SEPTEMBER 2017

Analyzing Genotoxic
Impurities in
Pharmaceuticals

Sponsored content from Presented in partnership with

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IntroDUCTION
G
enotoxic impurities (GIs)—which can induce genetic mutations— are
a major cause for concern among drug makers and regulators. GI
analysis is not easy, however, because it often requires methods that
offer excellent selectivity and sensitivity at subpart-per-million levels.
Therefore, providers of analytical techniques have been exploring new analytical
methods that can help improve and accelerate GI detection and analysis.

Analysts wanting to keep abreast of important developments in this area

have a new resource available to them: Analyzing Genotoxic Impurities in
Pharmaceuticals, an ebook sponsored by Waters Corporation and presented in
partnership with LCGC.

This compilation of articles starts with a piece authored by several small-

molecule pharmaceutical scientists at Genentech. They discuss several factors
that must be considered when developing an analytical method for GIs, and
suggest that hyphenated mass spectrometry methods are gaining popularity for
this purpose.

Next, we hear from a team of scientists from Ghent University and the Research
Institute for Chromatography of Belgium about new developments in robotic
sample preparation and ultrahigh-pressure liquid chromatography (UHPLC) and
gas chromatography systems with sub-ppm sensitivities.

The third piece in this ebook covers a methodology for selecting the best
techniques for the quantitative analysis of GIs in active pharmaceutical
ingredients at trace levels.

Last, a group of AstraZeneca scientists attempts to demonstrate how test

mixtures can facilitate both the development of UHPLC–MS methods and
automated performance monitoring of several instruments in various locations.

Given the importance of keeping GIs within acceptable safety limits, analytical
laboratories should keep abreast of the latest advancements and strategies for
detecting these impurities in pharmaceuticals.
TOC
Table of contents
Analyzing Genotoxic Impurities
in Pharmaceuticals

Analytical Technique Selection

Analytical Technologies for Genotoxic
Impurities in Pharmaceutical Compounds
Archana Kumar, Kelly Zhang, and
6 Larry Wigman

Chromatographic Trace Analysis

Determination of Genotoxic Impurities
in Pharmaceuticals
Frank David, Maria Ramble Alegre,
25 Gerd Vanhoenacker, and Pat Sandra

Methods Development
Method Selection for Trace Analysis of
Genotoxic Impurities in Pharmaceuticals
Frank David, Karine Jacq, Gerd Vanhoenacker,
36 Pat Sandra, and Andrew Baker

Performance Verification
A Test Mixture for Performance
Verification of Multi-User UHPLC–MS
Instruments
Tomas Leek, Niklas Falk, Ayda Babapour,
53 and Magnus Klarqvist

This eBook is sponsored by Waters Corporation and presented by LCGC.

V I E W I M P U R I T Y A N A LY S I S
IN A NEW LIGHT.
The demand to develop ground-breaking pharmaceuticals and
generics faster has never been higher. It’s a race to market where
the presence of harmful impurities simply cannot be missed.
Where mission-critical meets life-critical, you’ll find Waters–helping
you ensure the quality, safety and efficacy of drugs. Download the
latest methods in impurities analysis at impurities.waters.com

PHARMACEUTICAL Q
HEALTH SCIENCES Q
FOOD Q
ENVIRONMENTAL Q
CHEMICAL MATERIALS

©2017 Waters Corporation. Waters and the Science of What’s Possible are registered trademarks of Waters Corporation.
 Analytical Technologies
for Genotoxic Impurities
in Pharmaceutical
Compounds
Archana Kumar, Kelly Zhang, and Larry Wigman

Sponsored

Direct Quantification of Genotoxic impurities (GTIs) have gained

a GTI Using Convergence
Chromatography and considerable attention from health authorities
Click to Mass as well as from the pharmaceutical industry
view PDF
in recent years. Analysis and control of these
impurities in pharmaceutical compounds pose a
significant challenge, often requiring selective,
sensitive, and robust trace-level methods
for analysis. This article focuses on the
method development strategy and associated
Sponsored technologies for the analysis of GTIs. GTIs
typically have a wide range of physicochemical
Analysis of GTIs by Mass
Detection and Diverter Valve properties such as volatility, stability, presence
of UV chromophores, ionizable groups, and
Click to
view PDF
derivatizable functional groups. Various
separation and detection technologies are
available and can be used to identify and
analyze. Choosing the appropriate analytical
technique depends on the GTI physicochemical
properties, required sensitivity, and the
consideration of the matrix interference.

Genotoxic impurities (GTIs) belong to a class

shutterstock.com/Looker_Studio

of compounds that interact with DNA and

induce genetic mutations. These compounds
add significant risk without adding any benefit
to patients and are also known as mutagenic
impurities. Therefore, exposure to even low levels

6 | September 2017 | LCGC

 ANALYTICAL
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of such impurities may be of significant
toxicological concern.
Genotoxic assessment is required
throughout synthetic route development
and stability duration for the presence
of any GTI alerts. Demonstrating that
GTIs are controlled to safe levels is of the
utmost importance for patient safety (1).
In recent years, GTIs in pharmaceuticals
have become a topic of increasing
concern, consequently several review
papers have been published focusing on
the regulatory and toxicology assessment
(2–7), control strategy (7–9), and risk
assessment (10). Effective analytical
technologies for selective, sensitive, and
robust trace-level detection methods
are required for low-level quantitation.
Physicochemical properties of the GTIs
that need to be considered include
volatility, stability, presence of UV
chromophore, ionizable groups, and
derivatizable functional groups.
Regulatory Overview
GTIs are required to be controlled at
lower limits (11,12) compared to less-
toxic impurities controlled in new drug
substances (13,14) and drug products
(15). The concept of staged threshold of
toxicology concern (TTC) was developed
to allow higher limits for compounds in
development based on the duration of
exposure rather than lifetime exposure (16).
The International Conference on
Harmonization’s (ICH) M7 document
“Assessment and Control of DNA Reactive
(Mutagenic) Impurities in Pharmaceuticals
7 | September 2017 | LCGC
METHODS PERFORMANCE
DEVELOPMENT VERIFICATION
to limit Potential Carcinogenic Risk”
provides recommendations for toxicology
assessment, identification, categorization,
and control of actual and potential
mutagenic impurities that are likely to arise
during the manufacturing and long-term
storage of a new drug substance and drug
product (17).
Compound-specific calculations for
acceptable intakes may be applied where
sufficient carcinogenicity data are available
or if the impurity is similar to a known
carcinogen compound class. Performing
spiking and purge studies during the
development stage and justifying the
presence at higher limits for the impurities
may not only mitigate routine analysis at
trace levels, but also may mitigate the
need to specify control levels in drug
substance specifications.
ICH S9 document guidance supports
the concept that higher limits may
be appropriate for a drug candidate
or commercial product targeted for
advanced cancer with life-threatening
malignancies (18).
Structures of Commonly
Encountered GTIs
Structures of some of the impurities
commonly observed in pharmaceutical
synthetic routes that get structural alerts
are shown in Figure 1. These functional
groups are generally linked to genotoxicity
and are identified based on the chemistry
and structure–activity relationship (16).
Boronic acids were recently recognized
as mutagens, but do not have enough
 ANALYTICAL
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CI, Br, l O O O O O
A P S S
CI, Br, l OR A OR RO OR
Halo alkenes Primary halide Alkyl esters of phosphonates
and sulfonates

OH
NO2 NH2 N
A O
R R OOH
Aromatic nitro Aromatic amine N-Hydroxylamine Epoxide Hydroperoxide

OH
O O R N NH2
R B H
R H R CI OH
Aldehyde Acid chloride Boronic acid Hydrazine

A = H, alkyl, aryl; R = alkyl

Figure 1: Structures of commonly observed GTIs.

carcinogenicity data. Galloway and
colleagues (19) compared the data for 361
various chemicals used in the synthetic
route that had structural alerts and were
tested for Ames. They reported that 80% of
aromatic nitro and boronic acid derivatives
had positive Ames results, which was the
highest among the selected samples, and
50–60% of alkylating agents, acid chloride
derivatives and hydrazines, were positive.
Analytical Method Development
Strategy for GTI Analysis
GTIs were controlled at a TTC of 1.5 μg/
day at commercial stage. For a 1-g daily
dose of the marketed product, a GTI
would require control at a level of 1.5
ppm ([1.5 μg/day]/[1 g/day] = 1.5 ppm),
which is several hundred times lower
than the 0.05% (500 ppm) control level
per the ICH Q3 guidance for traditional
pharmaceutical impurities analysis.
Further, in case of the presence of three
or more mutagenic impurities in the drug
substance specification, total mutagenic
impurities should be limited to 5 μg/day
for clinical development and marketed
products. Considering such a low level,
robust and sensitive analytical methods
are a critical element of the control and
analysis of GTIs. Before developing a
method, it is important to understand a
few factors:
• the purpose of testing — whether
the method will be implemented for
API release testing or used during
development (spiking and purge
studies);

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Analytical
technologies

GC HPLC
(volatile, (limited volatility
thermally stable) thermally labile)

LC–UV LC–MS (SIM)

Direct Headspace Derivatization (high sample LC-MS/MS Derivatization
injection loading) (MRM)

Matrix interference removal To improve To improve

to enhance sensitivity volatility volatility

LLE
Analyte LLME To improve To improve
extraction SPE stability stability
SPME

To improve To improve
2D-GC ionization ionization
for MS for MS

Figure 2: Analytical method development strategy.

• the need for a limit test or quantitative
method — the requirements for
method validation vary;
• the physicochemical properties of the
analyte;
• and the end user of the method — it
can be a development laboratory, a
commercial user, or a manufacturing
laboratory.
With frequent changes in dose and
duration during the clinical trial stage, it is
often necessary to collect quantitative data
at the development stage. Quantitative
data collection requires a method with
lower detection limits than the analytical
specifications limit. After the process
understanding is gained and lower levels
are attained for each impurity compared
to the control level, a limit test can be
used rather than a quantitative test. Based
on physicochemical properties of the
analyte, gas chromatography (GC) or high
performance liquid chromatography (HPLC),
two of the most commonly used analytical
techniques, may be used (Figure 2).
Analysis of Genotoxic
Impurities by GC
GC is a preferred separation technique for
the analysis of volatile analytes. Direct liquid
injection and headspace are two commonly
used injection modes for GC analysis. The
specification limit calculated based on the
daily dose is a key factor in determining
the desired sensitivity of the method and
subsequently the selection of the detector.

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Table I: Literature references for sensitivity enhancement by removal of matrix interference

Sample Preparation Technique Detection Analyte DL (QL) Reference Year
Headspace GC FID Alkyl chlorides 0.8 (13.5) ppm Kaleemullah (23) 2011
Matrix deactivation MS Various <1 ppm Sun (24) 2010

Solid-phase microextraction MS (SIM) Sulfonates 5 ppm Colon (25) 2005

LLE direct injection (deuterated internal MS (SIM) DMS 0.3 (1) ppm Zheng (26) 2009
standard)
LLE direct injection MS Mesylates, besylates <0.1 ppm Wollein (27) 2012
SAX-SPE direct injection MS (SIM) 2-Chloroethanol 1.7 ppm Garcia (18) 2012
2D-GC MS Multiple <1 ppm Frank (28) 2010

GTI Analysis Using Direct Injection
GC analysis using direct liquid injection is
the primary choice for analysis of volatile
and thermally stable analytes such as alkyl
mesylates that lack the sufficient vapor
pressure required for the headspace
technique (20). The dissolve-and-inject
approach significantly simplifies the
sample preparation before analysis. Flame-
ionization detection (FID) is preferred
because of its easy availability and versatility.
FID may be used for higher control levels
that are justified by low dosage drugs;
however, sensitivity is somewhat limited (21).
The high sample loading technique may be
applied to obtain the desired sensitivity (17).
A hyphenated GC–mass spectrometry
(MS) technique in selected ion monitoring
(SIM) mode provides better sensitivity and
selectivity for impurities analysis at sub-
part-per-million levels. The most prominent
fragment ion is selected to obtain the
desired sensitivity and resolution. The
GC–MS method using SIM and repetitive
scanning has been reported for the analysis
of mesylates (20) and various alkyl halides
(22). However, this method has a restriction
of 1 μL for injection volume because of
the accumulation of active pharmaceutical
ingredients (APIs) and resulting matrix
interferences, which additionally require
frequent changing of the inlet. Inlet
contamination and resulting matrix
interference are the primary limitations
of the direct injection analysis method.
Most drug substances are nonvolatile,
and solutions might need to be prepared
at higher concentrations to achieve
sensitivity. This contamination may result in
irreproducibility and low recovery problems
during method validation and testing.
Enhancing Sensitivity by
Removing Matrix Interference
The presence of excessive matrix
interference may have a significantly
adverse impact on method sensitivity.
Using headspace GC for the analytes
with sufficient vapor pressure may be a
quick solution because most APIs are not
volatile. However, for complex samples,
the introduction of nonvolatile and reactive
materials in the GC inlet may be mitigated
by matrix deactivation, analyte extraction, or
two dimensional (2D)-GC. These techniques
may also be used to increase analyte
stability, for selective extraction, and for
enrichment of analytes. Table I summarizes

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Table II: Literature references for application of derivatization followed by GC for GTI analysis
Derivatizing Agent Detector Analyte DL (QL) Reference Year
Methanol MS Benzoyl chloride 0.2 ppm Raman (32) 2014
Ethanol MS Formaldehyde 3 ppm Raman (32) 2014

BSTFA MS 4-Chloro-1-butanol 0.05 (0.08) ppm Harigaya (33) 2014

Sodium thiocyanate FID MS (EI) Mesylates, DMS 5–10 ppm ≤0.05 ppm Lee (34) 2003
PFTP MS (SIM) Sulfonate esters <1 ppm Alzaga (35) 2007
PFTP MS Ethyl mesylate <0.5 (1) µg/mL Jacq (36) 2008
Benzaldehyde NPD Hydrazine <1 ppm Gyllenhaal (37) 1990
Acetone MS Hydrazine 0.1 ppm Sun (38) 2009
Benzaldehyde ECD Hydrazine <1 ppm Carlin (39) 1998
Acetylation NPD Aziridine 0.2 ppm de Haan (40) 1989
Acetylation ECD 2-Chloroethylamine 0.5 ppm de Haan (40) 1989
Acetylation ECD Chlorophenols 6–122 ng/L Morais (41) 2011

the various technologies used for the
removal of matrix interference and hence
increasing the sensitivity.
Analyte Extraction
Extraction methods such as liquid–
liquid extraction (LLE), liquid-phase
microextraction (LPME), solid-phase
extraction (SPE), and solid-phase
microextraction (SPME) help enhance
the analyte concentration and sensitivity,
thereby reducing matrix interference,
especially where the GTI and matrix have
diverse chemical properties. Small neutral
molecules, such as alkylating agents, have
high solubility in organic solvents and
may be extracted out from ionizable API
matrix, which will have better solubility in
water in an ionized form (14). The solvent
n-hexane is the best solvent for liquid
extraction since only sparse amounts of
API or potential impurities are extracted
from the matrix. n-Hexane does not
impact the sensitivity or selectivity by ion
depression being a nonpolar solvent (27).
However, this procedure is laborious and
may be prone to interferences because
of tough-to-break emulsions, the use
of organic solvents, and concentration
steps. Additionally, an internal standard
needs to be used to compensate for
any loss of analyte during the extraction
procedure. LPME offers the advantage
of much improved concentration
power as only microvolumes are used
for extraction. SPME is a solvent-free
extraction technique that involves the
use of a coated fiber to extract various
analytes that can be in liquid (direct
injection) or gas (headspace) phase (25).
The working pH range for the currently
available fibers is pH 4–9. That being said,
the adjustment of pH is a critical factor for
the ionization and extraction of potential
interferences. Polymeric ionic liquids
(PILs) with variable chemical properties
have also been evaluated as selective
SPME sorbent coatings for the analysis of
alkyl halides and aromatics. This allows
quantitation of GTIs in ultratrace level

