June 2020

Medical Mass Spectrometry Vol. 4 No. 1

Review

Utility of isotope-coded derivatization in gas chromatographic-mass
spectrometric analyses with special emphasis on metabolomics
Mahmoud H. El-Maghrabey

1,2,†

1

Abstract

1

1, Naoya Kishikawa *, Naotaka Kuroda

Graduate School of Biomedical Sciences, Course of Pharmaceutical Sciences, Nagasaki University,
1‒14 Bunkyo-machi, Nagasaki 852‒8521, Japan2
Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Mansoura University,
Mansoura 35516, Egypt

Gas chromatography (GC) is widely used in various ﬁelds. Among GC detectors, mass spectrometry detection(MS) provides highest selectivity and sensitivity, thus it is the most used detector especially in metabolomics studies. In
GC-MS analysis, derivatization is mostly unavoidable for most of the analytes in order to increase volatility, thermal stability, and detectability of the analytes. Nevertheless, chemical derivatization has some limitations including uncertainty inquantitation, diﬀerences in derivatization kinetics, and the stability for variable analytes. Thus, the GC-MS analysis strongly requires standardizing protocols. One of the most used protocols for standardization is isotope dilution strategy. However, available isotope-internal standards are limited in number and expensive. A cost-eﬀective and simpler alternative is isotope-coded derivatization (ICD), in which the standard or the control sample is labeled with a heavy isotope-coded moietyof the reagent and used as internal standards. On the other hand, the sample is labeled with the light form of the reagent.
Then, both mixed and analyzed by GC-MS(/MS) permitting the absolute quantiﬁcation of analytes and identiﬁcation of unknown ones. Due to the high importance of ICD, there are many reviews summarized its use in LC-MS analysis. However,there is no single review dedicated to summarizing its use in GC-MS. Until now, few types of reagents were designed for
ICD for GC-MS applications. These reagents can be classiﬁed into silylation, chloroformates esteriﬁcation, transmethylation, and aminooxyethyl propionates oximation ICD reagents. In this review, we will discuss every class of them and theirreported applications with special emphasis on metabolomics.
Key words: GC-MS, isotope-coded derivatization, standardization protocols, silylation, metabolite proﬁling

Introduction
Gas chromatography (GC) is a separation technique that†uses gases as mobile phases and liquid or solids as stationary phases. It is capable of resolving very complex mix-

The author is an International Research Fellow of Japan Soci-ety for the Promotion of Science (Postdoctoral Fellowships fortures of analytes based upon the diﬀerences in their boiling1)
point/ vapor pressure and polarity . GC is coupled with dif-

Research in Japan (Standard)).
*Corresponding author

ferent types of detectors and every one of them is selective

Naoya Kishikawa

for a special type of analyte. Among them, the most widelyused detector is ﬂame ionization detector due to its univer-

Graduate School of Biomedical Sciences, Course of Pharmaceutical Sciences, Nagasaki University, 1‒14 Bunkyo-machi,

2)

Nagasaki 852‒8521, Japan
Tel: ＋81‒95‒819‒2445, Fax: ＋81‒95‒819‒2446

sal nature and acceptable sensitivity .

Received: January 31, 2020. Accepted: April 15, 2020.

graphic analysis. MS is naturally compatible with the GC

Epub May 28, 2020.

analysis as there is no need for the sample vaporizationinterface, which is needed in the case of LC-MS. In addi-

Nowadays, mass spectrometry (MS) detection is being
used widely in particular when combined with chromato-

E-mail: kishika@nagasaki-u.ac.jp

DOI: 10.24508/mms.2020.06.007
24

June 2020

Medical Mass Spectrometry Vol. 4 No. 1

31,32)

tion, MS detectors oﬀer great sensitivity and selectivity
compared with all other GC detectors. Besides, MS detec-

methylsilyl)triﬂuoroacetamide (MTBSTFA).

tors not only record the presence of analytes, but also provides additional structural information regarding its chemi-

prior to GC analysis such as alkylation, esteriﬁcation or

In addition to silylation, there are various reactions usedacylation reactions; where an alkyl or acyl group replaces a33)
reactive proton . Based on the used reagents, these reac-

cal composition and molecular weight and chemical
1)
structure . All these factors lead to the wide use of GC-MS

tions may be performed in aqueous or organic medium.

