June 2019

Medical Mass Spectrometry Vol. 3 No. 1

Mini Review

GC/MS/MS-based targeted metabolomics forpathophysiological analysis of animal models
1,2
1,3
Kei Zaitsu *, Yumi Hayashi

In Vivo Real-time Omics Laboratory, Institute for Advanced Research, Nagoya University,
Furo-cho, Chikusa-ku, Nagoya, Aichi 464‒8601, Japan2
Department of Legal Medicine & Bioethics, Nagoya University Graduate School of Medicine,
65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466‒8550, Japan3
Department of Radiological and Medical Laboratory Sciences, Nagoya University Graduate School of Medicine,1‒1‒20 Daiko-Minami, Higashi-ku, Nagoya, Aichi 461‒8673, Japan
1

Abstract

Metabolomics is an essential tool for not only understanding the pathophysiology of various diseases but also

for searching for initial clues to unknown toxic effects of drugs. Mass spectrometry-based metabolomics has achieved
highly sensitive and selective analysis of metabolites, and gas chromatography mass spectrometry remains a gold standard
because of its robustness and usability. However, it is tedious to annotate metabolites with electron ionization (EI)-basedmass spectra; thus, gas chromatography tandem mass spectrometry (GC/MS/MS)-based metabolome analysis has played an
important role in metabolomics. In particular, the selected reaction monitoring (SRM) mode achieves higher selectivity andan improved signal-to-noise ratio, which leads to easier metabolite identiﬁcation. In this mini review, we concisely outline
the pros and cons of GC/MS/MS-based metabolome analysis and provide its applications to pathophysiological analysis of
disease and drug-induced toxicity in animal models based on our previous studies. Finally, future perspectives for newly
developed high-throughput metabolome analysis are brieﬂy described.
Key words: targeted metabolome analysis, gas chromatography tandem mass spectrometry

Introduction

lylation are required for gas chromatographic separation of
6,7)

analytes

Metabolome analysis is a powerful tool for investigating, the gas chromatography/mass spectrometry

the pathophysiology of various diseases and/or toxic effects
1‒4)
of drugs together with other omics techniques . Cur-

(GC/MS)-based platform has remained a gold standard fortargeted metabolome analysis because GC is a robust ana-

rently, mass spectrometry-based platforms are widely used

lytical system, and a capillary column shows high separa-

for metabolome analysis in various ﬁelds because of their
5)high selectivity and sensitivity . Although appropriate deri-tion capacity for analytes. However, metabolite annotation
using electron ionization (EI)-based mass spectra can bevatization methods such as methoxylation and trimethylsi-

tedious for analysts, even when automated annotation software is available for EI-based metabolome data, and there*Corresponding author

is also room for improving quantitativity, which will be

Kei Zaitsu

mainly achieved by increasing selectivity; the selectivity of
GC/MS is sometimes insufﬁcient when targets and contam-

In Vivo Real-time Omics Laboratory, Institute for Advanced
Research, Nagoya University, Furo-cho, Chikusa-ku, Nagoya,inants are co-eluted.

Aichi 464‒8601, Japan
Tel: ＋81‒52‒744‒2118, Fax: ＋81‒52‒744‒2121

To obtain a higher signal-to-noise (S/N) ratio and selec-

Received May 27, 2019. Accepted June 13, 2019.

tivity, gas chromatography tandem mass spectrometry (GC/
MS/MS)-based targeted metabolome analysis has been

Epub June 18, 2019.

increasingly used in medical and toxicological ﬁelds

E-mail: kzaitsu@med.nagoya-u.ac.jp

8‒15)

.