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 ANALYTICAL
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(29). The ability to modify their chemical
structures to achieve different analyte
solvation capabilities is an advantage.
However, this approach is still in an early
stage and further studies are underway to
understand the effect of various PILs.
2D-GC
Compounds such as epoxides and
Michael reaction acceptors do not
have sufficient volatility for headspace.
2D-GC with a Deans switching valve
may be used to divert the matrix to
waste and the fraction containing
desired GTIs to the detector. Using
this approach, the API, solvent, and
derivatization agents are not introduced
on the second column and MS detector.
Enhanced sensitivity is observed mainly
because of the removal of background
interference. Moreover, this technique
also results in the complete separation
of two analytes present in the same
sample (28).
GTI Analysis Using Headspace GC
Headspace analysis minimizes the
potential contamination of the injector,
column, and detector. In headspace
analysis, nonvolatile APIs do not partition
into the headspace and thus do not get
injected and enter the GC system (23).
High boiling solvents such as dimethyl
sulfoxide, N,N-dimethylacetamide, or
N,N-dimethylformamide are commonly
used for such analysis. Recently, the use
of ionic liquids has also been reported
for the analysis of high boiling analytes
12 | September 2017 | LCGC
METHODS PERFORMANCE
DEVELOPMENT VERIFICATION
(30,31). Ionic liquids offer an advantage
because they provide high thermal
stability and low volatility without causing
significant background noise at elevated
headspace oven temperatures. This
technique may be used for analytes
that have sufficient vapor pressure to
be injected using headspace GC and
that have demonstrated stability at high
temperatures.
GTI Analysis Using
Derivatization Followed by GC
Sample derivatization is an efficient way
to alter the physicochemical properties
of the analyte and increase the sample
stability, volatility, and ionization efficiency
needed for MS. Table II summarizes the
application of the derivatization technique
for various analytes, hence increasing the
stability and sensitivity.
Sulfonate esters are known DNA
reactive genotoxins and can be formed
from the reaction of residual alcoholic
solvents used in the manufacturing
process and sulfonic acids (equation 1).
O O
R1 OH + R2 S OH R2 S OR1 [1]
O O
Sulfonate esters do not have sufficient
vapor pressure to be analyzed using
headspace GC. Analysis using direct
injection is prone to artifacts because
of ester hydrolysis, decomposition of
API salts, and matrix interference.
Derivatization of sulfonate esters before
analysis not only stabilized the analyte,
but also increased volatility because of
 ANALYTICAL
CHROMATOGRAPHIC METHODS PERFORMANCE
TECHNIQUE
TRACE ANALYSIS DEVELOPMENT VERIFICATION
SELECTION

the low polarity of derivatized esters (34– O R2

36). For instance, pentafluorothiophenol H2N NH2 + N
N R2
R1 R2 R1
is a commonly used derivatization R1 = Ph,CH3 R1
R2 = H, CH3
reagent for sulfonate esters. As shown [3]
R2
in equation 2, sulfonate esters react with O
N R2
H2N NH2 +
pentafluorothiophenol to form volatile R R 1 2
R1 N
R1 = Ph,CH3 R1
sulfide: R2 = H, CH3

SH S R1
Derivatization reaction time is
O F F F F reported to be critical as an increase in
R2 S OR1 + [2] benzalazine has been observed if the
O F F F F
F F aqueous reaction sample is allowed to
However, this method does not stand for a longer time before analysis.
distinguish between different alkyl groups In situ derivatization of hydrazine using
(R2 in equation 2) and provides common acetone or acetone-d6 followed by GC–
derivatization products for the analysis of MS analysis of the resulting acetone azine
various alkyl sulfonates and sulfates, which has an advantage because acetone may
limits the selectivity. This approach was be used as a derivatizing agent as well as a
demonstrated by Jacq and colleagues (36) diluent (38).
to monitor the formation of ethyl mesylate Alkyl chlorides, which are alkylating
from ethanol and methanesulfonic acid in agents, may be formed because of the
the reaction mixture. use of various alcohols and aqueous
Hydrazine, a GTI, is a common hydrochloric acid during the API synthesis.
building block in the synthesis of A genotoxic impurity such as 4-chloro-
many drug substances and also is a 1-butanol may also be generated if
known degradation product of the tetrahydrofuran and hydrochloric acid are
antituberculosis drug isoniazid. The drug used during the manufacturing process of
is highly reactive with a high polarity, an API (equation 4)
low molecular weight, limited volatility,
and lack of UV chromophore, which O aq HCl Cl
HO
makes it challenging for the analysis of Reverse Williamson 4-chloro butanol
ether synthesis
residual hydrazine. Derivatization using THF
O aq HCl Cl
O aq HCl F
Cl
benzaldehyde to provide resulting HOWilliamson
Reverse
F F HO
4-chloro butanol [4] O
Reverse Williamson OH +4-chloro Si butanol Si Cl
benzalazine has been a favorite ether Clsynthesis
THF
ether synthesis N O
THF F
BSTFA
approach for the analysis of hydrazines
O aq HCl F
F
F
F
Cl
F
OS
followed by analysis using various Reverse Williamson OH + Cl Si
HO OH + Si
Si4-chloro butanol N Cl O
Si O Si Cl
Cl N O
detectors (equation 3) (37–39). THF The
ether synthesis
BSTFA
BSTFA
F
resulting benzalazine may be analyzed F F
O Si
using GC or HPLC analysis. Cl
OH + Si
N O
Si Cl

BSTFA

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 ANALYTICAL
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2
5.766.487.207.928.649.3610.080.80
Minutes
0.001
Absorbance (AU)
0.000
-0.001
-0.002
5.00 7.50
Figure 3: Overlay HPLC–UV chromatogram of bromopyrazole spiked in a drug substance. Column: 150 mm × 4.6 mm, 3.5-μm
Zorbax Eclipse XDB-C18 Rapid Resolution.
Because of low sensitivity and
a tendency to react back to
tetrahydrofuran, 4-chloro-1-butanol needs
to be derivatized before analysis. This
may be done easily by silylation using
N,O-bis(trimethylsilyl)trifluoroacetamide
(BSTFA, a common reagent for hydroxyl
group derivatization); the resulting
trimethylsilyl ether is analyzed by
headspace GC. To ensure accuracy and
precision, 3-chloro-1-butanol may be
used as an internal standard (33).
We also used derivatization with the
BSTFA technique followed by GC–MS
analysis in SIM mode to track the residual
amount of 4-chloro-1-butanol in the
final API as a limit test, with limits of
detection (LOD) of <1 ppm and sample
concentration of 1 mg/mL.
14 | September 2017 | LCGC
CHROMATOGRAPHIC METHODS PERFORMANCE
TRACE ANALYSIS DEVELOPMENT VERIFICATION
Bromopyrazole at 50 ppm; S/N = 16
10.00 12.50 15.00 17.50 20.00 22.50 25.00
Time (min)
Determination of low levels of small
alkyl aldehydes, such as formaldehyde,
poses an analytical challenge because
of their volatility and stability issues.
Formaldehyde is eluted very fast and
no specific ions are present for selective
and sensitive detection by GC–MS.
Derivatization is often applied to
enhance stability and sensitivity with the
change in its physicochemical properties
(32). Formaldehyde is derivatized to
diethoxymethane by addition of ethanol
in the presence of p-toluenesulfonic
acid as a catalyst. A similar approach
has been reported for the analysis of
unstable acid chlorides, aziridines (40), and
chlorophenols (41). A combined approach
of extraction followed by derivatization
has been developed for the determination
 ANALYTICAL
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Table III: Literature references for use of LC–MS for GTI analysis
Detector Analyte DL (QL) Reference Year
MS (SIM) Besylates, tosylates <0.1 ppm Taylor (21) 2006
MS (SIM) Nitro, chloro, and amino 0.2–0.6 ppm Liu (44) 2009

MS/neg. mode (SIM) Arylsulfonamine (0.2) ppm Liu (44) 2009

LC–MS (ESI)/IC Alkyl halides <1 ppm Lee (45) 2000

MS-MS (MRM) Methyl and ethyl mesylate 0.002 (0.01) µg/mL Kakadiya (46) 2011
MS-MS (SIM) 2-Chloromethyl-3,4-dimethoxy pyridine 0.1 (0.3) ppm Venugopal (47) 2012
LC–MS-MS-ESI (MRM) 4-chloro-1-hydroxy butane sulfonic acid 0.17 ppm Narayana (12) 2012
MS-MS (MRM) 4-DMAP 0.03(0.1) ppm Szekely (48) 2012
MS-MS (MRM) Substituted hydrazines 0.02 Reddy (49) 2014
(0.06) ppm
MS (ESI), HILIC Quaternary hydrazine derivatives (polar imp) 3 ppm Hmelnickis (50) 2008

of small, water-soluble amines such
as aziridine and 2-chloroethylamine in
drug substances at trace levels before
quantitation by GC (40).
Analysis of GTIs by LC
LC is the primary separation technique
for nonvolatile and thermally labile
analytes. LC techniques include reversed-
phase chromatography, normal-phase
chromatography, and hydrophilic-
interaction chromatography (HILIC).
Reversed-phase HPLC is typically the first
choice used for the analysis of nonvolatile
GTIs. HILIC may be used to retain and
analyze very polar analytes, which are
difficult to retain and are eluted at solvent
front with reversed-phase LC. Detection
by UV is the first choice for the GTI
analysis. However, MS is becoming a fairly
common and universal detection method
these days because of its enhanced
sensitivity and selectivity. Other detection
methods reported in the literature are
evaporative light-scattering detection
(ELSD) and charged aerosol detection
(CAD) for the analysis of GTIs that lack a UV
chromophore. Derivatization may be used
to introduce a chromophore for UV analysis,
to increase ionization for MS, or enhance
stability of the analyte.
Direct Analysis (No Derivatization)
GTI Analysis Using
High Sample Loading, LC–UV
Similar to GC, the control level of analytes
drives the required sensitivity of the method
and detector to be used with LC. UV
detection is typically less selective and less
sensitive compared to MS. However, at
the level of 100 ppm, for compounds with
strong UV chromophores and no matrix
interference, UV still remains the most
preferable detector for routine analysis
because of its robustness, ease of use, and
easy transfer (13–15,42,43).
The HPLC–UV impurity method for
drug substances may be used as an
initial platform followed by increasing
the sample load to increase sensitivity.
Similar technology has been used in our
laboratory for the determination of a GTI,

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Protonate 1. Isolate parent ion

100 Exact mass = 220.0 Exact mass = 221.0 2. Apply collision energy Exact mass 204.0
LC–MS 3 Monitor daughter ion LC–MS-MS
Loss of NH3
Relative abundance

50

Daughter ion of analyte

Exact mass 204

0
0 4 8 12 16 20 24 28 32
Retention time (min)

Figure 4: Representative chromatogram of hydroxylamine impurity MRM at 1 ppm level.

bromopyrazole, in a drug substance. In this of choice and is widely used for GTI
case, the GTI control level was determined analysis because of its high selectivity
to be 160 ppm based on the TTC approach. and sensitivity. Table III summarizes
First, the drug substance impurity method the applications of MS for the analysis
was assessed for the matrix interference of various analytes in pharmaceutical
and then the sample concentration was compounds. However, matrix interference
increased from 0.4 mg/mL to 1 mg/mL and is a higher risk than GC–MS and may
the injection volume was increased from cause analyte ion suppression or
10 μL to 20 μL to enhance the sensitivity. enhancement.
Figure 3 shows overlay chromatograms The recent advancement of MS
of API unspiked and spiked with instrumentation has established another
bromopyrazole at the 50 ppm level. This level and possibility to achieve much
method demonstrated excellent precision, higher sensitivity for GTIs (12,46,47,49–
spiked recovery, and linearity assessed up to 51). Switching from selected ion
180 ppm (R2 = 1.0). monitoring (SIM) to multiple reaction
monitoring (MRM) mode for the analysis
GTI Analysis Using LC–MS and LC–MS-MS of 4-dimethylaminopyridine resulted in
MS has become the detection method increased sensitivity and lower limits of

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Table IV: Literature references for use of derivatization before analysis by LC for GTI analysis
Technique or Derivatization Agent Detection Analyte DL (QL) Reference Year
2,4-DNPH UV diode array Formaldehyde 0.5 (1.5) ppm Nageswari (53) 2012
2,4-DNPH UV diode array Formaldehyde 0.1 (0.33) ppm Soman (54) 2008

Derivatization GC, ion, and Formaldehyde Manius (55) 1993

three different
2,4-DNP MS Aldehyde 0.1 ppm Raman (32) 2014
Benzaldehyde MS Methyl hydrazine- 1.2 ppm Raman (32) 2014
carboxylate
Aniline MS Ethyl chloroformate 10 ppm Raman (32) 2014
Triethylamine MS (SIM) DMS (0.5) ppm Liu (44) 2009
2-Mercaptopyridine MS (SIM) Chloroethyl 5 ng/mL Liu (8) 2010
chloroformate
DMA ESI-MS Epoxide 1 ng/mL Liu (8) 2010
DMAP-SPE (weak cation exchange) MS-MS (MRM) 4-Fluorobenzyl chloride 0.5 ng/mL Yang (56) 2005
DMAP-HILIC MS-MS Alkyl halides 1 mg/Kg Van Wijk (57) 2011
DMA MS Alkyl halides epoxide 1 ng/mL Bai (58) 2010
TMA-TEA-HILIC MS (SIM) Alkyl sulfonates; dialkyl 1–2 ppm An (59) 2008
sulfates
Acetonitrile-gas phase MS (SIM; MRM) Epoxides 1 ppm Wu (60,61) 2010,
Meerwein reaction 2011
Salicylaldehyde, TLC Hydrazine 1–20 ppm Kean (11) 2006
benzaldehyde-LLE
2-OH-1-Naphthaldehyde Hydrazine 20 ppb Mañes (62) 1988

quantitation (LOQ) (48). A highly sensitive mass spectrometer. The resulting

and precise analytical method using LC–
MS-MS was reported for the quantitation
of 2-chloromethyl-3,4-dimethoxypyridine
hydrochloride in APIs and the final tablet
form (47). Trace levels of ammonium
acetate were added to the mobile phase
to enhance ionization and detection. In our
laboratory, LC–MS-MS was also successfully
applied to quantify a hydroxylamine
impurity present at a low level in a drug
substance (52). LC–MS in SIM mode did not
provide the required sensitivity of 10 ppm.
To increase the sensitivity, the parent ion
obtained from LC–MS (SIM) was isolated
and a collision energy of 17 eV (optimized)
was applied using a triple-quadrupole
daughter ion was monitored, providing
an LOD of 1 ppm, excellent linearity, and
percent recovery of 91% at the 2 ppm
level. Figure 4 shows the representative
chromatogram for the daughter ion.
HILIC using MS-compatible mobile
phases in conjunction with MS has also
been used as an alternative to reversed-
phase and ion pair chromatography for
the analysis of polar analytes (50).
GTI Analysis Using
Derivatization Followed by LC
As is the case for GTI analysis by
GC, direct accurate analysis of some
of the genotoxic impurities by LC is

