evolved as a powerful tool for the
analysis of chiral compounds and is
now established as the method of
choice in almost all academic and
industrial laboratories dealing with
chiral molecules. Over the past 35
years intense efforts have been made
to obtain reliable tools for this purpose
and various methods and techniques
have been developed and optimized for
the accurate determination of the chiral
composition of enantiomeric mixtures
(Table 1).
The techniques in Table 1 do not all
have the same importance but all have
been applied. While the formation of
diastereoisomers prior to analysis was
the common practice in the past, today
the respective enantiomers of almost
any type of small chiral molecules can
be directly “discriminated”. Among all
techniques, liquid chromatography (LC)
and supercritical fluid chromatography
(SFC) are now the most used methods
for enantioselective analysis in the
pharmaceutical environment. Gas
chromatography (GC) is still used,
but more for volatile substances and
amino acids. There are numerous
applications of capillary electrophoresis
(CE), including drugs, but most of these
applications have been performed in
academic environments. It is used less
in the pharmaceutical industry, but might
still be useful and even the best method
for particular applications. Spectroscopic
methods, such as nuclear magnetic
resonance (NMR) spectroscopy,
vibrational circular dichroism (VCD)
spectroscopy, and mass spectrometry
(MS), are also occasionally used, but
not to the extent of the chromatographic
methods.
As many chromatographic techniques
are now available for the analysis of chiral
molecules, the choice is usually driven
by the application, that is, for volatile
compounds, GC might be preferred,
while for preparative purposes, LC or
SFC will be favoured.
The types of application covered
by the enantioselective analytical
techniques are quite broad and the
major ones are listed in Table 1. This
article focuses on the contemporary
enantioselective analytical techniques
with an emphasis on pharmaceutical
chiral compounds and, therefore, only
the most contemporary approaches are
discussed.
Analysis of Chiral Molecules
by HPLC
Over the past 30 years, HPLC on
chiral stationary phases has evolved
as a powerful method to separate
enantiomers. HPLC is applicable to
enantiomers of almost all types of small
chiral molecules, it is compatible with
almost all kinds of functional groups
(basic, acidic, neutral), and it can usually
be applied directly without previous chiral
derivatization to diastereoisomers. In the
classical pharmaceutical environment, it
concerns about 50% of the molecules,
the others being achiral or more complex
molecules, such as small and large
peptides, monoclonal antibodies, and
antibody–drug conjugates.
Hundreds of chiral stationary phases
(CSPs) have been commercialized for
HPLC, but the practical experience of
the last 25 years has led to a ‘“natural”
selection of the phases, and it is
generally recognized that a relatively
limited number of columns cover at
least 90% of all applications (Figure 1).
These “all-rounder” phases consist of
polysaccharide derivatives, particularly
from cellulose and amylose. It has
already been shown more than 40 years
ago that cellulose triacetate (CTA) has
unique chiral recognition properties
in enantioselective chromatography
(1), and the very first enantioselective
pharmaceutical application on this
polysaccharide phase was reported
more than 30 years ago (2). However,
it is the process elaborated by Yoshio
Okamoto and his group in Japan that
has made possible the preparation
of robust and high-performing
polysaccharide-based CSPs, which have
been commercialized and are suitable
for validated analytical processes (3).
Other column manufacturers have
produced generic versions of these
polysaccharide-based phases. Most
of the reported applications were
performed in the normal-phase mode,
but the CSPs can also be used in the
reversed-phase mode.
While normal-phase mode is generally
applied to determine enantiomeric
excesses of synthetic samples or for
preparative purposes, reversed-phase
conditions are usually preferred for
samples obtained from biological
matrices (stereoselective metabolism,
toxicity, and chiral stability). Figure 2
shows a typical example of chiral
separation, illustrating the increasing
complexity of the investigated drugs (with
multiple chiral centres) and the possibility
offered by the available tools to achieve
the separation of the four stereoisomers
under normal-phase conditions or under
Contemporary Analysis of Chiral MoleculesEric Francotte, FrancotteConsulting, Separation Sciences, Basel, Switzerland
The first high performance liquid chromatography (HPLC) column for enantioselective chromatography
was introduced commercially in 1981. This chromatographic mode has now become the method of
choice for the analysis of chiral pharmaceutical compounds, making previous approaches, such
as optical rotation, almost completely obsolete. However, supercritical fluid chromatography (SFC)
has been gaining increasing recognition as a complementary technique to HPLC for pharmaceutical
enantioselective analysis. Gas chromatography (GC) and capillary electrophoresis (CE) remain very
useful for particular applications.
31www.chromatographyonline.com
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polar organic conditions, which are
more appropriate to investigate aqueous
samples on polysaccharide-based
CSPs.
As a result of the complexity of
the mechanism of interaction with
polysaccharide-based phases, it
is almost impossible to predict the
chiral separation on these phases.
Small variations of the structure of the
analyte, of the polysaccharide CSP, or
of the mobile-phase composition can
dramatically affect the separation, and
even cause the inversion of the elution
order of the enantiomers. Therefore,
in most cases, method development
is required to identify appropriate
conditions to separate the enantiomers.
Many factors can influence the
separation, and it is common practice
to screen a broad combination of CSPs
and mobile phases. Numerous strategies
have been proposed by different groups
for screening chiral stationary phases
(4–6). However, because of the rapidly
evolving market for chiral columns,
the setup has to be regularly adjusted.
The screening is usually performed on
single columns, which are successively
tested, but this mode is not very efficient,
particularly if a large number of chiral
substances need to be processed. This
is still the most common approach, but
it is now widespread to use switching
valves sequentially connected to the
different columns in an automated
fashion, which has the advantage of
being a relatively low-cost setup.