. GC-MS is used for the analysis of many low
5,6)molecular weight compounds . GC-MS is widely used in

However, in situ aqueous derivatizations have several
advantages such as avoiding extraction step before derivat-

3,4)

analysis

7,8), plant bioactive

ization and unnecessity of excess reagents removal due to, and most importantly; biological.

their water solubility. In situ aqueous derivatization
included many reactions such as alkylation, acylation, ester-

GC analysis mostly needs chemical derivatization foriﬁcation, oxime formation, hydrazine formation, and Schiﬀ

analytes prior to their determination. The derivatization isneeded in order to increase the volatility and thermal stabil-base formation. These derivatization reactions were used
for proﬁling of many types of metabolites including alco-

ity of the analytes, improve their chromatographic behavior

hols, carbonyls, carboxylic acids, and amines

diﬀerent ﬁelds including food analysis
9,10)compounds analysis

6,11)

samples metabolite proﬁling

15,17,18,34).
However, in situ aqueous derivatizations requires an addi-

and suppress tailing by decreasing their polarity, improve
ﬁnally increase their detection sensitivity and selectivity.

tional step for sample introduction from the aqueous phaseto the GC injectors including liquid‒liquid extraction

Nevertheless, chemical derivatization has some drawbacks(LLE),and limitations. For instance, the derivatization reagent and, liquid-phase microextraction (LPME) ,(DLLME)38)
salting out assisted liquid‒liquid extraction (SALLE) ,the recovery of a compound from complex matrices, anddispersive

liquid‒liquid

microextraction

35,36)

procedure could cause potential uncertainty in quantitationand could introduce interfering by-products. In addition,

37)

39)
solid-phase extraction (SPE) , solid-phase microextraction

derivatization adds an extra stair in the sample treat-

40)

41)

(SPME) , and headspace sampling (HS) .

12‒15). Furthermore, most of the derivatization reagentsare hazardous and could harm the environment and the ana-

Furthermore, other alternatives to the conventional and inment

situ aqueous derivatizations, have been oﬀered. One of thealternative derivatization methods is the extractive derivat-

lyst which is not coping with recent green trends in analyti16‒19). Thus, it is better to avoid them if possical chemistryization, in which extraction and derivatization are com-

ble. However, the use of derivatization reagents isbined into one step where the analytes are transmitted froman aqueous phase to an organic one containing the derivat-

unavoidable in the case of nonvolatile compounds that are
15)

inherently not suitable for GC analysis . In this regard,
many reviews concerned with GC analytical derivatization

ization reagent (reactive organic phase) through pH control
42,43). Another alternative derivatization
or ion-pairing. The optimal derivatizing

method is solid-phase analytical derivatization (SPAD) inreagent for GC analysis should be highly reactive towards a
speciﬁc group of analytes and give only one thermally sta-

which the analytes are derivatized while adsorbed on theble and suﬃciently volatile product per each analyte. In
addition, if the derivatization reaction leads to the forma-

coating was also reported based on the same idea oftion of any by-products, there should be an easy way toremove them from the reaction mixture prior to GC analy-

also performed, where the analytes and derivatization

12‒15,20‒23)

have been publishedsis

12,15)SPE sorbent. Furthermore, derivatization in an SPME ﬁber
44)

SPAD . Lastly, derivatization in the GC injector port was
reagents are injected into the injector port at the same time.

.

The derivatization step and sample volatilization occur

The most commonly used derivatization reaction in GC

simultaneously through thermal decomposition in the GC

analysis is silylation as thermally stable and volatile silylated

heated injector

14,24). Several reagents
have been designed for this purpose including: N, O-

45,46).

From the above we can conclude that the derivatization
reactions can occur in various ways; either in organic sol-

derivatives can be produced easily

25,26), bis(trimethylsilyl)bis(trimethylsilyl)acetamide (BSA)
27,28), N methyl-N-(trimethylsilyl)
triﬂuoroacetamide (BSTFA)

vents media, in situ aqueous derivatizations, injector port insitu derivatization, or combined in one-step with theor N-methyl-N-(tert-butyldi-

extraction procedure . In principle, derivatization adds an

29,30)

triﬂuoroacetamide (MSTFA)

15)