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challenging because they react rapidly,
decompose, lack a UV chromophore
or ionizable functional groups, or
need to be derivatized before analysis.
Derivatizing agents are selected based
on the functional groups of the GTIs
and are focused mainly toward the
improvement of sensitivity, the generation
UV chromophores and are stable for
analysis (11,62). Determination of residual
hydrazine and isopropyl hydrazine
in our laboratory was performed by
derivatization followed by LC–UV
(equation 6).
OHC N
H N NH + N
2 2

of chromophores, and analyte stability OHC N

(32). Table IV summarizes the application H N NH

H
+
NOHC
NH
HN NH
+2
+
OHC
2
N
N NN
2 2 N 2
of derivatization for various analytes H
[6]
enhancing stability, introducing UV H N NH
OHC
+ OHC OHC
N
N
N
2 2
chromophore, and ionizable functional HN NH +HN NH + 2 N
N N
2
H
H
groups before LC analysis.
OHC
Aldehydes are prone to undergo partial HN NH + 2 N
N
H
aerial oxidation leading to inaccurate
determinations. A favorite derivatizing A solution of 5% benzaldehyde in
agent for the analysis of aldehydes methanol was added to the sample
has been 2,4-dinitrophenylhydrazine solution in water in an HPLC vial, and
(2,4-DNPH), which provides respective derivatization was carried out at 50 °C
hydrazone derivatives that have absorbing for 30 min. The derivatization time was
maxima at 360 nm (equation 5): optimized by comparing the peak areas
O
of the derivatized product at different
+ O N NH O N NH R
2

2
NH
2
N intervals. The detection wavelength
R H H
[5] was selected as 300 nm, which was the
2
NO NO2
R = H, alkyl
2,4-DNPH
absorption maximum of the derivatized
+ O N product, and LC analysis was carried out
O R
2
NH 2
O N NH
NH N
H 2
NO NO H using a Waters XBridge C18 column.
2 2
H, alkyl
2,4-DNPH Figure 5 shows a chromatogram of the
The resulting hydrazones are analyzed starting material spiked with hydrazine
either by HPLC–UV (53,54) or LC–MS (32). and isopropyl hydrazine. Linearity was
Derivatization has been one of the demonstrated over the 1–40 ppm range
main analytical techniques for the for hydrazine and the 5–100 ppm range
analysis of hydrazines as well because for isopropyl hydrazine (based on the
they are highly reactive, polar, and lack sample concentration of 10 mg/mL), with
UV chromophores. Aryl aldehydes are a correlation coefficient of >0.999 and
most commonly used to yield respective excellent precision.
benzalazine derivatives, which have The method was further optimized to

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Benzaldehyde
(Derivatizing agent)

5.208 - Hydrazone
50
bis-Hydrazone
Absorbance (mAU)

40

30
iPr-Hydrazone
20

3.879 - PH
10

0
0 1 2 3 4 5 6 7
Time (min)

Figure 5: A representative chromatogram for analysis of hydrazine and isopropyl hydrazine. Column: 150 mm × 4.6 mm,
3.5-μm Waters Xbridge C18; mobile-phase A: water; mobile-phase B: acetonitrile; temperature: 50 °C; injection volume: 5 μL;
flow rate: 1 mL/min.

determine the residual hydrazine in a retain polar derivatized products and

drug substance since the drug substance quantify the amount of derivatives.
was not aqueous soluble; the diluent HILIC using a mass compatible buffer
was changed to methanol and the provides a great advantage compared
concentration of the derivatizing agent to reversed-phase HPLC in which MS-
was changed from 5% to 10% in methanol incompatible ion-exchange or ion-pairing
to keep the same ratio in the derivatizing buffers are generally used. Another
mixture. The method demonstrated key advantage is that reagent-related
excellent spiked recoveries (98.9%). fragments produced for derivatives
Various alkyl and aryl halides, which are allow for low-level screening of alkyl
not sufficiently volatile and are thermally halides. A three-step weak cation-
labile, have been derivatized using exchange SPE procedure was used to
4-dimethylamino pyridine (4-DMAP) or remove excess DMAP and the resulting
dimethylamine (DMA) forming a stable solution was analyzed by HPLC–MS-MS
quaternary amine salt derivative before in MRM mode. Chloroformates tend to
LC analysis (equation 7) (56–58). be much more moisture sensitive and
N N
R hydrolytically unstable, so derivatization
+
[7] is used to stabilize the analyte. It was
R Cl +
N N reported that strong basic derivatization
HILIC–MS-MS has been used to reagents caused decomposition of the

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Table V: Literature references for use of innovative techniques for GTI analysis
Analytical Technique Analyte DL (QL) Reference Year
Matrix deactivation Various alkyl halides 0.4–4 ppm Sun (24) 2010
Molecular imprinting-scavenger resins Acetamide arylsulfonates Székely (51) 2012

2D-GC–MS Multiple <1 ppm Frank (28) 2010

Metal ion coordination ion spray MS Epoxide 7.5 ng/mL Bai (58) 2010
Coordination ion spray MS Various Bayer, Harvilla, and Gao (63–65) 1999,
2000, 2005
Meerwein reaction-MS Epoxides 1 ppm Wu (60,61) 2010, 2011
Capillary electrophoresis DMS, chloroacetyl chloride 3 (10) ppm Khan (66) 2012
Capillary electrophoresis Alkyl halides (0.05%) Hansen (67) 2005
Derivatization (dansyl hydrazine)–CE Formaldehyde 200 ppb Feige (68) 1996
2D HPLC, on-line reduction, 3-Nitrobenzanthrone 0.002 Hasei (69) 2012
fluorescent detector (0.006) ng
LC–ELSD, LC–CAD Yuabova (15) 2008
HILIC–CLND Hydrazines 0.02% Liu (70) 2009
Extractive ESI On-line reaction monitoring 2.5 ppm McCullough (71) 2011
Atmospheric-pressure p-Toluenesulfonate 0.1 ppm Devenport (72) 2013
thermal desorption-extractive ESI-MS
LC–MS (SIM) SFC–MS (SRM) Alkyl halides 0.03 (0.1) Huybrechts (75) 2007
ppm
Derivatization (3-iodobenzoyl chloride), 4-Chloro-1-butanol <1 ppm Harigaya (73) 2014
LC–ICP-MS
NMR Trifluoronitrobenzene 9 ppm Parmar (74) 2013

desired derivatives. Therefore, residual derivatization and LC–MS (SIM) in

chloroethylchloroformate (CECF) in API combination with a HILIC stationary
was derivatized using 2-mercaptopyridine, phase has been used for the analysis of
a less basic nucleophile (8). The authors a group of commonly encountered alkyl
suggested that both glassware and esters of sulfonates or sulfates present in
solvents needed to be suitably dried drug substances at low parts-per-million
to prevent interference from residual levels. Trimethyl and triethylamine have
moisture and achieve the desired been used as derivatizing agents and
sensitivity and reproducibility. a HILIC stationary phase was used to
Techniques similar to alkyl halides retain highly polar quaternary ammonium
can also be applied for the analysis of derivatization products and separate
sulfonate esters (equation 8) (44,59). them from the API peak.
O R R Chemical derivatization and coordination
+ R1
R2 S OR1 +
R
N
R R
N
R
ion spray ionization MS have been used to
O
R1 = Alkyl R = C2H5, CH3
[8] enhance the detection sensitivity of neutral
R2 = Aryl, CH3

O R R
+ R1
analytes such as epoxides (58). The chemical
R2 S OR1 + N N
O R R R R derivatization converts analytes into highly
A method comprising generic
R1 = Alkyl
R2 = Aryl, CH3
R = C2H5, CH3
ionizable, permanently charged derivatives,

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which become readily detectable by MS,
whereas the coordination ion spray MS
approach improves ionization by forming
neutral ion adducts with metal ions such as
Na+, K+, or NH4+, which are introduced into
the electrospray ionization (ESI) source.
Innovative Analytical Techniques
for the Analysis of GTIs
Because of the wide variety of GTIs
present and their physicochemical
properties, many innovative analytical
techniques have been developed and
reported in the literature to reduce matrix
interference and enhance sensitivity. Table
V summarizes these techniques for various
analytes. Matrix deactivation provided
an innovative approach to stabilize
reactive or unstable analytes (24). This
deactivation has been achieved chemically
either by protonating and masking the
basic functionalities or scavenging the
hypothetical reactive species in the
sample matrix. Molecularly imprinted
polymers have high affinity binding sites
for target analytes, whereas APIs have a
different size and structure and work as
scavenger resins. The efficiency of these
polymers has been demonstrated for the
quantitative removal of acetamide and aryl
sulfonates in the presence of APIs (51).
The analysis of neutral GTIs provides a
challenge because of their poor ionization
efficiency. Chemical derivatization
followed by coordination ion spray MS
improved sensitivity for some neutral
epoxides by forming neutral ion-
adducts with metal ions such as Na+,
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K+, or NH4+, which are then introduced
into the ESI source and analyzed by MS
(58,63–65). Formations of adduct ions in
positive or negative detection mode is
applicable. The gas-phase derivatization
via Meerwein reaction provided another
alternative for LC–MS analysis of trace-
level epoxides (60,61). Ethylnitrilium ions
generated by atmospheric pressure
ionization (during MS when acetonitrile
is used as mobile phase for HPLC) react
with epoxides, and then Meerwein
reaction products are analyzed by either
SIM or MRM modes. This technique is a
great alternative in cases when an analyte
is difficult to analyze directly because
it has poor ionization and is unstable in
solution-phase derivatization.
Genotoxins are mostly determined
using HPLC–UV–MS and GC–MS
techniques for their robustness. However,
the use of capillary electrophoresis (CE)
can complement the existing techniques
for analysis and open new horizons. CE
has been reported for the analysis of
dimethyl sulfate, chloroacetyl chloride
(66), formaldehyde (68), and alkyl halides
(67). It also appears to be more robust
to matrix effects and provides highly
efficient separations and consumes very
low amounts of reagents. However, it has
the drawbacks of poor sensitivity and
precision needed for trace analysis.
The potential applicability of supercritical
fluid chromatography (SFC)–MS in
selective reaction monitoring (SRM) mode
has been reported as a complementary
and orthogonal approach to HPLC for the
 ANALYTICAL
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analysis of a GTI at sub-part-per-million
levels (sample concentration 10 mg/mL)
in an unknown API (29). SFC may prove
to be an advantageous technique when
attaining a high sample concentration is
a significant challenge because of limited
solubility. A novel LC–inductively coupled
plasma (ICP)-MS method has been used
for the analysis of 4-chloro-1-butanol
(73). To enhance the selectivity, a 2D LC
system is further connected with ICP-MS.
Then, 4-chloro-1-butanol is derivatized
with 3-iodobenzoyl chloride in acetonitrile
and the resulting derivative is analyzed for
iodine (m/z 127). Methanol is selected as
the organic solvent for the mobile phase
of the second LC system, which is more
suitable for ICP-MS because of its lower
carbon content compared to acetonitrile.
Quantitative nuclear magnetic
resonance (NMR) spectroscopy (74) is also
feasible with recent developments of high
strength systems (such as 400–600 MHz)
combined with cryoprobe technology.
This technique has advantages of being
quantitative in nature, easy sample
preparation, and the ability to provide
quick answers. A comparison experiment
may be performed with and without
spikes at an appropriate level to provide
a quick answer if it is feasible to be
used for a particular analysis. 19F-NMR
is widely used for the quantitation of
fluorinated impurities. Because of the
high sample concentration (1 g in 300
μL DMSO-d6) and hence high viscosity,
analysis is done at 343 K to provide a
sharp signal, and 19F1 at -156 ppm is
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selected for quantitation because it
provides the simplest multiplet structure.
However, this approach does have some
notable disadvantages, such as being
less sensitive (requiring high sample
concentration) and more expensive.
Conclusion
Controlling GTIs in pharmaceutical
compounds is of the utmost importance
and is a requirement throughout the clinical
development and commercial stage. A
good process understanding and sensitive,
specific analytical methods are needed to
control these impurities below the TTC level.
Many factors, including physicochemical
properties of the analytes, need to be
considered as an effective analytical method
development strategy. Direct analysis
without extensive sample preparation is
preferred. However, sometimes sample
preparation becomes an integral part of
the analytical method if there is matrix
interference. Hyphenated techniques are
gaining popularity, and with advancements
in MS instrumentation, MS is becoming the
detection of choice because it provides the
required sensitivity and specificity in SIM
and MRM modes. Chemical derivatization
offers an alternative to achieve the needed
sensitivity and sample stability. Following
method development, the method is
validated to demonstrate linearity over
the desired range, spiked recovery, and
sensitivity.
Acknowledgments
The authors would like to acknowledge
 ANALYTICAL
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Drs. Christine Gu and C.J. Venkatramani
for their contribution to hydroxylamine
impurity analysis.
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At the time of publication, Archana
Kumar, Kelly Zhang, and Larry Wigman
are with Small Molecule Pharmaceutical
Sciences at Genentech Inc., in South
San Francisco, California. Direct
correspondence to: kumar.a@gene.com
This article first appeared in LCGC 33(5),
344–359 (2015).
 Determination of
Genotoxic Impurities in
Pharmaceuticals
Frank David, Maria Ramble Alegre, Gerd Vanhoenacker, and Pat Sandra

SPONSORED

Trace Analysis of GTIs by

The determination of genotoxic impurities
UPLC with UV and Mass (GIs) in drug substances and pharmaceutical
Detection
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products is an emerging topic in
to read pharmaceutical quality control. GIs are
the study
intermediates or reactants in the synthetic
pathway of a drug substance and should be
monitored at ppm (μg/g drug substance)
or even ppb (ng/g) levels. This is several
orders of magnitude lower than in classical
impurity analysis (0.05% or 500 ppm level)
SPONSORED or in residual solvent analysis. Analytical
Control of GTIs by UV and
methods for the determination of GIs
Mass Detection include gas chromatography (GC) and liquid
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chromatography (LC), both often combined
to read with mass spectrometry (MS) detection. Some
the study
typical examples of GIs trace analysis using GC
and LC are presented. The potential of on-line
reaction monitoring is also discussed.