The screening process can be
accelerated by using an isochronal setup
(7), but the use of HPLC systems capable
of working in parallel clearly shows the
highest effectiveness (8). Our group
developed this strategy several years ago,
and it is currently used as the standard
procedure for chiral method development
(8). Under the standard setup, using
columns of 15 cm × 0.46 cm and a flow
rate of 0.8 mL/min (20 min per run), up
to 20 different conditions (CSP or mobile
phases) can be tested each hour. The
system is totally automated. The parallel
setup might also be useful if the analysis
of many samples of the same molecule is
required, for example, for the screening
of catalysts in asymmetric synthesis, for
samples from biological investigations, or
for samples from formulation optimization
studies. In these cases, the same CSP is
used on all channels of the parallel unit.
The necessity of running efficient
parallel screening approaches for chiral
method development has become even
more essential since the introduction of
the immobilized polysaccharide phases.
Indeed, with this new generation of chiral
columns, it is possible to apply strong
solvating modifiers like ethyl acetate,
tetrahydrofuran, dichloromethane,
chloroform, or dimethoxymethane
that are not tolerated by the classical
non-immobilized polysaccharide phases,
considerably increasing the potential to
improve the separation by modulating the
mobile phase. This is a great advantage
because it could help to optimize the
retention times and selectivity, which are
crucial parameters for enantioselective
analysis.
Various approaches have been
developed for the preparation of
immobilized polysaccharide phases (9),
including a particularly simple process
that our group developed (7,8,10)
and which has led to the introduction
of several CSPs of this type on the
market (11,12). Screening approaches
incorporating these phases have been
described (8,13).
For racemic substances that cannot
be resolved on polysaccharide-based
CSPs, a few other columns are generally
tested in a secondary screening. All
these phases have been designed by
bonding small chiral molecules to silica
gel. These phases include an amino
tetrahydrophenanthrene-based phase
(14), the cyclodextrin-based CSPs (15),
the cinchona alkaloid ionic phases (16),
the cyclofructan (17), the macrocyclic
peptides (18), and glycoprotein phases
(19), which together share about 10% of
the applications of the enantioselective
analyses performed in the
pharmaceutical field. The quinine-based
phase (20), the α-1-acid glycoprotein
(AGP) (or orosomucoid) (21), and the
cyclofructan (22) phases might exhibit
excellent chiral recognition power for
polar or ionic compounds, which often
fail on polysaccharide-based CSPs.
Figure 3 shows the analytical separation
of the enantiomers of free amino acids on
the new quinine-based ionic CSP (20).
Thousands of pharmaceutical
applications have been reported
attesting to the effectiveness of the
enantioselective HPLC technique.
Modern enantioselective HPLC is very
efficient (≤5-μm particle size) and an
enantiomeric ratio lower than 99.9/0.1
can usually be easily determined.
The method is also routinely applied
for validated processes (23,24) and
for enantioselective drug metabolism
assessment (25). Figure 4 shows a
recent application of the simultaneous
quantification of the enantiomers of the
drug lacosamide and its chiral impurity
on an immobilized polysaccharide-
based phase (24). Another typical
application of a high enantiomeric
excess (ee) determination for the drug
Pemetrexed also exemplifies that even
polar analytes can be well resolved on
polysaccharide-based phases (26).
It must also be mentioned that a
considerable number of enantioselective
Table 1: Major techniques for enantioselective analysis and major applications fields.
Techniques Major Application Fields
Chromatography Asymmetric synthesis and catalysis
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t�0QUJDBM�SPUBUJPO Fragrances
t�.BTT�TQFDUSPNFUSZ�.4 Food
t��/VDMFBS�NBHOFUJD�SFTPOBODF�/.3 Pesticides (insecticides, fungicides)
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Sensor t�.FUBCPMJTN
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t�(SBWJNFUSJD�NBTT
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Advances in Pharmaceutical Analysis October 201632
Francotte
methods are developed in the context of
preparative separations or asymmetric
synthesis of drugs and drug intermediates.
For this purpose the polysaccharide-
based phases dominate again, with
more than 95% of the applications in the
pharmaceutical industry.
Recently, it has been shown that
ultrafast chiral HPLC can be achieved
within a few seconds, but no practical
applications have been reported so far
(27–28).
Analysis of Chiral Molecules
by SFC
Intensive research activities on
supercritical fluid chromatography
(SFC) in open tube capillaries and in
packed columns have been performed
since the early 1980s (29), but it took
many years until the technique was
really adopted. The poor acceptance
was mainly a result of the limited
robustness and low reliability of the
available instrumentation. From the
beginning of the new millennium,
the situation radically changed with
the significant improvement of the
new SFC instruments, and it was,
in fact, preparative enantioselective
chromatography that rescued packed
SFC (pSFC). Enantioselective separation
has been the major SFC application
for about 10 years and has now been
adopted by most pharmaceutical
companies for this purpose.
pSFC is very similar to normal-phase
HPLC—it “just” uses liquid CO2 as
the major component of the mobile
phase. However, in its supercritical
state, CO2 exhibits unique properties
that gives it some advantages in terms
of chromatographic performance
because of the low viscosity of the
fluid. Other advantages include
speed, efficiency, safety, costs,
and environmental impact (green
technology) (30).
Even though SFC is usually achieved
under sub-critical conditions, most
of these advantages remain. These
reasons have contributed to the rapid
establishment of packed SFC as a
powerful technique for enantioselective
analysis. Moreover, the switch from LC
to SFC was helped because the same
“chiral” columns can be used for either
LC or SFC.