25

June 2020

Medical Mass Spectrometry Vol. 4 No. 1

ICD Methods in the Field of GC-MS Analysis as Standardization Protocol with Special
Emphasis on Metabolomics

extra processing step to the sample treatment introducing
extra analytical error possibilities. As derivatization is often
unavoidable for GC-MS analysis of most analytes, a proce-

As mentioned previously, in GC-MS analysis, derivatization is mostly unavoidable for most analytes. However, arti-

dure called isotope dilution is often used to overcome theerrors introduced in the derivatization step and overcome
matrix eﬀect . Isotope dilution is based on the fact thatfacts can be introduced in crude samples exclusively with-many elements have two or more stable isotopes that have aout being formed in pure standard mixtures causing matrix

constant natural proportion. Examples of the isotope atoms

eﬀect . In addition, analytes with diﬀerent functional

47)

12

13

18

O/ O, and

14

15

63)

. A knowngroups react with diﬀerent derivatization kinetics and thestability of the formed derivatives can vary signiﬁcantly .
Thus, the GC-MS analysis strongly requires standardizing

13

N/ N

48)

amount of a stable isotope (d, C, O, or N) labeled stan-pairs are H/d,

C/ C,

16

18

15

64)

dard is added to a sample and the ratio between the isotopes
can then be measured by GC-MS. Isotope dilution could. One of the most used protocols for standardization is isotope dilution strategy as mentioned previ64,65)

protocols

provide very accurate and precise results, in addition to its
use as an essential tool for the validation of analytical pro-ously

47,56). The introduction of isotopically labeled internal

cedures and reference materials. Recently, many isotope
49‒55)
owing to theirdilution GC-MS methods were reported

standards at diﬀerent steps of sample preparation and using
quality control samples for evaluation of instrument perfor-

advantageous reduction of total analysis time and providing
47,56). However, isotope dilution
high-quality analytical data

mance is for sure a good practice for protocols standardiza64,66).
tion and identiﬁcation of derivatization biases sources
A cost-eﬀective and simpler alternative to isotope dilution

requires isotope-labeled standards that are either very
48,57)

expensive or unavailable. In addition, Choi and Jung

strategy for protocol standardization is ICD. Until now few
types of reagents were designed for ICD for GC-MS appli-

reported that the isotope dilution did not correct the matrix
eﬀect of cola and beer during the analysis of methyl-imid-

cations and most of them were applied to metabolomics or

58)

azoles . Cola and beer have less complicated matrix thanother biological samples, thus, this ﬁnding is very import-

have the potential for that. These reagents can be classiﬁed
into silylation, chloroformates esteriﬁcation, transmethyla-

ant and applies to other ﬁelds.

tion, and aminooxyethyl propionates oximation ICD

To overcome the limitations of isotope dilution, isotope-coded derivatization (ICD) was recently designed andreagents. In this review, we will discuss every class of them

introduced for overcoming problems of stable isotope dilution. For GC-MS analysis, ICD is available for the generalmetabolomics. A summary of the ICD method applied in
GC-MS analysis is presented in Table 1.

13
18
isotope-labeled reagents such as deuterium (d), C, O
15
and/or N-substitutions, and reacted with analytes for pre-Silylation reagentsand their reported and potential applications in the ﬁeld ofparing internal standard (IS). ICD could be used for diﬀerential analysis, absolute quantiﬁcation, and metabolic pro-Silylation reaction is one of the most used derivatization
approaches due to its universal reactivity to diﬀerent functional groups including alcohol, amines, and carboxylic

ﬁling through derivatization of a standard material orcontrol sample with the heavy isotope-labeled reagent andacids (Fig. 1). Thus, it was logic that the ﬁrst designed ICD
reagent was the silylation reagent, d9-N-methyl-N-(trimeth-derivatization of the targeted samples with the light isotope
factor of ICD is the commercial availability and the price of

ylsilyl)triﬂuoroacetamide (d9-MSTFA). This reagent was
67)
developed by Herebian et al. , where they used trimeth-the ICD reagent and when it is unavailable, how complex

yl-d9-chlorosilane to synthesize d9-MSTFA following thewould be its chemical synthesis. As ICD is a very important
tool to produce high-quality analytical data, there are many

procedure reported by Donike . Herebian et al. used
d9-MSTFA not to form heavy labelled internal standards,

(H,

12

C,

16

O, or

14

48,59,60)