Based on the threshold of toxicological concern

(TTC) concept, the European Medicines
Evaluation Agency (EMEA) published guidelines
on the limits of genotoxic impurities (GIs) in
pharmaceutical ingredients (1). Impurities that
contain a “structural alert functionality” (2)
getty Images/xxxxxxxx

must be quantified at levels below the TTC,

that corresponds to 1.5 μg daily intake (for
lifetime exposure). In practice, this means that

25 | September 2017 | LCGC

 ANALYTICAL
CHROMATOGRAPHIC
TECHNIQUE
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SELECTION
the GIs must be monitored in the drug
substance or pharmaceutical product
at levels far below classical impurity
determinations. Typically, methods
should allow the determination of GIs
(specific solute or group of solutes)
at levels between 10 ng/g and 1000
ng/g drug substance (10–1000 ppb).
Consequently, method development
for trace analysis of potential GIs is a
challenge in pharmaceutical analysis.
A comprehensive overview of strategies
to identify and control GIs is described
in a book recently published by Andrew
Teasdale (3). This work includes chapters
on regulatory guidelines, evaluation
of the TTC concept, risk assessment
and analytical methods, including
innovative use of nuclear magnetic
resonance (NMR) spectroscopy. It is
clear that methods such as NMR or
direct analysis in real time (DART) in
combination with mass spectrometry
(MS) will increase in importance in the
coming years. However, for the analysis
of trace impurities in a matrix that can
contain other impurities at concentration
levels that are several orders of
magnitude higher, the combination of
a chromatographic separation with MS
(providing high sensitivity and selectivity)
can still be considered as the present
state-of-the-art approach for genotoxic
analysis.
A short overview of chromatographic
trace analysis methods for GIs is given
and some typical cases are presented
that illustrate the potential of state-
26 | September 2017 | LCGC
METHODS PERFORMANCE
DEVELOPMENT VERIFICATION
of-the-art gas chromatography–mass
spectrometry (GC–MS) and liquid
chromatography–mass spectrometry
(LC–MS). Finally, the use of GC–MS
methods for on-line reaction monitoring
is also discussed.
Method Selection for
Trace Analysis of GIs
Driven by the EMEA and FDA guidelines
(1,4), the pharmaceutical industry has
developed several “compound-specific”
analytical methods to determine known
or suspected GIs in a pharmaceutical
intermediate or drug substance. A wide
range of methods is used, including
GC, GC–MS, LC, LC–MS, and NMR
methods (3). The development of these
methods is often time- and resource-
consuming. In 2009, we published a
method selection chart that can be
used as a guideline in the development
for trace-GI chromatographic methods
that would significantly improve both
the effectiveness and timeliness of the
method development process (5). This
chart was based on intensive research
on several classes of GIs. Since the goal
of this research was the development of
generic methods applicable in quality
control (QC) laboratories, we focused on
GC–MS and LC–MS methods applying
single-quadrupole MS systems. During
this study, it was observed that the main
challenge in GI determination is the
wide range of target solutes (volatile,
semivolatile, nonvolatile and highly
reactive) in combination with a wide
 ANALYTICAL
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SELECTION
Figure 1: Method selection chart for genotoxic impurity determination in pharmaceuticals.
range of matrices (active pharmaceutical
ingredients [APIs] and intermediates) with
different physicochemical characteristics
(water soluble, ionic, polar, nonpolar,
basic, acidic, and so forth). Consequently,
various sample preparation and analytical
methods are needed.
The method selection chart, shown in
Figure 1, is based on knowledge of the
physicochemical properties of the analyte
or matrix. Based on functionalities,
polarity and volatility of the GI (or class
of GIs) and solubility, functionality and
volatility of the matrix (API), a selection
can be made between GC–MS and
LC–MS methods. In addition, attention
was also paid to robust, automated
and hyphenated sample preparation
techniques, reducing solvent, and
27 | September 2017 | LCGC
METHODS PERFORMANCE
DEVELOPMENT VERIFICATION
sample consumption. This last aspect is
particularly important in GI analysis, since
some newly developed drug substances
are expensive and not available in large
quantities for testing.
The first question in the method
selection decision tree is choosing
between GC and LC. If the GI is
amenable to analysis by GC, and the
solute(s) has (have) sufficient vapor
pressure to be present in the headspace,
static headspace (SHS), headspace–
solid-phase microextraction (HS–SPME)
or dynamic headspace (DHS) can be
considered as the sample preparation
technique. The techniques are then
mostly performed on a concentrated
solution of the API in water, dimethyl
sulfoxide (DMSO), or another low volatile
 ANALYTICAL
TECHNIQUE CHROMATOGRAPHIC METHODS PERFORMANCE
SELECTION TRACE ANALYSIS DEVELOPMENT VERIFICATION

Figure 2: Analysis of Michael reaction acceptors by dynamic headspace–gas chromatography–mass spectrometry (DHS–GC–
MS). (a) Total ion chromatogram for promethazine (API) spiked at 1 ppm level with cinnamonitrile (peak 1) and 3-ethoxy-
2-cyclohexen-1-one (peak: 2); (b) Extracted ion chromatogram at m/z 129 for cinnamonitrile (peak: 1); (c) Extracted ion
chromatogram at m/z 140 for 3-ethoxy-2-cyclohexen-1-one (peak: 2).

solvent. SHS, HS–SPME, and DHS can
also be used if a nonvolatile GI can be
transformed into a volatile derivative. This
method was, for example, successfully
applied in the analysis of sulfonates, one
of the most important classes of GIs (6,7).
If the analyte is GC-amenable, but not
volatile enough for headspace analysis
(even after derivatization), direct injection
of a concentrated API solution can be
used. In this case, however, the API
matrix (or other impurities and eventually
thermally decomposition products) can
interfere with the determination of the
GI. Care should also be taken that no GI
is actually formed during injection in a
hot inlet, or during analysis, leading to
false positive results. For direct injection
of a concentrated API solution or extract,
the use of back-flushing or heart-cutting
two-dimensional techniques are very
useful in maintaining the analytical system
(column and MS detector) clean. This was
illustrated for the analysis of haloalcohols
and Michael acceptors (8).
For analytes not amenable to GC, LC–MS
is used. After selection of the ionization
mode (atmospheric pressure electrospray
[ESI] or chemical ionization [APCI], either in
positive- or negative-ion detection mode),

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 ANALYTICAL
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Figure 3: Analysis of 3-amino benzonitrile in bupivacaine (spiked at 0.1 ppm). (a) analysis of standard solution with
acetonitrile as mobile-phase B, (b) analysis of spiked sample with acetonitrile as mobile-phase B and (c) analysis of standard
solution with methanol as mobile-phase B, and (d) analysis of spiked sample with methanol as mobile-phase B.

the LC mode is chosen. Both reversed-
phase LC and hydrophilic interaction LC
(HILIC) are applied to separate the GIs
from the API. Solute derivatization can
also be considered in LC–MS methods to
increase retention and enhance detection.
This was used in the analysis of carbonyl
compounds (aldehydes and ketones)
(3), hydrazines (3), and in arylamines and
anilines (9).
Analysis of Michael Reaction
Acceptors by DHS-GC–MS
For GIs with sufficient vapor pressure to
be present in the headspace phase of a
concentrated solution of the API, SHS,
HS–SPME, or DHS can be considered
as “first-to-try.” Since most APIs have
relatively low volatility, the analytical
system (GC inlet, column, and detector)
will not be contaminated by the matrix
by applying these sample preparation
methods. The choice between SHS, HS–
SPME, and DHS depends on the volatility
of the analytes and the desired sensitivity.
In general, the sensitivity increases in the
order SHS < SPME < DHS.
The sensitivity that can be obtained
by DHS in combination with GC–MS is
illustrated by the analysis of some Michael
acceptors. The analysis was performed on
a MPS2 system equipped with the dynamic
headspace option (Gerstel GmbH). The
system was installed on a 7890 GC–5975
MSD combination (Agilent Technologies).
The sample (50 mg in 2 mL of DMSO/

29 | September 2017 | LCGC

 ANALYTICAL
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water placed in a 20 mL headspace vial)
was purged at 20 °C and 30 mL/min over
5 min. The volatiles were trapped on a
Tenax trap (Gerstel GmbH). The Tenax
trap was then desorbed at 250 °C and the
released solutes were analyzed by GC–MS.
Separation was performed on a
20 m × 0.18 mm i.d. × 1 μm DB–VRX
column (Agilent Technologies), using a
temperature program from 35 °C (2 min),
at 7 °C/min to 100°C, at 15 °C/min to
200 °C and at 25 °C/min to 260 °C, and
at a constant flow of 1.2 mL/min helium.
Detection was done in simultaneous scan–
selected ion monitoring (SIM) mode.
The DHS profile (total ion chromatogram
from scan-MS file) for the API promethazine
spiked at the 1 ppm level with Michael
acceptors cinnamonitrile (peak 1) and
3-ethoxy-2-cyclohexen-1-one (peak 2) is
shown in Figure 2a. By using extracted
ion chromatograms at m/z 129 for
cinnamonitrile (Figure 2b) and m/z 140 for
3-ethoxy-2-cyclohexen-1-one (Figure 2c),
respectively, the two target compounds
can be detected at the 1 ppm level with a
S/N > 100. If needed, higher sensitivity can
be obtained by operating the MS system in
the SIM mode. In this particular case, limits
of detection (LOD) were below 0.02 ppm. It
is clear that DHS is an excellent alternative
to SHS for problem cases (less volatile
solutes) and if extremely high sensitivity is
required.
Analysis of an Arylamine
30 | September 2017 | LCGC
METHODS PERFORMANCE
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GI by LC–MS–MS
As described previously, LC–MS methods
offer an alternative for solutes that are
not amenable to analysis by GC–MS or
for matrices (API) that could degrade into
GIs under GC conditions. In our previous
studies, methods based on LC combined
with single-quadrupole detection were
described (3,9). Selection was based on
the availability of instrumentation in QA/
QC laboratories. In the last couple of
years, triple-quadrupole MS systems
have become more accessible and, as a
result, higher sensitivities can be obtained.
However, the use of highly sensitive MS–MS
systems is still not a guarantee of success.
This is clearly illustrated by the analysis
of 3-aminobenzonitrile (arylamine class
of GI) in bupivacaine. The analysis was
performed on a 1290 ultrahigh-pressure
liquid chromatography (UHPLC) system
combined with a 6460 triple-quadrupole
mass spectrometer (Agilent Technologies).
The sample, dissolved in acetonitrile (100
mg/mL), was injected onto a 100 mm ×
2.1 mm i.d. × 1.8 μm Zorbax Eclipse Plus
C18 RRHD column (Agilent Technologies).
Mobile-phase A was 0.05% formic acid
(FA) in water and mobile-phase B was
either acetonitrile or methanol. The
gradient was 10% B for 0.5 min to 100% B
at 5 min, 1 min at 100% B, and back
to 10% B at 7.5 min (same gradient for
methanol and acetonitrile). The column
temperature was 40 °C using acetonitrile
and 45 °C when using methanol. The
triple-quadrupole MS instrument was
operated in multiple reaction monitoring
 ANALYTICAL
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TRACE ANALYSIS DEVELOPMENT VERIFICATION
SELECTION

Figure 4: Synthesis of ephedrine from norephedrine.

Figure 5: Scheme of on-line genotoxic impurity (GI) analysis instrumentation. (A) Left arm (liquid syringe), (B) Right arm (SHS
syringe), (C) Tray of 98 GC vials, (D) Flow cell, (E) Sample tray of 32 SHS vials, (F) SHS incubator, (G) 2 × 5 position wash stations
and (H) GC injector.

(MRM) mode using ESI ionization with Jet
Stream technology (Agilent Technologies).
3-Aminobenzonitrile was monitored
using m/z 119.1 to m/z 92.0 transition
(with 119.1 > 102.0 as qualifier) with 15 V
collision energy.
The chromatograms obtained for
a 10-ng/mL standard solution of
3-aminobenzonitrile analyzed using
acetonitrile as mobile-phase B is shown
in Figure 3a. The GI is eluted at 2.6 min
and is clearly detected. The extracted
ion transition from the analysis of a
bupivacaine sample spiked at 0.1 ppm
level (corresponding concentration
in extract = 10 ng/mL) under the
same analytical conditions is shown in
Figure 3b. In this trace, the GI could
not be detected. The analysis was
performed using diode-array detection

31 | September 2017 | LCGC

 ANALYTICAL
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SELECTION TRACE ANALYSIS
Figure 6: Plot of reaction kinetics for methyl iodide in norephedrine to ephedrine reaction.
(DAD) (Agilent Technologies) in-line with
MS and it was observed in the DAD
chromatogram that the API was eluted
at 2.35–2.70 min. The presence of the
matrix completely suppressed ionization
for the GI. The analyses were then
repeated, only changing mobile-phase
B to methanol (with slight increase of
column temperature). The extracted
transition chromatogram obtained for
the 10 ng/mL standard is shown in
Figure 3c. 3-Amino-benzonitrile now is
eluted at 2.4 min. In the chromatogram
of the spiked sample, the GI can now
be detected without problem (Figure
3d) since the use of methanol slightly
changed the chromatographic selectivity,
avoiding co-elution of the GI with the
API. This example clearly illustrates
that even when using state-of-the-art
mass spectrometry, the role of the
chromatographic separation is important
to overcome ion suppression. This
32 | September 2017 | LCGC
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example also showed that the availability
of UV–DAD in-line with MS detection can
help to diagnose problems.
On-Line Monitoring of
GIs During Synthesis
An interesting trend in pharmaceutical
analysis is to use analytical methods for
real-time monitoring of reactions, formation
or residual amounts of GI during a synthesis
process. This type of process analytical
technology (PAT) is now most often
performed by spectroscopic techniques
(UV, NIR, Raman), but for GI analysis
these techniques are often not specific
nor sensitive enough. Chromatography,
eventually combined with MS detection,
can be an interesting alternative. However,
speed is critical for PAT analysis. Recent
developments in fast GC instruments and
UHPLC can be applied to overcome this
problem and analysis times within a few
minutes are realistic.
 ANALYTICAL
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On-line and real-time monitoring of
GIs can also be used to study reaction
kinetics under different reaction
conditions, such as temperature, water
content, pH, ionic strength, and so on,
to provide interesting information on
conditions that favor or reduce formation
of GIs.
On-line GC–MS, applying robotics
for sampling, reaction stopping, and
derivatization was used to study reaction
kinetics for the formation of ethyl
methane sulphonate from ethanol and
methane sulphonic acid (7). Robotic
sampling and automated sample
preparation before GC–MS analysis
was also developed in a model study to
monitor the consumption and residual
level of methyliodide, a halide GI that
is used as methylating reagent for the
synthesis of ephedrine from norephedrine
(Figure 4). The reaction is performed
under alkaline conditions with ethanol as
the solvent.
On-line monitoring was done using
the instrument configuration shown in
Figure 5. A lab-scale reaction vessel
was used to mix the reagents. From
the reaction vessel, liquid sample was
pumped (via a downstream peristaltic
pump) through a dedicated flow cell
installed on a MPS2 autosampler (Gerstel
GmbH). One arm of the robot sampled
at dedicated times from the reaction
fluid, transferred this fraction to a 20-mL
headspace vial, and added dilution
solvent to stop the reaction and an
internal standard (1-bromopropane).
33 | September 2017 | LCGC
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The second arm then transferred the vial
to a headspace incubator oven (5 min
equilibrium at 80 °C) and finally 1 mL of
headspace was injected in the GC system
for analysis. GC separation was performed
on a 60 m × 0.25 mm i.d. × 1.4 μm DB-
VRX column (Agilent Technologies).
Analysis time was about 7 min. The
autosampler allowed additional sampling
during GC–MS analysis, so that every 2
min a sample was taken. From the relative
response of the peak area of methyl
iodide vs. internal standard, a reaction
kinetics plot could be obtained, showing
the consumption of methyl iodide in the
reaction, as illustrated in Figure 6.
A similar setup has been constructed
in the authors laboratories for on-line
monitoring by UHPLC–MS.
Conclusion
In recent years, methods have been
developed for trace analysis of
potential GIs in pharmaceuticals. New
developments in automated sample
preparation and state-of-the-art GC–MS
and LC–MS allow sub-ppm sensitivities.
Using robotic sampling combined with
fast GC or UHPLC offers interesting
possibilities for process monitoring.
Acknowledgment
The authors wish to thank Andrew
Teasdale and colleagues from Astra
Zeneca (Macclesfield, UK) and Roman
Szucs and Melissa Hanna-Brown from
Pfizer (Sandwich, UK) for their support in
this project.
 ANALYTICAL
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At the time of publication, Frank David
is the R&D manager at the Research
Institute for Chromatography (RIC) and
visiting professor at the Ghent University,
Ghent, Belgium. Maria Rambla Alegre
is a gas-phase specialist at RIC, Kortrijk,
Belgium. Gerd Vanhoenacker is a liquid-
phase specialist and LC product manager
at RIC, Kortrijk, Belgium. Pat Sandra is
thed Director of RIC and a professor at
Ghent University, Ghent, Belgium.
References
(1) European Medicines Evaluation Agency, Committee for
Medicinal Products for Human Use, Guideline on the Limits
of Genotoxic Impurities, CPMP/SWP/5199/02, London,
28 June 2006 (http://www.emea.europa.eu/pdfs/human/
swp/519902en.pdf)
34 | September 2017 | LCGC
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(2) Information on structural alert functionalities can be
found at: http://ecb.jrc.ec.europa.eu/documents/QSAR/
EUR_23844_EN.pdf.
(3) A. Teasdale, Ed, Genotoxic impurities: Strategies for
Identification and Control, (John Wiley & Sons, Inc., Hoboken,
New Jersey, USA, 2010) ISBN 978-0-470-49919-1.
(4) US Food and Drug Administration, Department of Health and
Human Services, Center for Drug Evaluation and Research
(CDER), Guidance for Industry. Genotoxic and Carcinogenic
Impurities in Drug Substances and Products: Recommended
Approaches, December 2008.
(5) F. David et al., LCGC Europe 22(11), 552–561 (2009).
(6) R. Alzaga et al., J. Pharm. Biomed. Anal. 45(3), 472–479 (2007).
(7) K. Jacq et al., J. Pharm. Biomed. Anal. 48(5), 1339–1344 (2008).
(8) F. David et al., Anal. Bioanal. Chem. 396(3), 1291–1300 (2010).
(9) G. Vanhoenacker et al., J. Chromatogr. A 1216(16), 3563–3570
(2009).
This article first appeared in LCGC
Europe, Advances in Pharmaceutical
Analysis Supplement, May 2013.
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 Method Selection
for Trace Analysis of
Genotoxic Impurities in
Pharmaceuticals
Frank David, Karine Jacq, Gerd Vanhoenacker, Pat Sandra, and Andrew Baker

