Wiener Tierärztliche Monatsschrift – Veterinary Medicine Austria
100 (2013)
From the Department of Primatology, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
LC-MS as a method for non-invasive measurement of
steroid hormones and their metabolites in urine and
faeces of animals
R. MURTAGH*, V. BEHRINGER and T. DESCHNER
received April 8, 2013
accepted July 5, 2013
Keywords: review, steroid hormones, bio-fluids,
non-invasive sampling, LC-MS.
Summary
This review presents the technique of liquid
chromatography-mass spectrometry (LC-MS). It
briefly outlines how LC-MS works, its different functions, the different types of instruments available, the
applications with which it is mainly used and the
costs involved. In particular, it highlights the effectiveness of LC-MS in measuring steroids and emphasizes its usefulness in measuring steroids noninvasively in animals, from matrices such as urine and
faeces. The advantages and disadvantages of
LC-MS in comparison to other methods of steroid
analysis such as immunoassays and gas chromatography-mass spectrometry (GC-MS) are discussed.
Abbreviations: APCI = atmospheric pressure and chemical
ionization; EIA = enzyme immunoassay; ESI = electro-spray
ionization; GC-MS = gas chromatography - mass spectrometry; HPLC = high performance/pressure liquid chromatography; HRMS = high resolution mass spectrometry; LCMS = liquid chromatography - mass spectrometry; MS/MS
= tandem mass spectrometry; OQ/PQ = operational qualification/performance qualification; RIA = radioimmunoassay;
SPE = solid phase extraction; ToF = time of flight; UPLC =
ultra performance liquid chromatography
Schlüsselwörter: Review, Steroidhormone, biologische Flüssigkeiten, nicht invasive Bestimmung, LC-MS.
Zusammenfassung
LC-MS als eine Methode zur nicht invasiven
Messung von Steroiden in Urin und Kot von Tieren
Dieser Übersichtsartikel gibt eine Zusammenfassung über die Technik der Flüssigchromatographie
gekoppelt mit Massenspektrometrie (LC-MS). Er beinhaltet eine kurze Einführung in die LC-MS und eine
detaillierte Beschreibung über deren Funktionalität.
Dabei wird auf die verschiedenen Instrumente eingegangen, die derzeit für LC-MS verwendet werden,
sowie auf die entstehenden Kosten und die Gebiete,
in denen diese Technik Anwendung findet. Besonders werden die Aspekte einer Steroidmessung mittels LC-MS angesprochen. Betont wird hierbei der
vorteilhafte Einsatz der LC-MS für die nicht invasive
Messung von Steroiden aus Urin und Kot. Des Weiteren werden die Vor- und Nachteile besprochen, die
eine Steroidmessung mit der LC-MS gegenüber einer Analyse mittels immunologischer Methoden
oder GC-MS hat.
Introduction
LC-MS is an instrumental method that combines
the separation capabilities of a high performance
liquid chromatography (HPLC) system with those of a
conjoined mass spectrometer.
Applications of LC-MS
LC-MS has a broad range of applications across
many different fields, including the petroleum industry (HSU et al., 2000), metabolomics (KOTŁOWSKA,
2012), proteomics (AEBERSOLD and MANN, 2003),
pesticide control (MATTERN et al., 1991), food safety
(NÚÑEZ et al., 2005), clinical and forensic toxicology
(MAURER, 2005, 2007), clinical diagnosis (RASHED,
2001; SHACKLETON, 2010), doping control (THEVIS
and SCHÄNZER, 2007; SHEN et al., 2009) and pharmacokinetics (BEAUDRY et al., 1998).
The usefulness of LC-MS in (steroid) hormone analysis of animals has become very well known over the
past decade. For example, LC-MS is widely used in
the livestock industry for the detection of illegal
growth hormones such as clenbuterol and other beta
agonists, which contribute to health risks in consumers (BLANCA et al., 2005). Other studies describe
methods developed for LC-MS to determine levels of
synthetic corticosteroids in bovine urine (VAN
POUCKE and VAN PETEGHEM, 2002; TÖLGYESI et al.,
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2010). Performance-enhancing drugs in race horses
and other equine athletes can be detected with a
method for measuring anabolic steroids in the serum
of horses (LIU et al., 2011). Misuse of testosterone for
doping purposes in human athletes can be determined by the simultaneous measurement of testosterone and its isomer epitestosterone in urine with
LC-MS (THEVIS and SCHÄNZER, 2007; DANACEAU
et al., 2008). LC-MS became the method of choice in
these research areas as it has the highest specificity.