The suitability of SFC for
enantioselective analysis of drugs has
been reviewed in a series of review
articles and book chapters, containing
an extended list of drug applications
(31–36). However, enantioselective
separations that can be achieved
on a particular CSP by HPLC do
not necessarily work under SFC
conditions (and vice versa). This is not
surprising because the mobile phases
are different and such effects are
observed in enantioselective HPLC on
polysaccharide-based stationary phases
when slight modifications of the modifier
composition are performed. Based on
our own experience, about 10% of the
chiral pharmaceuticals are better or
only resolved by HPLC, while the same
proportion is better resolved under SFC
conditions.
As for LC, any resolution of a new
racemic compound in SFC requires
method development. Several groups
have reported on their own process
flow (13,37–40), but because the chiral
column market is constantly evolving,
the setup has to be regularly adapted,
even though the preferred columns
33www.chromatographyonline.com
Francotte
CCCCChhhheeemmiccaallllyy iinnertt
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Hamilton Bonaduz AG
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Switzerland
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HDHT. First cement-free syringefor PAL Combi-xt headspace autosamplers
remain those based on polysaccharides.
Similar to HPLC, a parallel setup with
eight columns operating simultaneously
can be used for screening CSPs in
SFC (40,41). The instrument permits the
rapid identification of optimal conditions
(chiral column, mobile phase) to perform
the enantiomeric separations. Using a
standard configuration with columns of
15 cm × 0.46 cm, a flow rate of 3 mL/
min (7 min for each run), and a gradient
of 5–40% modifier within 5 min, up to
60 different conditions can be tested
within 1 h. In the primary screening,
eight polysaccharide-based CSPs have
been selected (40,41). Analogously
to enantioselective HPLC, almost
any combination of CO2 with organic
modifiers is feasible with the immobilized
polysaccharide-based CSPs because
they tolerate most organic solvents
(40,42).
Hundreds of pharmaceutical
applications have been published and
reported over the last 10 years and
surely many more have been performed,
in particular within the pharmaceutical
companies that apply SFC for hundreds
of new chiral molecules every year.
Although enantioselective SFC
is now well adopted and even the
method of choice in drug discovery
in most pharmaceutical companies,
there is only a very limited number of
validated methods (43,44) because
the SFC technique has not yet been
fully embraced by the pharmaceutical
development community. However,
considering the availability of robust and
high performing analytical instruments,
and the recent publication of numerous
papers demonstrating the possibilities
of SFC for pharmaceutical investigations
in a good laboratory practice/
manufacturing (GLP/GMP) environment,
it can be anticipated that this situation
will rapidly change in the next few years
(45,46).
Analysis of Chiral Molecules
by GC
GC was a major technique for
enantioselective analysis for two
decades (1980–2000). It was researched
intensively and reached a high level of
advancement (47). Numerous stationary
phases have been developed and a
few have been commercialized. The
most used phases are based on amino
acids and cyclodextrins (Figure 1). GC
is particularly appropriate for volatile
molecules, which is not often the case for
chiral drug compounds. However, it
is regularly applied to determine
the purity of volatile pharmaceutical
intermediates and it remains a standard
approach for enantioselective analysis
of amino acids. Nevertheless, the
introduction of the new cinchona
alkaloid ionic phases, which show a
wide application range, might change
this in the future (20). These latter ionic
phases have the advantage that they do
not require a derivatization of the amino
acids before analysis.
GC is very efficient and generally
shows a high separation power. Very
low selectivity values (alpha values
lower than 1.1) are sufficient to achieve
accurate ee determinations. Easy
coupling of GC to MS was also a major
Advances in Pharmaceutical Analysis October 201634
, ,
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Amylose derivatives1 - acid glycoprotein (LC) Cinchona alkaloids
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Saccharide chains (~45%)
N-acetylglucosamine
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Fucose
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183 amino acids
(LC, SFC, CE, CEC)
Cellulose derivatives
(GC, LC, SFC, CE, CEC)
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(GC, LC, SFC, CE)
L-Valine-tert.butylamide
(GC)
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Figure 1: Structures of the most used chiral selectors for enantioselective chromatography. Applied techniques are in brackets.
advantage compared to normal-phase
chiral separation for many sensitive
and biological applications. The
usefulness of GC for enantioselective
analysis has been recently reviewed by
C. Morrisson (48).
Recent applications include the chiral
analysis of nonprotein amino acids (49)
or pesticides in humans (50). The latest
examples of validated methods are the
enantioselective analysis for (L)-pidolic
(51) and the chiral GC–MS method for
the determination of free D-amino acids
ratio in human urine for gestational
diabetes studies (52).
Similar to LC and SFC, chiral
GC requires method development.
Although it is usually done by screening
GC columns one by one, Schafer
and colleagues proposed a parallel
chromatography screening system
(53). Interestingly, most of the current
applications are still performed on the
GC phases developed decades ago, but
there is still some research ongoing to
develop new and more efficient chiral GC
columns. Recent developments include
the utilization of cellulose derivatives
in coated open tubular capillaries,
which has been reported by Zhang
and colleagues (54). The chiral nematic
mesoporous silica column exhibits very
good chiral recognition abilities for a
broad variety of racemic compounds.
Enantioselective GC remains a popular
technique for the analysis of chiral
compounds and is the second technique
in importance after LC.
Analysis of Chiral Molecules
by Capillary Electrophoresis
(CE) and Micellar Electrokinetic
Chromatography (MEKC)
From the mid-1980s, enantioselective
capillary electrophoresis and micellar
electrokinetic chromatography have
been the focus of intensive research. CE
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according to charges and are true
orthogonal methods of analysis because
they work through different mechanisms
compared to GC, LC, or SFC.