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This article gives an overview of methods
that allow quantitative analysis of genotoxic
and potentially genotoxic impurities in
pharmaceutical ingredients at trace level.
A method selection chart is presented.
Depending on the solute characteristics,
either gas chromatography (GC) or liquid
chromatography (LC), both combined with
a single-quadrupole mass spectrometer
as detector are used. Different sample
preparation and sample introduction methods
were evaluated in terms of automation,
miniaturization, toxic solvent reduction and
hyphenation. These methods were applied
to different classes of genotoxic impurities
covering a broad range of volatilities and
polarities. Additional tools, such as capillary
flow technology in GC, allowing back-
flushing, and heart-cutting, offer interesting
possibilities for problem cases. Several
applications illustrate the potential of the
getty Images/xxxxxxxx

methods and the use of the method selection

chart.

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During the synthesis of active
pharmaceutical ingredients (APIs) and the
manufacturing of pharmaceutical products
in general, reactive intermediates,
catalysts, acids, or bases are used. These
compounds can potentially be present at
trace levels in final drug products. Due
to their chemical structure and reactivity,
some of these solutes are recognized
as genotoxic or potentially genotoxic.
Recently, the possible presence of these
genotoxic impurities (GTIs) or potential
genotoxic impurities (PGIs) in active
pharmaceutical ingredients (APIs) received
increased attention (1,2) and official
guidelines are issued (3,4). A threshold
of toxicological concern value (TTC) of
1.5 μg/day (daily intake) was proposed
as an acceptable risk (2,3). Based on
the daily intake of the drug (dose), this
translates into target limits of detection
for the determination of these potential
impurities in the drug substance below 1
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ppm (<1 μg/g API). This is about 500 times
lower than for classical impurity analysis
in pharmaceutical quality control (1 ppm
versus 0.05%).
As a very large number of solutes are
used or tested in drug synthesis and
development, no list of target solutes is
available, but rather a list with “structural
alert functionalities” is used (5). This
list includes sulfonates (for example,
ethyl methane sulfonate), alkylhalides,
arylamines, epoxides, Michael reaction
acceptors, and so on. It is clear that this
covers an extremely broad range of
polarities and volatilities. Moreover, some
solutes are reactive by nature and can
decompose or hydrolyze in GC or LC
analysis. In combination with the large
range of APIs and intermediates (water
soluble, apolar, ionic, and basic, acidic), the
determination of GTIs and PGIs poses a
real challenge to analytical laboratories.
Our laboratories have developed and
evaluated several methods for different
classes of impurities over the past few
years. Rather than developing specific
methods to determine a single (or a few)
target analytes in a specific (known) API,
the goal of our study was to develop a
number of generic methods that could
be applied to several impurities in a
wide range of APIs. These methods
form the basis for fine-tuned methods in
specific cases within drug development.
Developing each method on an individual
basis would be incredibly time- and
resource-consuming for AstraZeneca. The
project, therefore, aimed at developing a
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strategic knowledge and approach to the
whole area, which, when combined with
the methods developed, would deliver
significant efficiencies to the organization.
Based on our research, a method
selection chart (decision tree) was
constructed that is very useful to guide
pharmaceutical QC scientists through
the possibilities of trace analytical
methods that can be applied in GTI/
PGI analysis. Methods were developed
using either gas chromatography (GC)
or liquid chromatography (LC), both in
combination with a single-quadrupole
mass spectrometer as the detector
because these techniques are commonly
available in a routine QC environment. The
same separation methods can, of course,
also be applied using triple-quadrupole
instruments with multiple reaction
monitoring (MRM) or using ion trap and
time-of-flight (TOF) mass spectrometers.
Next, several sample preparation
methods were tested on different
classes of GTI/PGI in a selection of APIs,
with different properties (polarity and
functionalities). Attention is focused on
sample preparation methods that are
automated, robust, and on-line coupled
to GC or LC, while manual handling and
solvent use are largely reduced or avoided.
The result of this research is the decision
tree chart presented in Figure 1. Briefly,
this chart starts by asking if the GTI/PGI
is GC-amenable or not. If yes, the next
question is whether the GTI/PGI has
sufficient vapor pressure to be present in
the headspace phase of a concentrated
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solution of the API (in water, dimethyl
sulfoxide [DMSO], or other low-volatile
solvent). If yes, static headspace (SHS),
solid-phase microextraction (SPME) or
dynamic headspace (DHS) can be used. In
some cases, in situ derivatization can be
applied to generate a volatile derivative
of the GTI/PGI that can be analyzed by
SHS, SPME or DHS. If the solute is GC-
amenable (stable and eluted from the
GC column at moderate temperatures,
for example, <320 °C), but not volatile
enough for headspace techniques, a direct
injection of a concentrated API solution
in GC can be used. Attention should be
paid to the volatility of the solutes and the
matrix (API). Because the matrix is also
introduced, the API, other API impurities,
and API decomposition products (for
example, formed in the hot GC inlet) can
interfere with the target PGIs/GTIs or
can influence the system performance
(contamination). In this case, recently
introduced techniques such as capillary
flow technology (CFT)-based back-flushing
and two-dimensional GC (2D GC, GCxGC)
applying Deans switching can be used.
In our experience, several GTI/PGI were
found amenable to GC methods and
because of the availability of robust and
low-cost GC–MS systems, this option
should be evaluated first.
If the solutes are not amenable to GC
techniques, LC–MS instrumentation is
used. In selecting LC–MS, instruments
first a selection of the ionization mode
is needed. Both atmospheric pressure
electrospray ionization (AP-ESI) and
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Figure 1: Method selection chart for GTI/PGI in API determination.
atmospheric pressure chemical ionization
(APCI), either operating in the positive-ion
(PI) or negative-ion (NI) detection mode,
can be evaluated in terms of sensitivity and
selectivity. One mode can, for example,
result in a higher absolute response, but
combined with lower selectivity or higher
background, or both, the overall result
might be that another ionization mode
performs better for the given application.
Flow injection of solutions of the GTI/PGI
and MS acquisition in alternating + or –
scan mode and the use of a multimode
ESI/APCI source can be useful here.
Direct injection of a concentrated API
solution followed by the reversed-phase
HPLC method is tested first. At very low
retention of the solutes, hydrophobic
interaction liquid chromatography (HILIC)
or pre-column derivatization (into more
hydrophobic derivatives) can be used as
an alternative. All modes can be combined
with MS systems, using either ESI or
APCI. Although MS is often considered
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as a “universal” detection, some target
solutes can give very low or non-selective
response, either in ESI or APCI ionization.
In these cases, derivatization before
LC–MS analysis can also be useful. In
this article, the potential of the method
selection chart is illustrated with several
examples showing the trace analysis of
GTIs/PGIs in APIs.
Experimental
Chemicals: Various APIs, commercially
available from Sigma-Aldrich (Bornem,
Belgium) were used as test matrices.
Typically, the API is dissolved at 50 mg/
mL in an appropriate solvent (water,
DMSO, water–DMSO [1:1] for headspace
analysis, dichloromethane for direct GC
analysis, acetonitrile for LC, and so forth).
These solutions were spiked at a low
parts-per-million level with various GTIs/
PGIs (all available from Sigma). The GTIs/
PGIs were diluted at 0.1–1 mg/mL and
spiked in the API solutions.
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Figure 2: Analysis of organohalides in API (promethazine) by SHS-GC-MS (SIM). Sample spiked at 5 ug/g API. SHS at 80oC.
Solutes: (1) chloremethane, (2) vinyl chloride, (3) bromomethane, (4) vinyl bromide, (5) 1-chloropropane, (6) iodomethane,
(7) 2-chloropropane, (8) E-1.2-dichloroethene, (9) 2-bromopropane, (10) Z-1.2-dichloroethene, (11) 2-chloroacrylonitrile, (12)
1-chloro-2-methylpropene, (13) 1-bromopropane, (14) 2-iodopropane, IS. fluorobenzene, (15) 1-bromo-2-methylpropene, (16)
1-iodopropane, (17) E-1.2-dibromethene, (18) Z-1.2-dibromoethene, (19) 2-iodoethanol, (20) 3-bromo-2-methylacrylonitrile (cis),
(21) 3-bromo-2-methylacrylonitrile (trans), (22) 4-fluorobenzyl chloride, (23) benzyl chloride, (24) 4-fluorobenzyl bromide, (25)
benzyl bromide, (26) 4-methylbenzyl chloride, (27) 4-methylbenzyl bromide, (28) 4-chlorobutylether.

Instrumentation: Various systems were using a multipurpose sampler (MPS2,

used. Typical instrumental configurations
are described here. Experimental details
are given in the results and discussion
section with the specific examples.
GC–MS methods were developed and
validated on an Agilent 6890 or 7890
GC system combined with a 5975 MSD
system (Agilent Technologies, Wilmington,
Delaware). The GC instrument was
equipped with a split–splitless inlet and a
programmed-temperature vaporizing inlet
(CIS 4 PTV, Gerstel GmbH, Mulheim an der
Ruhr, Germany). Injection was performed
Gerstel), which allows liquid injection,
SHS, and SPME. The system could also be
equipped with thermal desorption (TDU)
and dynamic headspace (DHS) options
(Gerstel).
LC–MS methods were developed on an
Agilent 1100 or 1200 LC system combined
with a 1100 Series Quadrupole MSD
(version SL) system equipped with an ESI
source (Agilent Technologies, Walbronn,
Germany). The HPLC system consisted of a
binary pump, an automated liquid sampler,
a thermostated column oven equipped