Principle of LC-MS
HPLC separates compounds according to their polarity, resulting in different retention times on a stationary phase such as a silica-packed column (MEYER,
2010). In brief, the compounds are eluted from the
column via a liquid (mobile) phase. Different compounds vary in their affinities to the stationary phase
of the column and the mobile phase leading to different retention times (Fig. 1). Compounds differing in
polarity elute at different times, with unpolar substances eluting first when silica-packed columns are
used. If elution is carried out with an isocratic system,
consisting of only one mobile phase, elution times
can be unacceptably long, complicating the detection of later compounds as peaks become smaller
and broader. Alternatively, a gradient elution can be
applied whereby a second mobile phase of opposite
polarity is added to the system. The main purpose of
gradient elution is to move strongly retained components of the mixture faster by slowly mixing the mobile phases, changing the eluent from polar to nonpolar or vice versa. In the case of a reversed-phase
column, as on an isocratic system, substances elute
in order of decreasing polarity when the mobile phase
is gradually changed from polar to less polar. Using
elution gradients can improve peak form as well as
shorten run times.
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according to their mass/charge ratio (DE HOFFMANN,
2005). The solvent is vapourized in a heated spray
chamber as it enters the MS. Vapour is transported
through the chamber via a flow of carrier gas (e.g.
nitrogen) and then bombarded by an electron beam,
which ionizes the analytes within. Ions are created
either by transferring an electron to an analyte (forming a negatively charged ion) or by causing one to
be lost (forming a positively charged ion). The ions are
transported through a vacuum to the mass analyser,
where electro-magnetic forces differentiate them by
their mass and charge state. The information is
passed into a detector and a mass spectrum is produced. Ions are eventually neutralized, by colliding
with the MS walls and collecting electrons, and removed from the instrument by a vacuum pump. The
technique is very sensitive and has a high dynamic
range of measurement, up to four orders of magnitude. In combination, HPLC and MS separation mechanisms offer a very high compound specificity and
enable the simultaneous measurement of a large
number of substances.
Using MS/MS (tandem mass spectrometry) further
increases the specificity of a method. Not only is the
target analyte measured but in addition its precursor
ion is transferred to a chamber in the MS where it is
bombarded with a collision gas (e.g. argon, nitrogen
or xenon) and fragmented into product ions (Fig. 2),
the mass of which is then read in the detector. This
allows the identification of structural isomers that display similar chromatographic behaviour in the HPLC
column and that produce similar ions in the MS, as
different target analytes give different product ions
when fragmented.
Fig. 2: Schematic pathway of the MS/MS process from analyte
ionization, to parent ion formation, to fragmentation into product
ions, to detection.
Fig. 1: Elution of substances at different retention times from a
reverse-phase HPLC column. In such a system substances elute
in order of decreasing polarity.
The compounds are then transferred via a flow of
solvent to the MS, where they are further separated
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For example, the cortisol metabolite tetrahydrocortisol and its isomer allotetrahydrocortisol share a
common retention time on a HPLC column. They also
share a common molar mass and therefore the same
precursor ion mass when ionized. With MS/MS, the
precursor ion of each of the isomers fragments into
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Fig. 3: A = screenshot of the two most abundant product ions of tetrahydrocortisol. B = screenshot of the two most abundant product
ions of allotetrahydrocortisol. a, compound name; b, peak retention time; c, peak area; d, peak height; e, ionization mode; f, product ion
trace (precursor ion mass > product ion mass).
different product ions, which allows them to be distinguished from each other. The most abundant product
ion mass for tetrahydrocortisol is 301.15 m/z, whereas
for allotetrahydrocortisol it is 331.15 m/z (Fig. 3).
Instrumentation
In recent years, there has been a tendency to replace standard HPLC instruments with ultra performance liquid chromatography (UPLC) systems that,
like HPLC, can be conjoined to all types of MS
(SWARTZ, 2005). In contrast to HPLC, these systems
are designed to work with microbore® columns, which
can withstand higher pressures leading to faster flow
rates and elution times and shorter run times. Faster
separations reduce solvent amounts and running
costs. However, most standard HPLC instruments
are also capable of attaining the higher pressures and
flow rates required for microbore® columns.
Similarly, MS interfaces that are responsible for the
desolvation and ionization of the analytes have become increasingly sophisticated. There are two main
types of source interface:
1. electro-spray ionization (ESI) is more commonly
used and is better suited to ionizing polar compounds
such as steroid hormones (DEVENTER et al., 2006).