Much academic work has been
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a vast amount of original papers on this
topic. Various reviews compiling extensive
lists of pharmaceutical applications are
available (55–57). Interestingly, as for the
other separation techniques, the chiral
selectors derived from saccharides
have developed as the most widely
applicable. The cyclic oligosaccharides
cyclodextrins clearly cover the majority
of the applications. The method has
undoubtedly some advantages in terms of
cost and practical operation.
Although numerous applications
for a wide variety of chiral drugs have
been reported, most of the published
works have been performed in
academic environments and CE has
not reached the level of acceptance
of enantioselective LC analysis in the
pharmaceutical industry. The technique
35www.chromatographyonline.com
Francotte
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Figure 2: Method development for stereoselective drug metabolism analysis of a chiral bis-amido drug. Separation of the four stereoisomers under (a) normal-phase mode, column: 0.46 × 25 cm, 5-μm Chiralcel OZ (Chiral Technologies), mobile phase: heptane/ethanol/diethylamine 70/30/0.03, flow rate: 1 mL/min; (b) polar organic mode, column: 0.46 × 25 cm, 5-μm Chiralpak IE (Chiral Technologies), mobile phase: ethanol 5 mm + 0.03% diethylamine, flow rate: 0.5 mL/min. Elution order of the individual stereoisomers a–d is indicated.
O
O O
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O
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Time (min)
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Time (min)
(d)
(c)
NH
Figure 3: Separations of the enantiomers of selected α amino acids with cyclic and acyclic side chains. Column: 150 × 3 mm, 5-μm Chiralpak ZWIX(+) (Chiral Technologies); mobile phase: 50 mM formic acid + 25 mM diethylamine as additives in methanol/acetonitrile/water 49:49:2; detection: ELSD. Adapted with permission from reference 20.
is still suffering from its lack of robustness
and relatively low sensitivity, even
though some progress has been made
to improve this (58). The question of
the limited integration of CE in the
pharmaceutical industry was recently
discussed (59) and this trend is reflected
by the small number of CE publications
from this sector over the last three
years. Nevertheless, a few practical
applications have been achieved in the
industrial pharmaceutical environment,
including validated processes with UV or
MS detection (60–63).
Analysis of Chiral Molecules
by Spectroscopic Methods
Enantioselective analyses by
spectroscopic methods have drawn
the attention of many research groups
for a long time. In particular, NMR
using a chiral solvating agent (CSA)
(64) has been, besides optical rotation
(αD), a standard approach for the
determination of optical purity before
1980, at a time where no chiral stationary
phases were available. NMR using
CSA is a simple method and does
not need previous derivatization of
the analyte with a chiral molecule to
form diastereoisomers. It was much
used until the end of the century, but
because of its limited accuracy (± 3%)
NMR has been progressively replaced
by chromatographic techniques. The
value of the technique has recently
been reviewed (65) and applications to
pharmaceutical drugs are still regularly
published (66–68). A number of new
chiral solvating agents showing excellent
chiral discrimination properties were also
developed (69), giving accuracy values
approaching those of chromatography
on chiral stationary phases but still lower
(about 1% of the minor enantiomer).
Nevertheless, it is definitely less broadly
applicable than LC or GC.
MS has also been applied to
determine ee (70), and the promise of the
method has recently been reviewed (71).
The feasibility of using an MS instrument
solely for chiral recognition has
clearly been demonstrated and a first
practical application for the quantitative
determination of the chiral purity of an
antibiotic drug by flow-injection MS/
MS has been reported by Wu and
colleagues two years ago (72). However,
this approach is not broadly applicable
and it is likely that it will not reach the
level of popularity of the chromatographic
techniques.
VCD has also been reported to be a
suitable technique for ee determination
(73,74) and might be useful in particular
cases (75,76), but it cannot compete with
more generally applicable techniques for
daily use.
Enantioselective Sensors
The development and application of
enantioselective sensors is a topic of
increasing interest and might possibly
become a useful technique for ee
determination. Progress in this field
has recently been reviewed (77).
Very recent applications include the
discrimination of the enantiomers
of amino acids or ascorbic acid by
applying electrochemical processes
(78–80) or the enantiomers of naproxen
by recording change in the optical
properties (colorimetry) (81). Although
this approach requires the development
of a specific sensor for each particular
application, it can be of great interest
in diagnostic or environmental analysis
and has the advantage that it can be
miniaturized.
Conclusion
A broad variety of chromatographic and
spectroscopic approaches are available
for the enantioselective analysis and
determination of the chiral purity of
chiral drugs. Among all approaches,
chromatography is clearly the preferred
technique. Methods are easy to
elaborate with a relatively limited number
of chiral stationary phases, mainly
made from polysaccharide derivatives
in LC or SFC, and from cyclodextrins
or amino acids in GC, as the chiral
selectors. Interestingly, the number of
chiral selectors that have emerged as
the most widely applicable is relatively
limited, whatever the technique, and
even more fascinating is that the great
majority are composed of or contain a
large amount of saccharides. Compared
to the chromatographic methods,
spectroscopic approaches are usually
less accurate and are not so generally
applicable, even though their usefulness
has been demonstrated in some cases.
References(1) G. Hesse and R. Hagel, Chromatographia
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(3) J. Shen and Y. Okamoto, Chem. Rev. 116,
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Pharmaceutical Discovery 4(9), 26–32
(2004).