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with a column-switching valve, and a
diode array (DA) detector (optional). In the
six-port, two-position valve two columns
were installed allowing unattended
switching and column selection. A binary
pump with solvent selection valve was
used. Typically, the following mobile
phases were used: A1: 10 mM ammonium
acetate + 0.1% acetic acid in water; A2:
10 mM ammonium acetate in water; B1:
acetonitrile; and B2: methanol. In this
way, gradients could be programmed
combining A1/B1, A1/B2, A2/B1, and A2/
B2. This results in eight possible column–
mobile-phase combinations.
The MSD instrument was used in SIM
mode either using positive or negative ion
detection. During the elution of the API,
the LC column effluent could be diverted
to waste, not to contaminate the ionization
chamber.
Results and Discussion
Analysis of organohalides: A first
important class of GTI/PGI are the
organohalides, which are used as alkylating
agents in synthesis. Analytical methods
for these solutes were reviewed by Elder
and colleagues (6). Most of the target
solutes are stable, amenable to GC, and
also volatile enough to allow headspace
analysis. Typically, methods used for
volatile organic compounds (VOCs) in
environmental samples, can thus be used
as a basis for this class of GTIs/PGIs.
Using a dedicated column (60 m × 0.25
mm i.d. × 1.4 μm DB-VRX) and GC–MS
operated in simultaneous scan/SIM mode,
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a generic method was developed and
validated. A typical chromatogram (SIM
trace) obtained for an API (promethazine)
spiked with 28 solutes at 5 ppm (5 μg/g
API) level is shown in Figure 2. The API (50
mg) was dissolved in 2 mL of DMSO–water
(1:1) and static headspace analysis was
performed at 80 °C during 15 min. 1 mL
headspace was injected in split mode (split
ratio: 1/10).
From the chromatogram it is clear
that most solutes are well detected.
Good linearity and repeatability were
obtained and the limit of detection (LOD)
was below 0.5 ppm for most solutes. It
can also be observed that the relative
response of the late eluted, less volatile
compounds drops significantly, as can be
expected from their lower vapor pressure.
4-Methylbenzylbromide (solute 27) could
not be detected at this level and also a
more polar solute, such as iodoethanol
(solute 19) was not detected with the SHS-
GC–MS method.
Based on these results, however, it is
clear that a SHS-GC–MS system is the
first method to consider for the analysis
of halides. In our study, several other
GTI/PGI, such as epoxides (ethyloxirane,
epichlorhydrine), mustards (2-chloroethyl
methyl sulfide), and Michael reaction
acceptors (acrylonitrile and methacrolein)
could also be analyzed using this generic
SHS-GC–MS method.
To increase the sensitivity for some
solutes, headspace solid-phase
microextraction (HS-SPME) was also
evaluated. A 75 μm/85 μm Carboxen/
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Figure 3: Analysis of organohalides in API (promethazine) by headspace-SPME–GC-MS (top) and SHS-GC-MS (SIM) (bottom,
inversed). Sample spiked at 0.5 µg/g. SPME at 80oC. Solutes: see Figure 1.
PDMS fiber was used because this fiber is
recommended for the analysis of VOCs.
Headspace-SPME was performed at 80 °C
during 20 min. GC–MS conditions were
identical to the SHS-GC–MS method. The
analysis of promethazine, spiked at 0.5
ppm level, obtained by SPME and SHS
respectively are compared in Figure 3. It
is now clear that the late eluted solutes,
with favorable PDMS–air partitioning
coefficients (KPDMS/air), are concentrated
in the fiber and enriched, resulting in
very low detection limits. This is clearly
illustrated for the solutes eluted after 12
min (peaks 16–28). 4-Methylbenzylbromide
(peak 27) could now be detected. Also,
iodoethanol (peak 19) could be detected,
but repeatability was not good (see
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further). In any case, it is clear that SPME
is complementary to SHS and for solutes
typically eluting after toluene on an apolar
column (log KPDMS/air > 3), SPME leads to
higher sensitivity. It should, however, be
noted that for the very volatile solutes (for
example, chloromethane [1], vinylchloride
[2]), SHS is superior to SPME because no
enrichment on the fiber is obtained.
Another option to increase sensitivity
is the use of dynamic headspace (7). In
this method, the sample (50 mg in 2 mL
DMSO/water placed in a 20-mL headspace
vial) was purged at 20 °C during 5 min
at 30 mL/min. The volatile solutes were
trapped on a Tenax trap. Next, the Tenax
trap was desorbed at 250 °C and the
released solutes were analyzed by GC–MS
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Figure 4: Analysis of organohalides in API (promethazine) by dynamic headspace GC-MS (scan). Sample spiked at 0.2 µg/g. DHS
at 20oC. Solutes: see Figure 1.
methods. Separation was performed on a
20 m × 0.18 mm i.d. × 1 μm DB-VRX. The
profile (extracted ion chromatogram at
m/z = 105 from scan-MS file) is shown in
Figure 4. The bottom trace shows a blank
API (promethazine), the top trace shows
the chromatogram of the sample spiked
at 2 ppm level. 4-Methyl-benzylbromide
(peak 27) that was not detected by static
headspace GC–MS instrumentation (in SIM
mode) can now be detected in scan mode.
LODs in SIM mode were below 0.05 ppm.
Thus, dynamic headspace GC–MS can also
be considered as complementary to SHS
for problem cases (less-volatile solutes and
extremely high sensitivity required).
Analysis of sulfonates: Methyl-, ethyl-,
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and isopropyl esters from methane
sulfonic acid (mesylates), benzenesulfonic
acid (besylates), and tolylsulfonic esters
(tosylates) are probably the best known
class of GTIs. These esters can potentially
be formed from volatile alcohols (used as
solvent) and sulfonic acids used to produce
API-salts. The analysis of sulfonate esters
is not straightforward. Methods were
recently reviewed by Elder and colleagues
(8). Sulfonate esters cannot be analyzed
directly by headspace techniques because
the vapor pressure is too low.
Moreover, direct injection in GC or
HPLC is difficult and prone to artifacts
such as possible formation of the GTIs
by decomposition of the API-salts (false
positives), hydrolysis of the sulfonates
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Figure 5: Analysis of sulphonate esters in APl (ampicillin) by derivatization–SHS–GC–MS (SIM). Sample spiked at 1 μg/g.
Derivatization with pentafluorothiophenol. Solutes: (1). d3-MMS, (2). MMS, (3) d5-EMS, (4) EMS.
(for example, in aqueous media), and API
interference. An elegant method was
described by Alzaga and colleagues (9)
using in situ derivatization—SHS-GC–
MS. In this method, the sulfonates are
derivatized (and thereby also stabilized)
by reaction with pentafluorothiophenol.
The methyl-, ethyl-, or isopropyl-
derivatives are stable and volatile and can
be analyzed by SHS-GC–MS systems.
Isotope-labeled solutes, prepared in-
house from deuterated alcohols reacting
with sulfonic acids or sulfonyl chlorides,
are used as internal standards. This
method was completely automated on
a dual-rail autosampler (Gerstel) and
could also be applied to study sulfonate
ester formation kinetics (10). An example
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of a chromatogram obtained by the
derivatization—SHS-GC–MS method
for the analysis of ampicillin spiked
at 1 ppm level with methyl methane
sulfonate (MMS) and ethyl methane
sulfonate (EMS) is shown in Figure 5. The
isotope-labeled standards (50 ng) were
spiked in the headspace vial containing
50 mg API in 4 mL DMSO–water (1:1)
(concentration = 1 ppm). Derivatization
was performed by addition of 100 μL of
pentafluorothiophenol-solution (6.4 mg/
mL in 1 N sodium hydroxide). After 15 min
incubation at 105 °C, 1 mL of headspace
was injected (1/10 split) and analyzed on
a 60 m × 0.25 mm i.d. × 1.4 μm DB-VRX
column. Detection was done in SIM mode
at m/z = 214 (MMS-derivative), 217 (d3-
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Figure 6: Analysis of N–mustards in API (doxylamine) by derivatisation–SPME-GC–MS. Sample spiked at 0.5 μg/g. Derivatization
with ethylchloformate. Solutes: acyl-derivatives of trifluoro-ethylamine (CF3EtNH2), 2-chloroethylamine (Cl-EtNh2), 3–
chloropropylamine (IS) and bis(2–chloroethyl) amine (bisCIEtNH).
MMS-derivative), 228 (EMS-derivative) and
233 (d5-EMS-derivative).
Analysis of S- and N-mustards:
Mustards are β-halogenated dialkyl
sulfides (S-mustards—for example, bis[2-
chloroethyl]-sulfide) or β-halogenated
amines (N-mustards). These compounds
are also used as alkylating reagents in
chemical synthesis. Some of these GTIs,
such as 2-chloroethyl methyl sulfide, could
be analyzed by static headspace GC–MS
instruments. N-mustards (or “mustard-
like” solutes), with a primary or secondary
amine functionality, however, do not have
sufficient vapor pressure to be analyzed
by headspace techniques. For these
solutes, derivatization followed by SHS
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or SPME can be used. Successful analysis
of these solutes could be achieved by in
situ acylation using ethylchloroformate.
For this analysis, 100 mg API (doxylamine)
was dissolved in 2 mL solvent mixture
(water–ethanol–pyridine, 2:1:1). 50 μL
ethylchloroformate was added. Reaction
was performed at room temperature
during 15 min, followed by SPME
extraction during 10 min at 80 °C using
a 100 μm PDMS fiber. Analysis was
performed using split (1/10) injection at
250 °C, followed by separation on a 60 m
× 0.25 mm i.d. × 1.4 μm DB-VRX column.
Detection was done by MS in SIM mode.
The chromatogram (EIC of SIM) obtained
for an API sample (doxylamine) spiked at
0.5 ppm level is shown in Figure 6. The
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acyl-derivatives of trifluoroethylamine
(CF3EtNH2) (ion 144), 2-chloroethylamine
(Cl-EtNH2) (ion 102), 3-chloropropylamine
(IS) (ion 102), and bis(2-chloroethyl)amine
(bisClEtNH) (ion 164) can be detected.
Analysis of haloalcohols and epoxides:
As described previously, some more
polar halogenated compounds such
as iodoethanol could not be extracted
from an API solution using headspace
techniques, and bad peak shape and
low repeatability–linearity were obtained
(see compound 19, Figures 2 and 3).
In situ derivatization of these solutes
followed by headspace analysis was also
not successful. Because most of these
solutes are GC amenable, direct analysis
by GC was considered. In contrast to
HS techniques, the APIs are hereby also
introduced in the analytical system and
they (or their more abundant impurities
and degradation compounds) can
interfere with GTI/PGI determination and,
moreover, contaminate the analytical
system (GC column, MS detector). For this
reason, heart-cutting two-dimensional
GC was evaluated as an alternative (11). A
concentrated solution of the API is injected
on an apolar first dimension column. The
fraction containing the GTI/PGI is heart-cut
using a capillary flow technology Deans
switching device (Agilent Technologies)
installed in a 7890 GC system, to a second
dimension column, placed in a separate
low thermal mass column oven module
(LTM, Agilent Technologies). The target
solutes are further separated on the
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second dimension from interferences and
detected by MS (scan/SIM mode). The
main solutes (API, derivatization reagents,
solvent, and so forth) are not introduced in
the second column, avoiding overloading
and contamination of this column and of
the MS source. The use of the low thermal
mass oven enables an independent
temperature control of the second
dimension column and consequently more
flexibility in method optimization.
The application of Deans switching
is illustrated by the analysis of two
haloalcohols (bromoethanol, iodoethanol)
and an epoxide (glycidol, 2,3-epoxy-
1-propanol) in carbamazepine.
Carbamazepine was dissolved in dry
pyridine at 50 mg/mL. From this solution,
100 μL (= 5 mg carbamazepine) was
placed in a 2-mL vial and spiked with 5
μL of the PGI test mixture (= 5 ng PGI,
corresponding to 1 ppm). Then, 100 μL
of BSTFA was added. The vial was heated
at 70 °C for 30 min. After cooling, 500 μL
of dichloromethane and 500 μL of water
were added. The mixture was vortexed
and injection (1 μL in splitless mode) was
performed from the lower organic layer.
This sample preparation could be fully
automated on an MPS2 autosampler.
The first dimension separation was done
on a 30 m × 0.25 mm i.d. × 0.25 μm HP-
5MS column (in a GC oven). The profile
obtained on a monitor flame ionization
detector after this first dimension is shown
in Figure 7a. The PGI cannot be detected,
while solvent, residual silylating agent,
and API (also derivatized) overload this
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Figure 7: Analysis of haloalcohols and glycidol in API (promethazine) by 2D-GC–MS. (a) FID mobitor detector trace from first
dimension separation: detection of solvent, API (silylated) – fraction 8–14 min; heart-cut (b) Extracted ion chromatogram from
second dimension GC–MS analysis.

column. The fraction eluted at 8–14 min
(containing the target GTIs/PGIs) was
heart-cut and further analyzed on a 30 m
× 0.25 mm i.d. × 0.25 μm DB-17 column (in
LTM oven). The chromatogram in Figure
7b shows the detection of the haloalcohols
and glycidol free from interferences. The
2D GC approach is, therefore, very useful
for problem cases where no headspace
extraction was possible. Also some
less volatile Michael reaction acceptors
could be successfully analyzed using this