N) labeled reagent. The limiting

68)

48,59‒62).
However, there is no single review dedicated to summariz-

but for the identiﬁcation of unknown peaks in the chro-ing its use in GC-MS.

in case of analytes possessing the enolizable carbonyl

reviews summarized its use in LC-MS analysis

matogram. In silylation reactions, alkoxyamines are addedgroups to avoid the formation of multiple derivatives uponsilylation, as they form stable oximes with them. Thus, in26

June 2020

Medical Mass Spectrometry Vol. 4 No. 1

Summary of the GC-MS ICD reagents and their applications

Table 1.
Reagent

Analytes

Matrixd0/d9-MSTFA＋d0/d9-MHA

Amines, amino acids,
fatty acids, alcohol

Bacterial extract

Metabolite proﬁling of Corynebacterium
glutamicum67)

d0/d9-MSTFA

Sugars, amino acids,
and organic acids

Urine and serum

Absolute quantiﬁcation of metabolites

69)

d0/d6-MTBSTFA

Amino acids, fatty
acids, and organic acid

Serum

Relative quantiﬁcation and diﬀerential
metabolomics

70)

d0/d3-MCF in d0/d3-methanol

Amino acids andorganic acids

Urine and serum

Absolute quantiﬁcation of metabolites

74,75)NaOH in d0/d3-methanol

Fatty acids

Plant oil and yeast Quantitative comparison of the fatty acidcell lysatescomposition

Fatty acids

Yeast extracts

Identiﬁcation and classiﬁcation of microorganisms based on their fatty acids composition77)

S-fatty acylated
proteins

Zebraﬁsh liver

Monitoring changes of S-fatty acid acylation of
the liver proteins upon exposure to the DDT

78)

Fatty acids

Zebraﬁsh liver

Monitoring aberrant hepatic lipid composition
upon exposure to DDT

79)

C0-d3-AEP, C1-d3-AEP,
C2-d3-AEP
13

Aldehydes and ketones

Turmeric root

Triplex ICD with ZMI mass tagging for carbonyl proﬁling and absolute quantiﬁcations of two
methyl ketones

80)

C0-d3-MAP, C1-d3-MAP,
C2-d3-MAP

Aldehydes and ketones

Laboratory prepared mixtures

Upgrading aminooxyethyl propionates throughimproving reagent selectivity and adductstability and aided EI-induced ZMI reporter ion
generation

81)

13
13

13
13

13

Application

Reference76)

addition to d0/d9-MSTFA, Herebian et al. used d0/d3-o-methylhydroxylamine (d0/d9-MHA) for methoximation and iden-

strategies even when tested on serum and urine samples.
However, it was found that d9-MSTFA derivatives had vary-

tiﬁcation of metabolites possessing carbonyl groups. It was
found that d0/d9-MHA labeled metabolite pairs showed only

ing responses when spiked as IS in MSTFA derivatizedsamples. This fact pointed to a major challenge of MSTFA
derivatization, which is the requirement of optimizing deri-a small shift in retention time with appropriate changes in
the mass spectra. While metabolites pairs labeled with d0/
d9-MSTFA shown very similar retention times. In addition,

vatization condition to obtain complete reaction with stable69)

products . Furthermore, Huang and Regnier introduced
another silylation ICD reagent, d0/d6-N-methyl-N-(tert-bu-

d0-MSTFA and d9-MSTFA were found to have very similar

tyldimethylsilyl)-triﬂuoroacetamide (d0/d6-MTBSTFA) (Fig.
1c). They used d0/d6-MTBSTFA as an ICD reagent in com-reactivity as proved by the similar numbers of reacted
metabolites to each one of them. Using d0/d3-MHA and d0/

d9-MSTFA, Herebian et al. were able to elucidate many

bination with GC-MS (time-of-ﬂight; TOF) analysis for rel-derivatization groups and molecular weights of various
unknown metabolites. They could identify about 31 metab-

ative quantiﬁcation and diﬀerential metabolomics study .
They used d0/d6-MTBSTFA due to the greater hydrolytic

olites in Corynebacterium glutamicum hydrophilic extracts

70)

for the ﬁrst time . Silylation and methoximation reactions

stability of its derivatization products than those obtained
from MSTFA derivatization, thus, preventing the scram-

schemes are presented in Fig. 1a and 1b, respectively.
Afterward, Lien et al. used also d9-MSTFA, but this time

bling of isotopomers in the course of GC-MS analysis.
Using diﬀerential coding with d0/d6-MTBSTFA, the authors

for synthesizing internal standards . They found that thewere able to compare the concentration of amino and fatty

normalization of data by ICD signiﬁcantly increased theacids between two diﬀerent samples simultaneously. The
authors conﬁrmed that the derivatization eﬃciency and sta-

67)

69)

precision when compared with traditional derivatization27

June 2020

Fig. 1.