Since selected reaction monitoring (SRM) mode can dra-

DOI: 10.24508/mms.2019.06.007
11

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Medical Mass Spectrometry Vol. 3 No. 1

MS-based method for oxidized and nitrated oleic acid in
9)biological samples . Generally, negative ion chemical ion-matically improve selectivity and S/N ratio, GC/MS/MS,
especially SRM mode analysis, provides not only highoperability for metabolite annotation, which is achieved bysimplifying the results, but also higher relative quantitativ-

ization dramatically improves the S/N ratio, which isity. Indeed, our team successfully applied GC/MS/
MS-based targeted metabolome analysis to drug-induced

ionization selectivity

mainly due to background noise reduction and improved
18,19). In their study, pentaﬂuorobenzyl−(PFB) ester derivatization was used to produce [M-PFB]−
and [M-H] ions, which were selected as precursor ions,acute intoxication in animal models to estimate unknown
toxic effects of drugs and understand their pathophysiol-

improving the selectivity of the method.

. As described later, GC/MS/MS-based targeted

Unlike CI, EI provides many fragment ions during ion-

metabolome analysis is extremely useful for exploratory

ization because high internal energies are supplied to theinvestigation of initial clues to unknown mechanisms ofvarious diseases and drug-induced toxicity.

ionized molecules created by EI. Since the intensity ofSome vendors currently provide ready-to-use GC/MS/
MS-based targeted metabolome platforms, and such com-analysis, analysts sometimes need to select fragment ionsas precursor ions for collision-induced dissociation. Speciﬁ-

mercially available platforms can greatly contribute to thecally, the GC/EI-MS/MS-based method is more complex

widespread use of metabolomics in various ﬁelds. GC/MS/
MS-based metabolome analysis has also become morewith respect to optimizing analytical conditions, such as

4,16,17)ogy＋•

molecular ions (M ) is generally weak or undetected in EI

familiar to scientists who will use metabolomics in theirselecting SRM transitions and setting the collision energy(CE), than that with GC/CI-MS/MS, though a large variety

research. Moreover, technological advances in ionizationof SRM transitions can be selected for each metabolite,

for mass spectrometry will provide researchers with a new
approach to high-throughput metabolome analysis, andallowing the selectivity of the method to be dramaticallyenhanced. Tsugawa et al. reported a GC/EI-MS/MS method

ambient ionization/mass spectrometric techniques havebeen extended to various scientiﬁc ﬁelds including metabo-using SRM analysis with TMS derivatization, achieving
11)

simultaneous detection of 110 metabolites . Also, Hirata et
al. used a GC/EI-MS/MS-based metabolome method andlomics.
In this mini review, we concisely outline the pros anddetermined sensitive metabolic biomarkers for pancreaticcons of GC/MS/MS-based targeted metabolome analysis
and introduce its application to drug-induced toxicity andcancer in human blood .
To develop a GC/EI-MS/MS-based metabolome method,animal disease models for pathophysiological analysis

an enormous amount of time is required to select appropri-

based on our previous studies. Additionally, we provide

ate SRM transitions, optimize the CE for each transition,

future perspectives using new analytical techniques,

and allocate scheduled SRM. Therefore, some vendors havenamely ambient ionization/mass spectrometry,high-throughput metabolome analysis.

provided commercially available methods for GC/MS/
MS-based metabolome analysis, which are optimized in

13)

for

advance and are thus highly practical for users who want to

GC/MS/MS-based Targeted Metabolome Analysis

begin using metabolomics.

GC/MS/MS-based targeted metabolome analysis has

In our previous studies, we used an EI-based method for
12,14,15), which is commercially availmetabolome analysis

been performed not only in chemical ionization (CI) mode

able from Shimadzu Corporation (Kyoto, Japan). With this. From the viewpoint of method

method, appropriate SRM transitions are selected inadvance, and the CEs for each SRM transition and sched-

but also in EI mode

8‒15)

development, the CI-based method is generally simpler
than the EI-based one because CI normally generates pro-uled time are sufﬁciently optimized. Currently, ca. 500
metabolites are pre-registered for the method and can be

tonated or deprotonated ions; thus, precursor ions are easily
selected for each metabolite. Xu et al. used GC/positive
chemical ionization (PCI)-MS/MS to analyze 37 fatty acids

simultaneously detected in a single run, and quantiﬁer andqualiﬁer ions for each metabolite are selectable, which
enhances the identiﬁcation accuracy for the targeted metab-

derived from methyl esters in the plasma of coronary artery10)
disease patients . Tsikas et al. reviewed a GC/electron cap-

olites. Additionally, the retention indices of each metabolite
are pre-registered in the method, and the retention times ofture negative ion chemical ionization (ECNICI)-MS/
12

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Medical Mass Spectrometry Vol. 3 No. 1

each metabolite can be easily adjusted by analyzing

instance, we performed metabolome analysis on serotonin
syndrome (SS) rat models, where hyperthermia and abnor-

20,21)

alkanes.