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Figure 8: Analysis of aziridines in API (vitamin C) by RPLC/HILIC, Sample spiked at 1 μg/g. Solutes: AZIR 1: 1-(2-cyanoethyl)
aziridine (CAS number 1072-66-8, ion 97.2), AZIR 2: 1-isobutyrylaziridine (CAS 20386-12-8, ion 114.2), AZIR 3: cls-2,3-diphenyl-1-
propylaziridine (CAS 314062-46-9, ion 238.1), AZIR 4: 2-aziridin-1-yl-1-(4-nitrophenyl)-ethanol (CAS 21719-28-8, ion 209.1).
approach (11).
Analysis of aziridines, arylamines
and aminopyridines: For solutes not
amenable to GC, LC–MS methods were
developed using a single-quadrupole
mass spectrometer with electrospray
ionization and operating in the selected
ion monitoring (SIM) mode. Of course,
the same methodology can be applied
using triple-quadrupole instruments with
multiple reaction monitoring (MRM).
For a known API with only one or a
limited number of GTIs/PGIs possibly
present, selective sample preparation
methods based on (micro-) liquid–liquid
extraction or solid-phase extraction can
be developed. For generic methods,
however, a concentrated solution of the
API is directly injected. The major problem,
hereby, is the elution of the (main) API
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that can interfere with the GTIs/PGIs and
suppress detection (ionization). For this
reason, it is not possible to develop a
single HPLC method that is applicable to
all analyses. We therefore developed in
parallel a number of HPLC methods based
on two columns, each with (minimum)
two mobile phase combinations, resulting
in four methods that can be applied in
an automated sequence during method
selection–optimization. In this way,
scouting runs can be made to select
the optimal column–phase combination
for a given set of PGIs/GTIs in a given
API. Either two reversed-phase HPLC
columns (for example, C18 + phenyl) or
a reversed-phase liquid chromatography
and hydrophobic interaction liquid
chromatography (HILIC) method can be
combined. HILIC is very complementary
to reversed-phase liquid chromatography,
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Figure 9: Analysis of aldehydes in API (metoclopramine) by derivatization - RPLC, Sample spiked at 1 μg/g. Derivatization: DNPH,
Solutes: formaldehyde (ion 209.1), proplonaldehyde (ion 237.1), hexanal (ion 279.1), nonanal (ion 321.1).
especially for more polar solutes that are
eluted after the (less polar) API.
The reversed-phase liquid
chromatography–HILIC approach
is illustrated for the analysis of four
aziridines. For this application, the HPLC
system was equipped with a Alltima HP
C18 column (15 cm × 3 mm i.d. × 3 μm,
Grace, Lokeren, Belgium) and a Prevail
Silica HILIC column (15 cm × 4.6 mm
i.d. × 3 μm, Grace). Mobile-phase A was
0.1% ammonium acetate + 0.1% acetic
acid in water, and mobile-phase B was
acetonitrile. The reversed-phased liquid
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chromatography method used a gradient
from 10% B to 100% B in 10 min (5 min
hold) at 0.5 mL/min. The HILIC method
used a gradient from 100% B (2-min hold)
to 50% B in 13 min (1-min hold) at 1 mL/
min. Detection was done by MS using
ESI in positive ion monitoring mode.
The chromatograms (EIC from SIM) for a
vitamin C sample, dissolved at 100 mg/
mL in acetonitrile–water (9:1), and spiked
at the 1 ppm level with the four solutes,
are given in Figure 8. Test compounds
1 and 2 (most volatile, lowest mass) are
not retained on reversed-phased liquid
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chromatography and are measured by the
HILIC method. Test solutes 3 and 4 show
more retention in reversed-phased liquid
chromatography and can be separated
from the API. For all solutes excellent
signal-to-noise ratios are obtained, no
interference from API was observed, and
the method could be validated.
A similar approach was applied to the
analysis of arylamines and aminopyridines
(12). In this case, direct analysis of target
solutes was possible using two reversed-
phased LC methods. For the solutes with
low retention, pre-column derivatization
was used.
Analysis of aldehydes and ketones: Some
GTIs/PGIs can be analyzed either with
GC or with LC methods. In these cases,
method selection is based on ease-of-use
of the method, robustness and possible
automation. A typical group of solutes
that can be analyzed with GC and LC
techniques are aldehydes and ketones.
For these solutes, the separation itself
is not critical but detection is difficult.
Formaldehyde is eluted very fast in
GC–MS and no specific ions are present
for sensitive and selective detection.
Therefore derivatization is often applied,
both in GC and LC instrumentation.
In our study, pre-column derivatization
using 2,4-dinitrophenylhydrazine
(DNPH) was applied for solutes with
carbonyl functionality. This method
allowed the analysis of relatively volatile
solutes (for example, C1–C10 aldehydes)
and less-volatile solutes (for example,
50 | September 2017 | LCGC
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4-hydroxybenzaldehyde) using the same
method. Isotopically labeled aldehydes
were used as internal standards to improve
the repeatability and accuracy of the
method. The separation was performed
on a Zorbax Stablebond Phenyl column (15
cm × 3 mm i.d. × 3.5 μm, Agilent), using
a 10 mM ammonium acetate in water
(solvent A)–acetonitrile (solvent B) gradient
from 36% B to 100% B at 12.5 min with
a 0.65-mL/min flow rate. Derivatization
was automated in the 1100 series ALS
autosampler. The sample was dissolved in
a mixture of methanol–acetonitrile–water
(2:1:1) at 50 mg/mL. Internal standard was
added at 0.5 ppm level. From the sample
solution, 4 μL was drawn in the sampler,
followed by 1 μL of DNPH solution (500
mg of 2,4-dinitrophenylhydrazine is
dissolved in 10 mL of methanol, 1 mL
of concentrated sulfuric acid is added,
and the solution is diluted to 50 mL with
methanol, with a resulting concentration
of 1%). Detection is done by MS using
electrospray ionization in the negative-ion
monitoring mode.
The analysis is demonstrated on a
metoclopramine sample spiked with
a set of aldehydes at the 1 ppm level.
The resulting chromatogram is shown
in Figure 9. Metoclopramide is eluted
before 4 min. The derivatized aldehydes
are clearly detected in the extracted
ion chromatograms. The most volatile
aldehyde (formaldehyde) can also
be detected at this level without API
interference.
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Figure 10: Analysis of hydrazines in API (vitamin C) by derivatization–reversed-phase LC. Sample spiked at 1 μg/g. Derivatization:
hexylchloroformate, Solutes: 1. 1-methylhydrazine (EIC 175), 2. acetchydrazide (EIC 203), 3. 2-hydrazinoethanol (EIC 205), 4.
phenylhydrazine (EIC 237).
Analysis of hydrazines: The same
methodology was applied for the
analysis of hydrazines. In this case,
hexylchloroformate was used as pre-
column derivatization reagent, resulting
in N-acyl derivatives showing higher
retention in reversed-phase LC and good
detectability by MS. An example is shown
in Figure 10. Vitamin C was dissolved
at 100 mg/mL in acetonitrile and spiked
with 1-methylhydrazine, acetohydrazide,
2-hydrazinoethanol, and phenylhydrazine
at the 1 ppm level. To the sample, 7 mL of
borate buffer (15 mM sodium tetraborate,
pH 9.5 in 10% acetonitrile) was added,
followed by 0.5 mL hexylchloroformate
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solution (2% in acetonitrile). After reaction
for 15 min at room temperature, the
reaction was stopped by adding 0.5
mL phosphoric acid (17% in water) and
injection was performed. The separation
was performed on a Zorbax Eclipse Plus
column (15 cm × 3 mm i.d. × 3.5 μm,
Agilent Technologies), using a 0.05%
formic acid in water (solvent A)–acetonitrile
(solvent B) gradient from 15% B to 100%
B at 19 min with a 0.5-mL/min flow rate.
Detection was done by MS in SIM mode
using ESI in positive ion monitoring mode.
Conclusions
The chart shown in Figure 1 offers an
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interesting tool for method selection
for the determination of GTIs/PGIs in
APIs. Static headspace in combination
with a GC–MS system can be used for
the most volatile organohalides. SPME
and DHS techniques can be used to
extend the headspace extraction to less-
volatile solutes and also offer very high
sensitivities. For nonvolatile solutes, such
as sulfonate esters and N-mustards,
derivatization-SHS or derivatization-SPME
can be used.
Haloalcohols were analyzed after
derivatization and direct injection. Heart-
cutting 2D GC was used to avoid solute
interference and MS-source contamination.
Nonvolatile compounds are analyzed by
LC–MS instrumentation. An automated
selectable column approach using two
reversed-phase LC tools or a reversed-
phase LC–HILIC approach was used for
aziridines, arylamines and aminopyridines.
Solutes with low retention in reversed-
phased LC can also be analyzed after
pre-column derivatization. Derivatization is
also useful to increase detectability in MS
analysis, as demonstrated by the analysis
of aldehydes and hydrazines.
Acknowledgments
The authors would like to thank all the
individuals and technical groups within
AstraZeneca who contributed to the set
up and running of this project, in particular
Hans Jurgen Federsel and the members
of Global Process R&D External Sciences
Leadership Group.
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At the time of publication, Frank David
was an R&D manager at the Research
Institute for Chromatography (RIC) and
visiting professor at the Ghent University,
Belgium. Karine Jacq was a GC specialist
at RIC, Belgium. Gerd Vanhoenacker was
a liquid phase specialist and LC product
manager at RIC, Belgium. Pat Sandra
was the director of RIC and professor at
the Ghent University, Belgium. Andrew
Baker worked for AstraZeneca R&D within
Analytical Chemistry at Loughborough,
UK.
References
(1) T. McGovern et al., Trends in Analytical Chemistry 25(8), 790
(2006).
(2) L. Muller et al., Toxicol. Pharmacol. 44, 198 (2006).
(3) European Medicines Evaluation Agency, Committee for
Medicinal Products for Human Use, Guideline on the limits
of genotoxic impurities, CPMP/SWP/5199/02, London,
28 June 2006 (http://www.emea.europa.eu/pdfs/human/
swp/519902en.pdf)
(4) Guidance for Industry. Genotoxic and Carcinogenic
Impurities in Drug Substances and Products: Recommended
Approaches. US Department of Health and Human Services,
FDA, Center for Drug Evaluation and Research (CDER),
December 2008.
(5) Information on structural alert functionalities can be found at:
http://ecb.jrc.ec.europa.eu/documents/QSAR/EUR_23844_
EN.pdf.
(6) D.P. Elder, A.M. Lipczynski and A. Teasdale, J. Pharm. Biomed.
Anal. 48, 497 (2008).
(7) DHS brochure Gerstel GmbH, http://www.gerstel.com/en/
dynamic-headspace.htm
(8) D.P. Elder, A. Teasdale and A.M. Lipczynski, J. Pharm. Biomed.
Anal. 46, 1 (2008).
(9) R. Alzaga et al., J. Pharm. Biomed. Anal. 45, 472 (2007).
(10) K. Jacq et al., J. Pharm. Biomed. Anal. 48, 1339 (2008).
(11) F. David et al., Anal. Bioanal. Chem., 2009, submitted.
(12) G. Vanhoenacker et al., J. Chrom. A 1216, 3563 (2009).
This article first appeared in LCGC Europe
22(11), 552–561 (2009).
 A Test Mixture
for Performance
Verification of
Multi-User UHPLC–MS
Instruments
Tomas Leek, Niklas Falk, Ayda Babapour, and Magnus Klarqvist

Sponsored

Analysis of GTIs by LC-MS

Chromatographic techniques with mass
spectrometric detection are important
Click to
view PDF
enablers in modern drug discovery. With the
development of robust instrumentation and
implementation of user-friendly software
(or software packages), nonexpert users can
now walk up to easily accessible advanced
chromatographic systems and perform
experiments at their own convenience.
Although remarkable improvements in
robustness and ease of use have happened since
the introduction of the first high performance
liquid chromatography–mass spectrometry
(HPLC–MS) systems, the instrument
performance still needs to be qualified and
monitored to ensure consistent high-quality
getty Images/xxxxxxxx

results. This article will demonstrate how

a simple test mixture of carefully selected
compounds can facilitate both the development

53 | September 2017 | LCGC

 ANALYTICAL
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of generic ultrahigh-pressure liquid
chromatography–mass spectrometry
(UHPLC–MS) methods and automated
performance monitoring of multiple
instruments located in separate
laboratories and buildings.
Medicinal chemistry is an important
part of early drug discovery where novel
compounds are designed and synthesized
to develop treatments for unmet medical
needs (1,2). The iterative optimization
of biological activity and compound
properties is typically described by a
design–make–test–analyze (DMTA) cycle
(3). The consequences of progressing an
erroneous sample in the DMTA cycle can
be severe, leading not only to the waste
of valuable project resources by diverting
project efforts in the wrong direction,
but also potentially causing ambiguity
on ethical and intellectual property
issues. One widely adopted technology
for the analysis of reaction mixtures
and final screening compounds is
ultrahigh‑pressure liquid chromatography
KEY POINTS
• A representative and stable test mixture for a walk-up
UHPLC–MS platform in a drug discovery setting has
been developed.
• The test mixture was used for method development and
instrument set-up to improve the value of the walk-up
platform, that is, performance versus time and cost.
• A novel application has been developed to enable
remote performance monitoring of multiple low-pH mobile phases. As in every
UHPLC–MS instruments located in separate laborato-
ries and buildings at a low cost.
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(UHPLC) in the reversed-phase mode
with mass spectrometric (MS) detection.
Following the development of more
robust instrumentation and user-friendly
software packages, multi-user UHPLC–
MS instruments are now a common
feature of many laboratories for organic
synthetic chemistry (4). Walk‑up (which in
this article is used as a generic term for
a multi‑user system with a user-friendly
sample login, standardized methods,
and automated report generation)
solutions provide the end user with the
convenience of performing standardized
high‑performance analytical experiments
in close proximity to their workplaces
without the need of direct support
from separation and MS experts. This
article will present an example of how
a simple test mix can facilitate both
method development and function as
a system suitability test (SST) mix in a
UHPLC–MS-based walk‑up platform. The
initiative to develop this new generic
test mix originated in a technology shift
where seven UHPLC–MS systems were
replacing 17 high performance liquid
chromatography (HPLC)–MS instruments
in the walk-up platform for medicinal
chemistry. The main benefits from the
technology shift were expected to be
an increase in performance, including
a higher sample capacity and a higher
flexibility by enabling each UHPLC–MS
system to operate with both high- and
analytical procedure, a relevant SST
protocol must be implemented to ensure
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adequate instrument performance
before running actual samples. Given the
complexity of designing a generic test
mix, the benefits of using multivariate
analysis (MVA) became clear early in the
process and MVA was found to be a
valuable tool in the selection process of
test mix compounds.
After implementation, the SST protocol
evolved rapidly from monitoring the
performance of individual instruments to
automatically monitoring and visualizing
the performance of all UHPLC–MS
instruments in the platform. The aim
of the extended routine was to ensure
interchangeable systems where data
acquired on one of the UHPLC–MS
systems should be the same as that
obtained on any of the other systems. To
efficiently visualize the results from the
expanded protocol, a novel and efficient
visualization of SST data is described and
the benefits are discussed.
Experimental
Instrumentation: All experiments
were performed on Acquity I-Class or
Acquity Classic UPLC systems (Waters
Corporation). The systems consisted
of a binary pump, an in-line degasser,
an autosampler, a heated column
compartment, a diode-array UV detector,
and Waters SQD or Waters SQD2 MS
detectors with electrospray ionization.
Software: Instrument control and data
acquisition: MassLynx 4.1 software
(Waters Corporation); multivariate
data analysis and visualization: SIMCA
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13 (Umetrics); data evaluation and
visualization: TIBCO Spotfire 6.5 (TIBCO
Spotfire); calculation of logD and pKa
ADC/Labs 2012 (Advanced Chemistry
Development, Inc); php-scripting and
visualization of performance monitoring:
pChart 2.0 (www.pchart.net).
Chemicals and Columns: Acetonitrile
(HPLC grade) was purchased from
Fisher Chemical. Water was obtained
from an in-house Millipore system
(resistivity: 18.2 MΩ × cm at 25 °C).
Formic acid, ammonia-solution (26%),
ammonium hydrogen carbonate,
trifluoroacetic acid, and final test
compounds 3-acetaminophenol,
glyburide, amitriptyline (hydrochloride),
and (DHQD)2PHAL were purchased
from Sigma-Aldrich at analytical grade
or higher. Test compounds dissolved in
dimethyl sulfoxide (DMSO) were acquired
from company internal compound
management. Method development
was made on 50 mm × 2.1 mm and
30 mm × 2.1 mm, 1.7-μm BEH C18 or
1.8‑μm HSS C18 Acquity columns from
Waters Corporation.
General Mobile Phases and Stock
Solutions: Two different generic systems
for gradient elution were applied, one at
pH 3 and one at pH 10 (apparent pH as
measured in the weak mobile phase). In
both cases, mobile-phase A consisted
of 5% acetonitrile and 95% water, and
mobile-phase B was 95% acetonitrile
and 5% water. The pH was adjusted as
described below. Low pH-system: 10
mM formic acid and 1 mM ammonia in
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Figure 1: Visualizations of chemical space to facilitate the screening of test mix candidates and test mix prototypes. Here the
initial candidates (colored circles) and final compounds (blue stars) are positioned relative to a reference drug library in (a) PCA-
based virtual global space map and (b) according to lipophilicity and molecular weight.

both A and B. High pH-system: 6.5 mM detection was at 210 nm and 230 nm for
ammonium hydrogen carbonate with 47 impurity profiling. Peak capacity studies
mM ammonia in both A and B. were performed with an initial hold of
The test compounds were dissolved 1 column volume (CV) followed by the
and diluted to 0.2 mg/mL in DMSO or gradient elution at a different length.
in reference mix (30:70 w/w acetonitrile– The gradient ended with a 3-CV wash
water), and were dispensed into glass and 5-CV equilibration of the column
vials and sealed with a pre-slit silicone at starting conditions before the next
septum. injection.
General UHPLC–MS Setup and Core Procedure for Virtual Screening: A
Methods for Test Mix Development: virtual global space map (5) was used
Generic 2.5-min 5–95% acetonitrile to position test compounds relative to
gradients at a flow rate of 1 mL/min at the compounds in a diverse reference
45 °C were used at low and high pH. library. In the first step the variability
For the analysis of test mix stability, in chemical space was described using
the gradients were extended to 5 min. a principal component analysis (PCA)
All other conditions were constant. UV of calculated common physiochemical