2. atmospheric pressure chemical ionization (APCI)
is used to measure thermally stable, less polar substances such as some hydrocarbons (MARVIN et al.,
1999).
MS instruments can be divided into two main types:
MS (e.g. quadrupoles and ion traps) and high resolution mass spectrometry (HRMS, e.g. LC-MS time of
flight (ToF) and orbitrap). The principle difference between these instruments is that MS separates ions
via electro-magnetic forces in a mass analyser,
whereas HRMS separates ions via their acceleration
through a flight tube. For example, LC-MS ToF
measures the mass/charge ratio of an ion via the time
it takes it to reach the detector. MS instruments can
only scan for a target range of masses, whereas
HRMS instruments can scan for masses of both
target and non-target substances. MS instruments
such as quadrupoles are currently more sensitive and
therefore mainly used for quantitative analysis but
HRMS instruments, which are mainly used for qualitative purposes, are catching up. There are already
HRMS instruments available that allow for higher resolution and sensitivity (WU et al., 2012).
Steroid hormones and LC-MS
Measuring steroid hormones is of huge importance
to investigating stress levels in the fields of animal
welfare (MÖSTL and PALME, 2002) and behavioural
ecology, where steroid hormones influence animal
behaviour and vice versa (DUFTY et al., 2002; GODWIN
et al., 2002; WINGFIELD et al., 2006; ANESTIS, 2011).
Steroids comprise a wide group of natural and
synthetic organic compounds that share a similar
chemical structure of 17 carbon atoms arranged in a
four-ring system. They are largely produced in specific glands such as the ovaries, testes and the adrenal
cortex (BORON and BOULPAEP, 2009). They are
involved in a variety of endocrinological functions
ranging from metabolism and digestion to the
development and functioning of the sexual organs
and include the sterols, bile acids, corticosteroids
(glucocorticoids and mineralcorticoids) and the sex
steroids (progestogens, androgens and oestrogens;
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GOMES et al., 2008). Steroid hormones are present in
their active form mainly in the blood, metabolized in
the liver and kidney and excreted via urine and faeces
in a multitude of modified, inactive forms that can be
conjugated to substances such as glucuronides and
sulphates. However, the metabolites of a steroid
hormone in urine or faeces can originate from different sources. For example, while testosterone is
largely produced in the testes, the testosterone metabolites that are abundant in urine can also derive from
the adrenal glands. Therefore, if researchers are
primarily interested in testicular function it is unwise
to rely on the concentration of metabolites that also
originate in the adrenal glands (HAUSER et al., 2011).
An LC-MS can be used to develop methods to
measure either conjugated or non-conjugated forms
of steroid hormone metabolites, as required. A large
number of steroid conjugates are possible. Often it is
preferable to measure the de-conjugated form than
to measure all possible conjugated forms. The
measurement of conjugated steroids can be more
complex due to the different ionization properties of
conjugated steroids. In-source dissociation of
glucuronides from steroids, for example, can
potentially interfere with accurate determinations
(KAKLAMANOS et al., 2009). Furthermore, reference
standards used to identify and quantify the target
analyte are not always available for some conjugated
forms. Care must be taken to ensure that chemical
de-conjugation does not lead to degradation and/or
transformation of the target analyte (HAUSER et al.,
2008a).
Non-invasive measurement of hormones in
animals
Blood collection from animals, especially wild
animals, is highly invasive and difficult to perform and
can be very stressful for the animal (WILSON et al.,
1978; BEERDA, 1996). It has become increasingly important to develop methods to measure steroid
hormones in samples obtained non-invasively. Over
the last few decades there has been increased
research into enzyme immunoassay (EIA)-, radio
immunoassay (RIA)- and HPLC-based techniques
(e.g. fraction collection) to measure hormones in
samples from humans and domestic, wild and captive animals. Examples include steroid hormone analysis in the urine and faeces from livestock (PALME et
al., 1996), faecal steroid analysis for monitoring reproductive status in farm, wild and zoo animals
(SCHWARZENBERGER et al., 1996), analysis of
salivary cortisol in squirrel monkeys (FUCHS et al.,
1997), pregnancy determination in faecal samples
from felids (DEHNHARD and JEWGENOW, 2013) and
measurement of urinary reproductive hormones in
captive apes (SHIMIZU et al., 2003). In addition,
methods to analyse non-steroidal hormones such as
oxytocin in the urine of chimpanzees (CROCKFORD
250
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et al., 2013) and thyroid hormones in urine and faecal
samples from birds and mammals have been described (WASSER et al., 2010). In wild chimpanzees,
for example, urine samples (MULLER and LIPSON,
2003; EMERY THOMPSON, 2005; SOBOLEWSKI et
al., 2012) and faecal samples (EMERY THOMPSON,
2005; MUEHLENBEIN, 2006) can easily be collected
for the purpose of non-invasive study.