Based on these procedures, we can easily change themal muscle contraction such as myoclonus are the main
15,22). We applied GC/MS/MS-based metabsymptoms of SSscheduled time and optimize dwell times for each transition
using Excel-based software provided by the vendor. Partic-

olome analysis to plasma, liver and muscles of control andthe SS rats and readily identiﬁed 144‒195 metabolites inularly, adjusting and optimizing scheduled times for each
SRM transition are time-consuming unless analysts can use

plasma and the tissue samples. Many plasma metabolites
were signiﬁcantly increased, though these have no remark-such optimization software. Owing to such user-friendly

able networkability (Fig. 1a). Signiﬁcantly changed metabolites in liver and muscles showed respective networkabil-

tools, commercially available methods facilitate widespreaduse of metabolomics in various ﬁelds.
As described above, methoxylation and trimethylsilyla-

ity (Fig. 1b‒d), and these changes were strongly related totion are generally used for metabolite derivatization in GC/
MS- and GC/MS/MS-based metabolome analyses, though

the SS symptoms. Consequently, these results suggest thatsome researchers used other derivatization techniques tomodel rats were caused by the metabolome changes in thetarget tissues. We also observed site differences in metabo-signiﬁcant changes in the plasma metabolome of the SS

improve sensitivity or selectivity. Kvitvang et al. reported

lome changes in muscles, which were well matched to supporting results obtained from gene-expression analysis ofthat methyl chloroformate (MCF) derivatization wasapplied to 67 principal metabolites including amino acids
and non-amino organic acids, achieving signiﬁcant sensitiv-

uncoupling protein-3 (UCP-3). Finally, we revealed the8)

ity for these in urine and serum samples . This suggested

pathophysiology of SS based on metabolome analysis asthat there is a high potential to improve the selectivity or

shown in Fig. 2.

sensitivity by changing derivatization methods in GC/MS/
MS-based targeted metabolome analysis. However, a lim-

Exploratory investigation of unknown toxic effects ofdrugs in toxicology

itation of GC/MS/MS-based metabolome analysis is that it

In forensic toxicology, abuse of new psychoactive sub-is generally difﬁcult to sensitively detect highly polar
metabolites such as phosphate metabolites, and these can-23)

stances (NPSs) is a social problem worldwide . To date,various NPSs have been distributed in underground mar-

not all be detected by GC/MS/MS. Thus, compensatory
analytical techniques such as liquid chromatography tandem mass spectrometry (LC/MS/MS)-based-metabolome

kets, and cathinones and cannabinoid receptor (CBR) ago24‒26). It is essential to evalnists are most frequently abused

analysis are also necessary to expand the coverage of target

uate the acute toxicity of NPSs in forensic toxicology,

metabolites.

though minimal information is available on their toxicity.
This is because they are generally synthesized in clandes-

Application of GC/MS/MS-based Targeted
Metabolome Analysis to Pathophysiology ofAnimal Models

tine laboratories, and it is too difﬁcult for forensic toxicologists to obtain information on either their chemical struc27)tures or pharmacokinetics . Nevertheless, some NPSs are
28,29)

Pathophysiological analysis of animal disease models
by GC/MS/MS-based targeted metabolome analysis

highly toxic and sometimes fatal to humans. Although

it is too difﬁcult to extrapolate phenotypic changes in

As is well known, metabolome analysis is a useful tool

human from the metabolome changes in animal models,for investigating initial clues concerning the pathophysiol-

metabolomics is a useful technique for understanding
NPS-induced toxicity in animal models to search importantogy of various diseases animal models. To investigate
potential biological markers, however, metabolome analy-

clues related to their toxic effects. In particular, the results

ses on blood samples from animal disease models were per-obtained are easily understandable as phenotypic changes,

formed in most studies, while more information is obtainedby tissue metabolome analysis to achieve pathophysiologi-

which are pivotal to estimating the unknown toxic effects

cal understanding of diseases models, because the bloodin toxicological ﬁelds for estimating or predicting theadverse effects of drugs on animal models, and thus metab-30‒32)of NPSs on animal models

metabolome is easily changed depending on metabolomechanges in pathological and/or toxicity-affected organs. For. This idea is also accepted

olomics has been widely applied not only to forensic toxi13

June 2019

Fig. 1.