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descriptors (lipophilicity, hydrogen bond
properties, polar surface area) for the
reference library. In the next step the
test mix candidates and a library of
bioavailable compounds (for comparison)
were positioned via PCA‑score prediction
in the virtual global map. As new test mix
candidates were identified they could
be included by the same procedure and
visualized in the chemical space without
changing the underlying t-scores.
Procedure for Test Mix Stability: Test
compounds were dissolved in DMSO to
0.2 mg/mL or in an acetonitrile–water mix
(30:70 w/w). The sample solutions were
dispensed into amber glass vials sealed
with pre-slit silicone septum and stored
at three different conditions: 1: in the
UHPLC autosampler (20 °C and dark); 2:
in the laboratory (ambient temperature
and protected from light); and 3: in the
refrigerator (8 °C and dark).
Stability samples were analyzed at
both pH 3 and pH 10 and evaluated
regarding peak area of the respective
test compound and the number of
total impurities formed at 3 days, 5
days, 8 days, 15 days, and 30 days and
compared with the initial data point. The
test mix stability was investigated both in
vials stored continuously at the different
conditions and in vials cycled in and out
of storage
General Procedure for Performance
Monitoring (PM): The PM procedure
was initiated by an automated creation
of a batch-file job in MassLynx, including
several blank injections to clean and
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condition the system before running the
test mix. The raw data were processed
in openlynx and the results were
moved into a repository file server. The
chromatographic channels (UV and MS)
were extracted together with instrument
metadata. The data were presented
using pChart 2.0 in an internal web
page, easily accessible for both analytical
experts and service engineers. The test
chromatograms (with UV lamp usage
time and number of injections) were
visualized in a primary panel to obtain a
swift overview of the overall instrument
performance. The processed data (RT,
peak area) were stored in a database.
Data from the most recent 30 days can
be visualized to detect drift and aging in
trend plots. When needed the content
in the database can be accessed and
visualized with descriptive statistics in the
software (6).
Results and Discussion
To secure adequate system performance
and ensure the quality of the generated
data, SST should be part of any analytical
method (7). However, the inherent
nature of medicinal chemistry means
that content and composition of future
samples is unknown and therefore the
strategy used to select representative
test mix compounds was to cover most
of the chemical space used by drug
projects. Ideally, the test mix should also
be applicable to performance testing of
the entire chromatographic system. With
this approach the same test mix can be
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Table I: Final test mix for method development and basic, and acidic compounds. When
system suitability of generic UHPLC–MS at low and considering a minimum SST protocol,
high pH (calculated properties)
the primary aim is to ensure that only
logD logD
Compound Structure [M+H]+
pH 3 pH 10 systems with adequate performance are
3-Acetaminophenol 152.1 0.7 0.2
available for walk-up analysis. Based on
experience and the literature (8), frequent
errors and unacceptable variance in
Glyburide 494.2 3.7 1.8
retention and peak area were associated
with mobile-phase composition, flow
Amitriptyline 278.2 1.8 4.9 rate, and injected sample amount. Since
the function of the separate instrument
modules is tested after service and repair,
(DHQD)2PHAL 779.4 3.5 9.1 the SST protocol can be reduced to
monitor variance in retention time and
peak area of test compounds and not the
Table II: Summary of test mix stability in solution at function of individual instrument modules.
studied conditions
In addition, the drift caused by aging of
DMSO Ref Solvent
stationary phases and contamination of
Autosampler (20 °C) > 30 days 5 days
Bench (ambient) > 30 days 8 days
the MS ion source needs to be captured
Fridge (8 °C) > 30 days* 15 days
and monitored to facilitate preventive
*Not exposed to freeze-thaw cycling maintenance.

used both for method development and Test Mix Development

as a mix of performance indicators for
SST. Although the future compounds
for biological testing are unknown,
the chemical space occupied by
these compounds and the synthetic
intermediates leading to them will cover
a vast chemical space. The final walk-up
methods and hence the test mix should
therefore cover the range from polar
low-molecular‑weight (MW) compounds
to lipophilic high‑molecular-weight
intermediates. As a guideline for test mix
development, a lipophilicity range of logP
0–10 and a molecular weight of 100–1000
should be covered with a mix of neutral,
The initial test mix candidates were
performance indicators found in literature
for chromatographic test mixtures (9–13)
and in unpublished internal protocols
for chromatographic testing. To obtain
an overview of the candidate’s relative
location in chemical space, a virtual
navigation map was created using a PCA-
based global space map (Figure 1a).
Advantages with this approach were the
swift positioning of test mix candidates
in the virtual map and the possibility to
evaluate the coverage of combinations
of candidates before performing any
additional experimental work. Promising

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combinations of test mix candidates
were thereafter combined into test mix
prototypes for experimental validation.
The prototype mixtures were prepared
in DMSO, analyzed at both pH 3 and
pH 10, and then evaluated for retention,
peak shape, and UV–MS responses.
After several iterations, a final test mix
of compounds could be selected (Table
I). In summary, all the compounds in
the test mix are visible both in UV
and electrospray ionization (ESI) and
resolved at both high and low pH using
the described methodology. Glyburide
(a weak acid) was selected primarily to
monitor the performance of the detectors
with good ionization in both positive
and negative electrospray and good
visibility in UV. Amitriptyline (lipophilic
base) was selected as a test probe of
chromatographic performance. Polar
starting materials were represented by
3-acetophenol and high-molecular-weight
lipophilic compounds by the catalyst
(DHQD)2PHAL.
After defining the test mix compounds
the next step was to investigate the
stability of the compounds in solution.
An experimental study was therefore
performed to study the effects of sample
solvents and the storage conditions.
Ideally, the sample solvent should have
an elution strength lower (or similar) to
the weak eluent in the gradient to achieve
good chromatographic performance,
while at the same time the sample should
be readily soluble in the solvent. An
acetonitrile–water mixture (30:70 v/v) was
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found to provide a promising compromise
of solubility and performance. As an
alternative to water-based mixtures,
neat DMSO has been found to be
a useful sample solvent capable of
dissolving a wide range of compounds
without causing severe chromatographic
artefacts. DMSO is known to have mild
oxidizing and hygroscopic properties
(14–15), however, and may compromise
the stability of the test mix. The stability
of the test mix compounds in DMSO and
in the acetonitrile–water (30:70 v/v) mix
was therefore investigated. The criteria
for stability was arbitrarily set to ± 5% of
the initial area (content). Interestingly, no
major chemical degradation (no single
impurity was above 2%) or reduction in
peak area could be observed in DMSO
samples stored for 30 consecutive days
and protected from light (Table II).
However, as the freezing point for neat
DMSO is around 19 °C, a freeze‑thaw
sequence is introduced when transferring
the DMSO solution in and out of the
refrigerator. Therefore a repeated
freeze‑thaw cycling was studied, showing
increasingly lower peak areas with each
cycle, indicating a dilution or compound
precipitation because of water uptake
in the DMSO (16). Freeze-thaw cycling
of the test mix in DMSO solution should
therefore be avoided. The stability
(content) in the acetonitrile–water mix was
in general terms lower, which is probably
a consequence of the higher volatility of
acetonitrile compared to water. Since the
vials are not perfectly sealed, acetonitrile
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Figure 2: Examples of how the test mix can be used to evaluate the influence of (a) column length and (b) mobile-phase
conditions on peak capacity. In (c) the estimated peak capacity to solve a generic separation problem with n components is
illustrated.
can evaporate and gradually reduce the
total sample volume and consequently
also alter the solvent composition with
decreased solubility of the lipophilic
(DHQD)2PHAL.
Test-Mix-Driven Method Development
From a user perspective, there are several
demands on a high‑performing walk-up
system. The analysis time should be short
to ensure a high sample capacity and the
peak capacity (PC) should be as high as
possible to separate as many peaks as
possible within any given analysis time.
Further, the instrument configuration
and methods should be robust and give
comparable results between systems. In
reality, compromises are necessary and
to maintain a data-driven optimization
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the test mix was utilized for method
development with different methods and
instrument setups were compared and
evaluated in terms of speed, performance,
and robustness. One example of how
the test mix was used to steer method
development can be found in the process
of finding a suitable combination of
column dimension, flow rate, and gradient
times. Performing chromatographic
separations at elevated temperatures
and high flow rates are known factors to
achieve high PC of small molecules (17).
To mitigate performance with the risk
of over-pressuring the instrument, the
maximum operational pressure was limited
to 80% of maximum system pressure.
Consequently, the flow rates were limited
to 1 mL/min for 2.1 × 50 mm column and
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1.25 mL/min for 2.1 × 30 mm columns.
The PC for the test mix components
was thereafter monitored when varying
column length, mobile phase conditions,
and gradient times. For simplistic reasons,
PC was here defined as the gradient time
divided with peak width at 5% of peak
height. When evaluating the performance
obtained on 30 mm and 50 mm columns
(Figure 2a), a gradient time of 1 min was
required to elute the test mix on 50-mm
columns. Although shorter analysis time is
possible on the 30-mm column, the PC is
dramatically reduced and so the 50-mm
columns were selected for further method
development. Next, the effect of gradient
time on PC was studied at pH 3 and pH
10. The observed general increase in PC
with longer gradient time was expected,
but an unexpected observation was made
when studying the separation between
amitriptyline and (DHQD)2PHAL at pH 3.
The two compounds are well separated
with short gradients, but as the gradient
time lengthens the separation of the
studied compounds decreases to zero at
13 min (coeluted peaks). This observation
was further investigated by repeating
the experiments with an addition of
trifluoroacetic acid to the mobile phases;
the pair now remained well separated up
to and above 13 min, indicating a more
homogeneous retention mechanism
possibly because of ion-pairing with
trifluoroacetic acid. Although the
separation was preserved and the peak
shape was improved with the addition
of trifluoroacetic acid, the PC was not
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nearly as good as at pH 10 (Figure 2b).
In the next method development step,
an estimate of the required PC (and
gradient time) for the general walk-
up methods was made with the aim of
balancing performance and capacity. To
picture the complex relationship between
gradient time, peak capacity, and the
capability of resolving “n-components”
in a hypothetical sample, the empirical
relationship PC = n1.5 was adopted from
reference 18. The solution shown in Figure
2c was used to obtain a general overview
of how the separation power develops
(resolving n components) with increasing
gradient time. Assuming simple or
moderately complex samples (where the
number of components are <20), relatively
short gradients (1.5–2 min) should provide
sufficient separation power to resolve
most of the hypothetical future samples.
The power of MS detection should
also be considered because it gives a
secondary dimension capable of resolving
ionizable compounds even if they not are
fully separated in the chromatographic
dimension. Studying the shape of the
graph (Figure 2c) shows the benefit of
providing slightly longer methods in
the walk-up platform. To balance the
separation power with sample capacity
it was therefore decided to implement
core methods with 1.5-min gradients for
reaction monitoring (2-min total runtime)
and 3.5-min gradients for more-complex
samples (total 4 min). Consequently, if
more-extensive method development (pH,
solvents, columns) is required to resolve
 ANALYTICAL
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Figure 3: Details of the web page showing (a) overview of daily test chromatograms from automated performance monitoring.
The partial trend plot of overall system performance at high pH shown in (b) clearly revealed the deviating system (green).
the components, the optimization should
be done on other systems and performed
by separation scientists to use the walk-
up instrumentation in the most efficient
manner.
Performance Monitoring of Walk-Up
UHPLC–MS Systems
To ensure appropriate quality of the
results generated on walk‑up instruments,
an automated daily procedure was
developed where the SST mix was
injected and the performance results
were visualized in a company internal
web page. By using this approach
instruments on different floors and even
in different buildings can be tested and
their performance monitored at a very
62 | September 2017 | LCGC
METHODS PERFORMANCE
DEVELOPMENT VERIFICATION
low cost. The strategy of presenting the
most recent test chromatograms in a
single visualization panel was found to be
an effective way of identifying a deviating
system for operators without an in-depth
knowledge of chromatographic theory.
Ideally, all test chromatograms, obtained
with a given LC–MS method, should
appear very similar and a diverging
system should be easily detected when
compared to another. Shortly after
implementation, it became evident that
the SST mix could be further simplified
to a performance monitoring (PM)
test mix as the major system variance
(especially in retention time and general
peak shape) was captured with the
test probes amitriptyline and glyburide
 ANALYTICAL
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(Figure 3a). Complementary to the
panel of daily test chromatograms, the
trends for representative calculated data
are visualized in a secondary panel to
detect drift in performance over time.
One of the first examples was the value
of harmonized performance monitoring
(Figure 3b), where the retention times
began to deviate for one of the systems.
The erratic system was taken out of
service but no obvious explanation
to the drifting retention times could
be identified until the UHPLC column
compartment was disassembled and
accumulations from a microleakage
were detected. The combination of the
leakage’s concealed location and the
elevated temperature in the compartment
made detection of the leakage difficult.
The problem was, however, effectively
brought to the attention of the service
engineer and therefore the use of a
malfunctioning walk-up instrument was
avoided. The performance monitoring has
also been used with significant success
to validate suggested improvements and
simplifications in the walk-up platform.
One initiative leading to improved
robustness regarding peak alignment
between UV and MS signals was achieved
by replacing the typical T-split between
the diode-array detector and the MS
system with a standardized length of
fused-silica capillary. Although the
mobile-phase flow is now suboptimal
for MS detection, the perceived value of
improved robustness in peak alignment
and general detection fidelity exceeds
63 | September 2017 | LCGC
METHODS PERFORMANCE
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the extra efforts of cleaning the MS
inlet. In addition, small changes to the
operational protocols, such as preparing
the mobile phases in 30-L barrels instead
of 1-L bottles, effectively reduced the
variance between systems. Increasing
the initial acetonitrile content in the
gradients from 5% to 10% was shown
to give a major improvement in system
availability because sample precipitation
and contamination are less prone to occur
with the higher organic content.
Conclusions
A test mixture with carefully selected
compounds representative for the
chemical space to be analyzed has
been shown to be an asset to control
both the quality of data produced and
to develop and improve the analytical
walk-up LC–MS platform. Combined with
daily performance monitoring, this has
resulted in increased system availability
and allowed for a change from 17 HPLC–
MS systems to seven UHPLC–MS systems.
These improvements have decreased
the need for maintenance and service,
increased robustness and efficiency of
the platform, decreased the cost, and
ultimately increased the value of our walk-
up LC–MS platform to the business.
Acknowledgments
The authors would like to thank our
colleagues at Medicinal Chemistry,
AstraZeneca Gothenburg for their
contributions to the development and
continuous improvements of the UHPLC–
 ANALYTICAL
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MS walk-up platform and for valuable Support Engineer at TIBCO Spotfire,

discussions. We would also like to thank Sweden.
Dr. Charles Elmore for valuable editorial Ayda Babapour joined AstraZeneca
comments and suggestions on the work. in 2013 and worked as an analytical
chemist with UHPLC–MS techniques in
References early research in drug discovery as well
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(3) S. Andersson et al., Drug Discovery Today 14(11–12), 598– works with analytical and preparative
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(7) J.W. Dolan, LCGC Europe 17(6), 328–332 (2004).
(8) V.J. Barwick, J. Chromatogr. A 849, 13–33 (1999). in 2003 and currently holds a position
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as Associate Director heading the
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(12) 14 SRM 870, CoA Issue date 30 Oct 2000, National Institute
of Standards and Technology (http://www.nist.gov/srm/) AstraZeneca. Magnus received his
(13) A. Marin et al., J. Liq. Chrom. and Rel. Tech. 31(1), 2–22
(2007).
Ph.D. in 1998 from Umeå University,
(14) D.H. Rammler and A. Zaffaroni, Ann. NY Acad. Sci. 141(1), Sweden, and joined the European
13–23 (1967).
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a research group in the Department
of Environmental Chemistry, Umeå
Tomas Leek is currently an Associate University, between 2000 and 2003.
Principal Scientist at Respiratory, Magnus is the author or co-author of
Inflammation, and Autoimmunity IMED over 20 peer-reviewed articles.
Biotech Unit, AstraZeneca, developing
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separations.
Niklas Falk joined AstraZeneca in
2000 as a senior research scientist
in cheminformatics, providing IS/IT
solutions to drug discovery projects.
Since 2015 Niklas has been a Senior

64 | September 2017 | LCGC

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