In addition to these immunoassay-based methods
of quantification, a number of LC-MS methods have
been developed to measure steroid hormones and
their metabolites in matrices obtained non-invasively,
such as urine and faeces. This has been very
beneficial in clinical contexts (KRONE et al., 2010;
LUTZ et al., 2006) as well as in the field of primatology
(HAGEY and CZEKALA, 2003; HAUSER et al., 2008a;
WELTRING et al., 2012).
Immunoassays versus LC/GC-MS
Immunoassays are based on the binding of a specific antibody to an antigen of the target analyte (RAEM
and RAUCH, 2007). The specificity of any given assay
depends on how exclusively the antibody binds to the
target analyte. Cross-reactivity occurs when the antibody of an assay also binds to other substances that
are similar in their structure to the target analyte.
Many potentially cross-reactive substances must be
tested during the development of an assay. Most
commercial assays are developed for a specific compound in serum. As the concentration of potentially
confounding metabolites and/or degradation products found in serum is generally rather low and target analytes such as testosterone and cortisol are
highly abundant, cross-reactivity does not play such
an important part in these assays. However, in
matrices such as urine or faeces the metabolites are
more abundant than the target analytes and so the
potential confounding effects of cross-reactivities are
dramatically increased (PREIS et al., 2011; WELTRING
et al., 2012). Successful attempts to derive meaningful measurements of steroid hormones and their
metabolites in matrices such as urine and faeces,
despite the confounding effects of cross-reactive
substances, have led to the use of validated groupspecific assays (MÖSTL et al., 2005; PALME, 2005;
HEISTERMANN et al., 2006) or a chromatographic
separation of sample extracts prior to assay
(ZIEGLER, 1996; MÖHLE et al., 2002).
LC-MS techniques do not suffer from problems
related to cross-reactivity. A high compound specificity is achieved by the combined separation based
on specific characteristics in terms of polarity during
liquid chromatography separation, as well as the
mass/charge ratio of the parent ion in combination
with the mass/charge ratio of fragments of the analyte during MS and MS/MS separation steps. A
growing number of studies point out the advantages
of LC-MS compared to techniques such as EIA and
Wiener Tierärztliche Monatsschrift – Veterinary Medicine Austria
RIA (ROSNER et al., 2006; FANELLI et al., 2011; KOAL
et al., 2012).
Although they are less accurate and versatile,
immunoassays have the benefit of being faster and
cheaper than LC-MS (RAUH, 2009; THIENPONT et al.,
2008). An additional advantage is that there is no need
to develop methods, as many commercial assays for
many different hormones already exist. However,
the application of an assay in a species or matrix
other than the one for which it was developed
necessitates careful validation (PALME, 2005). LC-MS
can be used to estimate the degree of interference by cross-reactivity and therefore can have an
important role in the analytical validation of EIA or RIA
for specific hormones in a variety of matrices
(KUTSUKAKE et al., 2009; BEHRINGER et al., 2012;
PREIS et al., 2011). LC-MS can be used to ensure that
assays reliably measure the target analyte. Whatever
method is used, a thorough physiological validation is
mandatary to ensure that the measured steroids reflect something biologically meaningful (PALME, 2005).
LC-MS allows the simultaneous measurement of
several hormones and metabolites in any given
sample, leading to high throughput capabilities. This
renders the method particularly interesting for studies
where a number of metabolites are of interest
(HAUSER et al., 2011; KOAL et al., 2012; KUTSUKAKE
et al., 2009; PREIS et al., 2011; SURBECK et al.,
2012a,b). Particularly interesting for behavioural
endocrinology research is a method that allows the
simultaneous measurement of 23 steroid hormones
and their metabolites in small volumes of urine
collected non-invasively from wild primates (HAUSER
et al., 2008a).
Another method of chromatographic separation in
connection with mass spectrometry is GC-MS. The
detection principle is similar to that of LC-MS, using
the mass fragments that result from ionization of the
molecules. Before analysis, analytes of interest must
be extracted from the matrix into a liquid solvent
phase. The extract is injected into the GC where it is
swept onto a separation column by an inert carrier
gas. The analytes in the mixture are carried through
the column by the carrier gas and separated from one
another by virtue of their interaction with the coating
(stationary phase) on the inside wall of the column.