Medical Mass Spectrometry Vol. 3 No. 1

Networkability of the signiﬁcantly altered metabolites in each tissue.
(a) Plasma, (b) liver, (c) gastrocnemius muscle, and (d) trapezius (reprinted from ref. 15 with permission).

Fig. 2.

Pathophysiology of the metabolome alteration observed in serotonin syndrome rat models (reprinted from ref. 15
with permission). Abnormal contraction of skeletal muscles induces up-regulation of anaerobic respiration, resulting in hyper thermogenesis via UCP-3 activation. Various metabolites were also accumulated in blood, which ﬁnallyleads to urea cycle disruption in the liver and increase of BUN.

14

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Fig. 3.

Medical Mass Spectrometry Vol. 3 No. 1

Score plots of projection to latent structures discriminant analysis for control and cannabinoid receptor agonist
administered mice
mice (reprinted(reprintedfrom
fromref.
ref.1414
with
withpermission).
permission).
Blue
Blue
(blackplots,
in monochrome
5 mg/kg administeredprinting) group;
plots, 5 mg/kg
red plots,
ad15
ministeredmg/kg administeredgroup; red (dark)group;plots,
green15
plots,
mg/kg
controladministeredgroup. These
group;
groupsgreenwere(bright)
separatedplots, alongcontrolthegroup.
ﬁrst principle
These groups
com-dependence
were separated
ponentaxis, which
along
shows
the ﬁrst
a doseprinciplecomponent
effect ofaxis,
thewhichagonist.
shows a dose-dependence effect of the agonist.

Future Perspectives for a Newly Developed
Metabolome Analysis

cology but also to toxicological assessment using animals.
A concise review on applying metabolomics to the adverse

As described above, GC/MS/MS-based targeted metabo-

17)

effects of drugs has already been published .
In our study, a high dose of CBR agonist can induce seizure-like abnormal behavior in rodents, though the mainlome analysis has played a pivotal role in metabolomics
because of its selectivity, operability, and robustness. Chro-mechanisms of this have not been elucidated; we applied
GC/MS/MS-based metabolome analysis to a CBR agonist

matographic separation is necessary for simultaneous analysis of a vast number of metabolites, though it leads to a
low-throughput analytical run. Particularly, some derivat-

administered to rat cerebrum to investigate clues to abnor14)

mal behavior . In this study, rat cerebrum was pretreatedized metabolites are known to be unstable even at roomby the Bligh and Dyer extraction method, and an internal
standard (IS) correction method was used for relative quan-temperature, and their degradation can bias the ﬁnal results.
Thus, there is a pressing and underlying need to develop ahigh-throughput analytical method for metabolomics.

tiﬁcation. Various normalization methods such as IS-based
and quality control-based techniques have been reported forAmbient ionization techniques such as direct analysis inmetabolome analysis, and it is necessary to select an appropriate correction method for matching experimental condi-

real time (DART) and desorption electrospray ionization. Based on the pre-experi-

many reports have been published on their applications tomental results, GC/MS/MS-based targeted metabolome
analysis showed high repeatability for extracts from rat. Ambient ionization techniqueslack chromatographic separation, resulting in lower selec-

cerebrum samples. Finally, we applied projection to latent
structures discriminant analysis (PLS-DA) to the obtained

tivity, though their high-throughput nature is advantageous.
Our team also developed high-throughput analysis of

metabolome data, demonstrating the dose-dependent effectof the drug (Fig. 3). Based on the loading plots, the CBR

metabolites using probe electrospray ionization tandem
mass spectrometry (PESI/MS/MS) and applied this to ani-

agonist can induce energy metabolism and glutamatergic

mal model tissues

excitatory neurotransmission disorders in the cerebrum.