(5) R. Sneyers, T. Vennekens, T. Huybrechts,
I. Somer, G. Torok, and S. Vrielynck, LCGC
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(12) +��-FF �8�-��8BUUT �+��#BSFOEU �5�2�:BO �:��
Huang, F. Riley, M. Hardink, J. Bradow, and
P. Franco, J. Chromatogr. A 1374, 238–246
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(13) P. Franco and T Zhang, in Chiral
Separations: Methods and Protocols, (�,�
Scriba, Ed. (Methods in Molecular Biology,
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Advances in Pharmaceutical Analysis October 201636
Francotte
0.25
0.030
0.025
0.020
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0.00
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Column: Chiralpak-IC
MP: n-Hexane: Ethanol
85:15 (v/v) (R)-
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-1
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LAC (lacosamide)
0.15% spiked chromatogram
Imp-1 (impurity)
NH0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
Time (min)
Time (min)
Figure 4: Separation of the enantiomers of the lacosamide drug and its chiral impurity on 250 mm × 4.6 mm, 5-μm Chiralpak-IC column (Chiral Technologies); mobile phase: n-hexane/ethanol 85:15 (v/v); flow rate: 1.0 mL/min; oven temperature: 27 °C. Adapted with permission from reference 24.
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Eric Francotte received his Ph.D. in
organic chemistry from the University of
Louvain in Belgium, and spent two years
as a postdoctoral fellow at the University
of Geneva in Switzerland. He joined
former Ciba-Geigy (Novartis) in 1980
where he established a centre of expertise
for the development and application of
chiral polymers for the chromatographic
resolution of chiral compounds. After 10
years he moved to Novartis pharma as an
executive director and was responsible
for a global technology platform dealing
with separation sciences. His major
achievements include the invention of
an innovative process to immobilize
polysaccharide derivatives as chiral
stationary phases. Several phases
arising from this technology have been
marketed as the result of an external
collaboration and are considered as the
new gold standards in enantioselective
chromatography. He was also a
pioneer in the implementation of new
chromatographic technologies, such
as SMB and SFC. He is the author or
co-author of numerous publications,
patents, and editor of several books.
He has chaired various international
symposia in the field of preparative
chromatography, chirality, and SFC. He
is now an independent consultant for
separation science.
37www.chromatographyonline.com
Francotte
The ever-growing problem of drug
purity and counterfeit medicine needs
to be addressed. It is estimated that
an important portion of all world trade
in branded pharmaceutical products
is counterfeit, leading to a great health
risk and public health concern. As
witnessed by numerous official sources,
including the World Health Organization
(WHO), the proportion of counterfeit
medicines has tremendously increased,
representing more than 50% in some
developing regions of the globe (1). The
primary reported cases of counterfeits
are affiliated to emerging countries, where
economically middle-income markets are
most represented (that is, Sub-Saharan
African countries, as well as Asian and
Latin American regions). According to
health-related government agencies,
the most prevalent therapeutic category
affected by counterfeits is anti-infectives
(21.1%), with the much needed antibiotic,
antiparasitic, and antiretroviral medicines
(2). Discovering and determining
counterfeit drugs is mandatory not only
for these countries, but also for others,
particularly when considering the internet
market (3). In order to characterize
counterfeit and substandard medicines
(Figure 1), different levels of action can be
applied. The evaluation of the packaging
based on a comparison with the genuine
sample and the search for manipulation
signs is appropriate for detecting
counterfeits, whereas chemical methods
should be implemented to authenticate
the samples and provide the chemical
composition of substandards (7).
Characterization of Counterfeit
and Substandard Medicines with
Chemical Analysis
There is an urgent need to set up tools
and workflows to fight against counterfeit
and substandard medicines and their
associated health risks. Evaluating
the quality of active pharmaceutical
ingredients (APIs) and excipients usually
requires a broad variety of modern
analytical methodologies, allowing the
qualitative and quantitative determination
of the content and the purity of the drugs
(8).
Methods for qualitative analysis include
basic but critical physical, organoleptic,
and haptic tests, as recommended
by international Pharmacopoeia.
Colorimetric assays using chemical
assays reacting with functional groups
is another traditional way to identify drug
compounds, although false-positive
results may be obtained because of
the low specificity of such assays,
circumventing the determination of
structurally similar molecules, such as
impurities. These tests are therefore
mainly implemented as a first and
rapid screening to establish only the
presence versus absence of an API in
a medicine. Spectroscopic techniques,
such as Raman, near infrared (NIR),
and nuclear magnetic resonance (NMR)
spectroscopy, are used to control the
quality of pharmaceuticals because
they provide a wealth of information on
the molecular structure of the APIs and
excipients, as well as on their spatial
distribution. Separation techniques, such
as thin-layer chromatography (TLC), high
performance liquid chromatography
(HPLC), gas chromatography (GC),
and capillary electrophoresis (CE), are
more selective assays and can be used
for qualitative as well as quantitative
determination. They are fully established
and have been added to almost all
monographs of major Pharmacopoeia for
quality control (QC) purposes. The higher
selectivity of hyphenated techniques
(that is, with mass spectrometry [MS])
offers detailed pictures of an analyte or
an analyte mixture, while the structure
of unknown contaminants can be
elucidated and determined in very small
quantities. In modern QC laboratories
these analytical instruments are mostly
available, allowing for an extensive
product testing during production
and during the distribution within the
respective supply channels. However,
they need trained people to use them
and samples generally need to be
brought to the laboratory to be analyzed.
In emerging countries, comprehensive
QC of circulating medicines is hardly
achieved because of relatively
young healthcare systems, restricted
laboratory capacities, weak analytical
infrastructures, and chaotic distribution
logistics. Therefore, a sequential
Characterization of Counterfeit and Substandard Medicines Using Capillary ElectrophoresisJulie Schappler and Serge Rudaz, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU,
Geneva, Switzerland
The proportion of counterfeit medicines has increased dramatically. Combatting this issue is complex, and
various levels of action are necessary. The quality control (QC) of imported batches using simple, reliable,
and cost-efficient analytical approaches is vital. Capillary electrophoresis (CE) is becoming important
because the analysis is achieved in a capillary with small dimensions, and is usually filled with an aqueous
buffer. No organic solvent is required and injection volumes are in the nanolitre range, which is convenient
because of the low availability of reference substances and reduces the environmental impact. CE is now
recognized by numerous Pharmacopeia and can be used for counterfeit and substandard characterization
as a validated analytical procedure that adheres to international guidelines.