Each analyte interacts with the stationary phase at
different rates, resulting in different retention times. In
the case of steroids, derivatization is necessary as
GC-MS requires volatile substances. In contrast, LCMS does not require a lengthy derivatization that
could result in analyte losses (GOMES et al., 2009).
Nonetheless, GC-MS has the advantage that
fragmentation patterns of substances can be reproduced. The patterns are instrument independent,
allowing databases and libraries to be created and
shared between users (HALKET, 2004). Furthermore,
internal standards can be used to monitor efficiency
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of derivatization so that, once produced, a calibration
curve can be used repeatedly. This is in contrast to
the situation in LC-MS, where ion suppression can
occur with ESI or APCI so a new standard curve has
to be created for each sequence of samples (WUDY
and HARTMANN, 2004).
Before hormone analysis of bio-fluids with LC-MS,
sample clean-up is essential to remove unwanted
matrix compounds that could potentially affect the
measurements. Often solid phase extraction (SPE) is
employed. Depending on whether the study is
interested in measuring conjugated or unconjugated
forms of metabolites, further steps may also be required, such as hydrolysis or solvolysis procedures
(HAUSER et al., 2008b). Although extraction procedures can be time consuming, the sensitivity and
reliability of LC-MS instruments is constantly increasing with the development of new instruments
and thus it is highly likely that less time will need to be
spent on clean-up procedures in the future.
There are a few other disadvantages when it comes
to using LC-MS, including the two most common
complaints, matrix effects and ionization issues. The
matrix effect is interference from molecules in the
sample that cannot be removed during the clean-up
steps. Unwanted matrix can cause ion suppression or
enhancement and ultimately interfere with the quantification of analytes (VAN EECKHAUT et al., 2009).
However, this can usually be controlled for with the
use of synthetic hormones as internal standards,
something that cannot be done with EIA or RIA.
Costs of measuring hormones via LC-MS
The cost of new LC-MS instruments ranges from
€ 100,000 to more than € 600,000. Costs depend on
the features, such as whether an HPLC or UPLC
system is required, the presence of on-line extractions, the type of MS interface, ionization mode,
sensitivity and detection limits. In addition to the
initial purchase cost, machines require an annual
operational qualification and performance qualification (OQ/PQ) and maintenance work, which are
often performed by an external company, usually the
vendor of the instrument. Maintenance contracts
currently cost roughly € 10,000 per year, including an
annual OQ/PQ visit and year-round technical
support. Contracts are recommended as there can
be frequent troubleshooting involved for instrument
upkeep. However, with qualified personnel much of
the work can be carried out in-house, greatly
reducing the costs. It is estimated in our endocrinology laboratory that analysis of one analyte per urine/
faecal sample costs an average of € 15. The figure
includes consumables, reagents, extraction costs,
hydrolysis (urine only), solvolysis, LC-MS analysis,
equipment maintenance contracts and personnel
costs. It is evident that LC-MS is only cost-efficient
when multiple compounds per sample are analysed
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and when the machine is frequently in operation, i.e.
it is used for routine analysis.
The level of training and expertise varies amongst
personnel working with LC-MS. System operation
and data analysis can be carried out by anyone with a
reasonable amount of training. Maintenance of the
instruments and troubleshooting requires the technical, mechanical and electronic skills of a scientist/
engineer. Interpretation of data and the development
of methods requires in-depth knowledge of both
organic and inorganic chemistry.
Conclusion
LC-MS can be very useful for the measurement of
hormones obtained non-invasively in a variety of
matrices from many different species. Furthermore, it
can be used for the analytical validation of other
measurement techniques such as EIA and RIA. The
advantages of specificity and the ability to perform
100 (2013)
simultaneous measurements can outweigh its
biggest disadvantage, which is its cost, when
multiple substances are routinely analysed per
sample. The continual development of instruments
hints at an exciting future in metabolite analysis
across many fields of research.
Acknowledgments
The authors gratefully appreciate invaluable input
from Anja Weltring and two anonymous reviewers.
We thank Carolyn Rowney for editing the manuscript
and acknowledge all the help and support from the
endocrinology group in the Primatology Department
of the Max Planck Institute for Evolutionary Anthropology in Leipzig. We especially thank Rupert
Palme for the invitation to participate in the ISWE
Conference in Vienna and for his support with this
manuscript.
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*Corresponding author’s address:
Róisín Murtagh,
Max Planck Institute for Evolutionary Anthropology,
Deutscher Platz 6, 04103 Leipzig, Germany,
e-mail: [email protected]