this uses a thin probe needle for sampling and ionization
46)
units . Our team combined PESI and tandem mass spec-

tions and/or sample features

33‒35)

38‒41)(DESI) are currently widely used in various ﬁelds
42‒45)

metabolome analysis

42,44)

Interestingly, these results are consistent with otherresearchers ﬁndings that CBR agonists can induce mito36,37). PESI was developed in 2007, andtrometry, resulting in higher selectivity and S/N ratio. Particularly, our system can directly analyze tissue samples

chondrial dysfunction by stimulating CBRs on the outermitochondrial membrane

, and

.

such as liver and brain without sample pretreatment,
15June 2019

Medical Mass Spectrometry Vol. 3 No. 1

achieving total ﬂow rapidity for metabolite analysis. Thus,

tential of metabolomics as a functional genomics tool.

our system was introduced as the fastest method in a review

Trends in Plant Science 9: 418‒425, 2004.

47)

article by Zampieri et al. . We have extended the method

4) Robertson DG, Watkins PB, Reily MD: Metabolomics in

to one hundred metabolites and completed direct tissue

toxicology: Preclinical and clinical applications. Toxicol

analysis within 5 min with no tedious sample pretreatment.
Ambient ionization/mass spectrometry will be a new ana-

Sci 120: S146-S170, 2010.
5) Fiehn O: Metabolomics by gas chromatography‒mass
spectrometry: Combined targeted and untargeted proﬁl-lytical tool for metabolome analysis.

ing. Curr Protoc Mol Biol 114: 30.34.31‒30.34.32, 2016.

Conclusion

6) Dettmer K, Aronov PA, Hammock BD: Mass spectrome-

In this mini review, we outline GC/MS/MS-based targeted metabolome analysis and its application to patho-

try-based metabolomics. Mass Spectrom Rev 26: 51‒78,

physiological analysis of animal disease models and inves-

7) Rawlinson C, Kamphuis LG, Gummer JPA, Singh KB,

tigation of unknown toxic effects of drugs on animals. GC/
MS/MS-based targeted metabolome analysis generally

Trengove RD: A rapid method for proﬁling of volatile andshows high selectivity and improves S/N ratio, providinguser-friendly platforms for metabolome analysis. In our pre-derivatisation and GC-MS. Metabolomics 11: 1922‒1933,

vious studies, GC/MS/MS-based metabolome analysis8) Kvitvang HFN, Andreassen T, Adam T, Villas-Bôas SG,

strongly helped us to interpret the pathophysiology in the

Bruheim P: Highly sensitive GC/MS/MS method foranimal models. For the serotonin syndrome model rats, wequantitation of amino and nonamino organic acids. Anal

were able to systematically understand biological changes
in the model rats. We also easily found that the abused can-Chem 83: 2705‒2711, 2011.

2007.

semi-volatile phytohormones using methyl chloroformate
2015.

9) Tsikas D, Zoerner AA, Jordan J: Oxidized and nitrated

nabinoid receptor agonist can cause energy metabolism disruption in rat cerebrum. Although it is difﬁcult to extrapo-

oleic acid in biological systems: Analysis by GC-MS/MS

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Cell Biol L 1811: 694‒705, 2011.

and LC-MS/MS, and biological signiﬁcance. BBA-Mol

we could estimate the phenotypic changes such as toxicity
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10) Xu Y-J, Ho WE, Xu F, Wen T, Ong CN: Exploratory in-tionally, we discuss future perspectives of new analyticaland eicosanoids in coronary artery disease patients. Pros-

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S, et al: Highly sensitive and selective analysis of widely

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Conﬂicts of Interest

A preliminary study on postmortem interval estimation of
suffocated rats by GC-MS/MS-based plasma metabolic

There are no conﬂicts of interest to declare.

proﬁling. Anal Bioanal Chem 407: 3659‒3665, 2015.

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