Ph
oto
Cre
dit: H
ero
Im
ag
es/G
ett
y Im
ag
es
Advances in Pharmaceutical Analysis October 201638
approach is recommended, according to
the goal of the analysis, the localization of
the laboratory, and the overall technical
skills of the analyst (Figure 2). In a first
approach, a large screening can be
performed with a nondestructive method,
for example, vibrational spectroscopy
(Raman, NIR). These methods give rapid
and low false-negative results, can be
brought to the field where the samples
can be tested, are sufficiently easy to
handle by people with little training, and
can be conducted without special, or
reduced, sample preparation (9). When
a sample does not comply in terms
of identity or quantity, a confirmatory
analysis should be done with an
orthogonal approach, such as that
afforded by separation techniques. As
previously mentioned, separation-based
devices including GC, LC, or CE are often
not portable, therefore the suspicious
samples are generally brought to the
laboratory where the analysis takes place
by trained people (10).
Rodionova et al. applied the
sequential approach for the
detection of counterfeited ampoules
of dexamethasone (11). First, NIR
measurements with chemometric
data processing were performed
directly in the closed ampoules of the
injectable formulations. The technique
was able to detect small differences in
the samples and emphasized reliable
discrimination between genuine and
counterfeit samples, using a modelling
set that included samples from various
batches (Figure 3[a]). Samples were then
subjected to GC–MS, HPLC coupled to
a diode array detector (DAD), and CE
coupled to an ultraviolet (UV) detector
for confirmation. GC–MS did not find any
differences between genuine and fake
samples, avoiding the discrimination
between both samples. HPLC–DAD
revealed that samples from different
batches were identical, but for some
impurities they differed in quantity. A
new impurity (marked with an asterisk)
was found in a fake sample (Figure 3[b])
and MS was further applied for its
identification. According to the high
efficiency and orthogonal selectivity of
the separation, CE–UV reliably found
three impurities in the fake sample that
were not present in the genuine sample
(Figure 3[c]). In this study, the CE method
showed the most informative results
for discriminating samples with close
chemical composition.
As can be seen from this study,
chromatographic approaches can be
applied for the confirmatory analysis,
but they suffer from several drawbacks
that often hamper their use in emerging
countries: (i) they are expensive to
acquire, maintain, and run; (ii) high
amounts of reference material and
organic solvents are needed; (iii) the
consumables and the chemicals may be
prohibitive and difficult to obtain.
In this context, CE has a role to
play in this fight because it offers
a low consumption of sample and
reference material, little use of organic
solvent, affordable consumables and
maintenance, and a basic requirement of
chemistry knowledge.
Characterization of Counterfeit
and Substandard Medicines Using
Capillary Electrophoresis
The well-established CE technology can
be applied for the characterization of
counterfeit and substandard medicines.
In this article we present several CE
applications, as well as developments
that were made in this context towards
the instrumentation and the methods.
Applications: Vidal et al. developed
a method based on CE coupled to
capacitively coupled contactless
conductivity detection (C4D) for
the determination of an important
counterfeited medicine, sildenafil, used
for treating erectile dysfunction (12).
Experiments were performed with a
laboratory-made CE device equipped
with two C4D detectors in series. The first
detector afforded a low efficiency but
fast detection, while the second detector
was used to obtain higher efficiency
results. The background electrolyte
(BGE) was made of acetic acid 0.5 M
and the capillary was coated prior to a
series of analyses with a cationic coating
to prevent analyte adsorption on to the
capillary wall. An interesting feature of
C4D detection was the possibility to
detect in the same run, not only sildenafil,
but also its associate salt (citrate).
Thus, commonly counterfeited tadalafil
or vardenafil tablets (which should be
exempt of citrate) containing sildenafil
could be easily detected.
39www.chromatographyonline.com
Schappler and Rudaz
Counterfeits Su
bst
an
dard
s
Fails quality standards
IllegalLegal
Meets quality standards
Illegitimatebad treatment
Legitimatebad treatment
Legitimategood
treatment
Illegitimategood
treatment
Figure 1: Categories of medicines. The medicines failing quality standards are considered “substandards” and they can be legitimate or illegitimate. The unapproved medicines are considered “counterfeits” and they can meet or not meet the quality specifications.
Laboratory
On-site
Pharmacopeia QC
2. Confirmatory assays
1. Large screening
Sequential approach for the characterization ofcounterfeits and substandardsin emerging countries
Governmental
authorities
for example, separation techniques,
hyphenated techniques
for example, colorimetric assays,
spectroscopic techniques
Figure 2: Proposed hierarchic testing of medicines using different analytical approaches.
Antimalarial drugs are another major
class of counterfeit medicines that are
dramatically present on the African
market. Lamalle et al. developed a
CE–UV method for 15 antimalarials (13).
As these molecules cannot be ionized
at the same pH, micellar electrokinetic
chromatography (MEKC) was preferred
over capillary zone electrophoresis,
allowing the separation of neutral
compounds. Preliminary experiments
were first performed to select the
most crucial factors (including pH,
surfactant concentration, acetonitrile
proportion, and temperature). Next,
an experimental design methodology
was applied to enable the prediction
of optimal conditions for best analyte
separation in less than 8 min. Finally,
the method was successfully applied to
the quality control of African antimalarial
medicines for their qualitative and
quantitative content. At the same time,
another European laboratory developed
a MEKC–UV method for artesunate and
amodiaquine in fixed-dose combination
tablets (14).
Capillary electrophoresis was also
used to determine adulterants in
pharmaceuticals. De Carvalho et al.
applied CE–C4D for the determination
of adulterants in herbal weight loss
products collected in pharmacies
located in different Brazilian states.
A first study emphasized that either
anxiolytics, diuretics, or laxatives were
added to 4% of the weight products
collected (15). In a second study, CE
analyses revealed that more than 30% of
the analyzed formulations were found to
contain diuretics in the final composition
(16). These drugs have a low incidence
of side effects, but are associated
with several metabolic disorders
when administrated in high doses.
The developed CE procedure, which
enables a rapid and selective screening,
is now considered by governmental
organizations for inspecting commercial
dietary supplement formulations.
In another study, Cianchino et al.
developed a CE method with a
basic BGE to obtain a characteristic
fingerprint of Hedera helix L., used as
a cough treatment in Argentina (17).
The developed approach enabled the
determination of adulterants, such as
synthetic drugs used in respiratory
diseases (for example, ephedrine,
codeine, diphenhydramine) in
phytopharmaceutical formulations. The
fingerprint could also help to distinguish
differences between Hedera helix L.
from various sources.
Instrumental Advances:
Introducing high-quality separation
analysis into the daily fight for major
health and public safety issues and doing
it at an affordable cost is challenging.
Taking into account the limited technical
and financial resources in most
developing countries, a few initiatives
have emerged to provide sustainable
and affordable CE instruments that can
be easily implemented at a low cost by
organizations within these countries. For
example, a collaboration between Swiss
academic institutions and hospitals was
implemented to build a low-cost CE
device and help transitional countries
with limited means to fight against
counterfeit and substandard medicines
(18). A series of iteratively refined and
field-proofed CE units were built. The
first prototype addressed most of the
mechanical and electronic issues, and
included an original UV detection device
based on a LED technology. The second
generation improved the software and
the apparatus ergonomics, while the
last prototype included modifications
to improve the system robustness.
Methods were developed for drugs
selected from among a list provided by
several African partners. Marini et al.
evaluated the analytical performance
of this low-cost CE equipment for drug
quantification (19). A complete validation
study with reduced requirements
regarding calibration purposes was
Advances in Pharmaceutical Analysis October 201640
Schappler and Rudaz
(a)
(b)
(c)
4.0
3.0
2.0
5500 7000
5
5 6 7 8
10 15 20 25
8500 10000
1.0
1000
800
600
400
0.8
0.6
0.4
0.2
0.0
-0.2
-0.4
200
0
0.0
cm-1
Time (min)
Time (min)
F2
G2
G1
G1
Absorbance
units (AU)
Absorbance
units (m
AU)
Absorbance
units (m
AU)
F2
Figure 3: Detection of counterfeited ampoules of dexamethasone using several analytical approaches. (a) A raw NIR spectra of 30 genuine samples (blue lines) and 15 fakes (red lines); (b) HPLC–DAD chromatograms of a fake (F2) and genuine (G1 and G2) samples; (c) CE–UV electropherograms of a fake (F2) and genuine (G1) sample. Adapted with permission from reference 11.
performed on medicines representing the
most targeted pharmacological groups
by counterfeiting, that is, antimalarial,
diuretic, and anti-infective products.
A comparison study was performed
with commercial CE equipment and
equivalent quantitative performance
was obtained between both devices,
demonstrating the great potential of this
simple and low-cost device (19). Finally,
concrete positive results in emerging
countries (mostly in Africa and in Asia)
were obtained, thanks to cooperation at
a geographically well-distributed series
of sites, implementing the prototype
instruments there and successfully
training the local personnel to accomplish
reliable identification and quantification of
pharmaceuticals. Sarr et al. implemented
the device at the Senegalese National
Medicines Control Laboratory where
a CE method was developed and
validated for the quality assessment
of metronidazole-based drugs (20).
Eleven metronidazole samples were
taken from various markets in Benin and
Senegal and analyzed by CE–LED/UV.
Fortunately, all collected samples were
compliant with USP specifications. The
results obtained with the low-cost CE
apparatus were confirmed with an official
chromatographic method described in
the US Pharmacopoeia. Similar results
for both methods with comparable
precision were obtained, demonstrating
that low-cost CE analysis could be a real
alternative in developing countries for
drug QC to protect population health.
It should be noted that although
portability of the device is not afforded
by the main suppliers, CE can also
present a great potential in point-of-care
diagnostics, where the speed of the
analysis, the device compactness, and
the sample volume are critical factors.
Gregus et al. developed a homemade
novel, simple, portable CE instrument
equipped with C4D for the analysis
of small volumes of biological fluids
(21). The instrument is light (<5 kg),
all necessary parts including a tablet
computer are accommodated with
small dimensions (20 × 33 × 17 cm),
hydrodynamic injection is performed
with less than 10 μL sample volume, and
the device can continuously operate for
at least 10 h. Several applications were
demonstrated, including the diagnostics
of respiratory tract diseases (for example,
asthma, chronic obstructive pulmonary
disease, cystic fibrosis).
Recent Method Developments: To
analyze a high number of compounds
and benefit from CE with basic chemistry
knowledge and training, simple and
generic methods should be applied to
determine counterfeit and substandard
medicines. In this context, Schappler
et al. developed a strategy based on
multiple injections to simultaneously
characterize and quantify APIs in
medicines in one single run, using an
external calibration and an internal
standard (22). Two generic CE–UV
methods were developed, using a BGE
at an acidic or a basic pH for basic or
acidic compounds, respectively. More
than 80 drugs from the list of the 200
essential medicines defined by WHO
could be analyzed with this approach.
Both methods were fully validated
according to international guidelines for
two important APIs (metronidazole and
ampicillin). They were further applied
for QC of formulations and detection of
substandards in samples from Tanzania.
This methodology was also implemented
for the simultaneous identification and
quantification of insulin formulations,
obtained from regular and parallel
markets, by CE with time-of-flight mass
spectrometry (TOF-MS) detection (23).
Particular attention was paid to the BGE
composition and acetonitrile added to the
BGE to enable direct MS coupling as well
as reduction of the protein adsorption.
The multiple injection approach offered
an alternative way to compensate for
ionization variability and matrix effect in
the absence of stable isotope-labelled
compounds for insulin. Two injections
were performed in the same analytical
run, the first one with a standard of insulin
at a known concentration and the second
one with the sample to be identified and
quantified. Figure 4(a) shows the total
ion electropherogram (TIE) obtained
with this multiple injection approach. The
41www.chromatographyonline.com
Schappler and Rudaz
(a) 2
x106
a
a
b
b
x103
1
1
0
x105
4
2
0 2 4 6
1000 1500
6 7 8
2000
Counts versus Acquisition Time (min)
Counts versus Acquisition Time (min)
Counts versus Mass-to-Charge (m/z)
8
[M+3H]3+
1452.921
[M+4H]4+
1936.557
7.349
6.518
(b)
(c)
Figure 4: Analysis of insulin by CE–TOF-MS. (a) Total ion electropherogram (TIE); (b) extracted mass spectrum used for identification purpose; (c) extracted ion electropherogram (XIE) used for quantification purposes. Adapted with permission from reference 23.
(M+3H)3+ and (M+4H)4+ multicharged
ions were detected as the major extracted
ions (1937 and 1453 m/z, respectively,
(Figure 4[b]) and enabled identification
of insulin in unknown samples. The
extracted ion electropherogram (XIE)
was reconstructed using both ions and
integration was achieved for quantification
purposes (Figure 4[c]). It should be
noted that this approach is currently not
adapted for emerging countries because
of the expense and the need for highly
trained MS coupling.
Lamalle et al. alternately used
MEKC–UV to distinguish between human,
bovine, and porcine insulins in less than
12 min and easily determined if human
insulin was replaced by bovine or porcine
insulin in counterfeited pharmaceutical
formulations (24).
Conclusions
The expanding use of counterfeit drugs
in developed and emerging countries
is a growing problem and is associated
with challenges in QC. CE features high
performance (qualitative and quantitative
methods can be implemented and
validated), high sustainability (one
analysis consumes less than 1 μL
of sample, reference material, and
solvent), and low costs (analyses and
maintenance are affordable), and so it
is currently recognized as a contributing
technique that helps health organization
laboratories to spot check medicines
in an effort to monitor counterfeit and
substandard drugs and therefore deliver
high-quality medicines to vulnerable
populations.
A few issues still remain to fully
implement CE technology in emerging
countries. Because CE relies on a high
electric field, the input electric power has
to be reliable, which is, unfortunately,
not the case, with many countries
experiencing unpredictable power
breakdowns. Further improvements
should therefore be implemented in
CE equipment to render the device
more autonomous, sustainable, and
accessible, at even lower costs: (i) a
renewable energy source to supply high
voltage regardless of the local electricity
facilities; (ii) a universal detection system
to broaden the analysis range to any
pharmaceutical drug; (iii) a reduction of
the whole device dimensions to obtain
a disposable and portable system; and
(iv) integrated software to simplify data
treatment and reporting by the user. In
addition to continuous improvements
of the methodological and technical
aspects of the analytical device,
educational programmes should also
be developed and implemented (for
example, with e-learning tools) to provide
adequate initial and continuing guidance,
as well as sustainable and readily
deliverable training.
In this battle, a big stake is the share
of the information. Counterfeit medicines
are distributed through international
routes and it can be difficult to predict
them. Efforts should be made to quickly
and accurately detect points of entry and
locate where to perform the first step of
the analysis. The contributions of forensic
scientists will be decisive in all technical
aspects, including profiling seizures
with chemical and packaging data. The
interpretation of data on the production
and the distribution of counterfeits has
the potential to be a reliable approach to
aid in the understanding of the organized
crime phenomenon behind counterfeiting
and to enable efficient and operational
strategies for decision making (25). In
this context, citizen science projects
(for example, Hackathon at CERN [26])
are emerging that collect information
on medicines with devices that can be
used by anyone (such as smartphones
to take pictures, geolocalize products,
and create and curate databases).
This approach is particularly attractive
and complementary to chemical
analysis because it involves the global
population in the constant battle against
counterfeiting. Another important aspect
of medicines relates to the trust in drugs
by the patients themselves. Blockchain
databases, when combined and fully
integrated with analytical approaches,
offer an additional tool to strengthen
the trust and monitor the quality in drug
distribution chains, and should be
investigated further.
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pier71
Julie Schappler is a pharmacist and
holds a Ph.D. from the University of
Geneva. She is currently a lecturer and
research associate. She heads the
unit of capillary electrophoresis and
sample preparation, which works on
developing methods to improve analysis
performance, while reducing analysis
time and cost.
Serge Rudaz is an associate professor
at the University of Geneva. He is a
research group leader and member of the
management board of the Swiss Centre
for Applied Human Toxicology (SCAHT)
Foundation. He is also President of the
Competence Center in Chemical and
Toxicological Analysis (ccCTA).
Advances in Pharmaceutical Analysis October 201642
Schappler and Rudaz
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