Medical Mass Spectrometry Vol. 4 No. 1

Silylation and combinatorial methoximation reagents and their reaction schemes where (a) silylation reaction of d0/
d9-MSTFA with diﬀerent functional groups, (b) reaction scheme of d0/d3-MHA with carbonyls and (c) the structureof d0/d6-MTBSTFA. The isotope-coded parts of the reagents are referred to as “X”.

The method was modiﬁed and optimized for the determination of both amino and non-amino organic acids including

bility of the product are identical for the derivatization
70)
reagent and its deuterium-labeled analog . Thus, d0/
d6-MTBSTFA are perfect candidate ICD reagent pair for
metabolomics studies. However, for absolute quantiﬁca-

di- and tri-carboxylic acids, through modiﬁcation of reac-tion, spiking of internal standards in all samples and the
60,71). In conclucalibration curves standard series is required

chloroformate, including methyl chloroformate (MCF), ission, silylation ICD GC-MS analysis is among the most frequently used methods in the ﬁeld of metabolomics, how-and organic acids metabolites. In addition, when they arecompared with MSTFA derivatization, the reaction prod-

ever, there are some drawbacks inherited in this technique.

ucts of MCF derivatization are more stable and reproduc-

For instance, the instability of MSTFA products and theible . Also, esteriﬁcation reaction could be performed
using in situ aqueous derivatization, contrast for the silyla-

73)tion medium and conditions . Hence, esteriﬁcation withvery useful in metabolomics studies concerned with amino

24)

scrambling caused by this. Thus, this ICD approach is moresuited for target metabolite proﬁling and not for non-target

tion reactions that necessitate the use of hazardous organic

approaches. In addition, the high price and limited avail-solvent. Thus, esteriﬁcation with chloroformate ICD

ability of isotopically labeled silylation reagents is also
another obstacle. Thus, more development and further

reagents seems to be a better alternative to the ICD strategy
done with silylation. The formed derivatives are more sta-

research is strongly needed in this area.

ble and carboxylic and amino functional groups are labeled
diﬀerently with the alcohol solvent (usually methanol) and

Esteriﬁcation using methyl chloroformate

Esteriﬁcation with chloroformates is a general derivatiza-

the chloroformate (usually MCF), respectively (Fig. 2).
Kvitvang et al. established an absolute quantitative GC cou-tion approach for the carboxylic functional group in
GC-MS analysis due to the rapid nature of the esteriﬁcationpled with tandem MS (GC-MS/MS) for analysis of about
seventy metabolites upon ICD with d0/d3-MCF in d0/
d3-methanol, where the heavy labeled standards are spiked

72)

reactions in aqueous media forming stable derivatives .
28

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Medical Mass Spectrometry Vol. 4 No. 1

Fig. 2.

Reaction scheme for esteriﬁcation reactions with d0/d3-MCF where the isotope-coded parts of the reagents arereferred to as “X”.

Fig. 3.

Reaction scheme for IFAT derivatization where (a) is the general IFAT for free fatty acids and (b) IFAT for S-fatty
acylated proteins.

The isotope-coded parts of the reagents are referred to as X .

into light-labeled samples. Moreover, they used positive

more, to overcome shipment restriction for MCF, d0/d3-methanol was used in combination with ethyl chlorofor-

chemical ionization (PCI) instead of the traditional electronmate. These methods yielded lower sensitivity than theoriginal d0/d3-MCF method, due to the unavoidable use of

impact ionization. PCI has the privilege of preserving themolecular ion even for high molecular weight compounds,
thus, enabling selective and unique selected reaction moni-less intensity SRM transitions to maintain selectivity. How-

toring (SRM) in the tandem mass mode even between
74)
co-eluting metabolites . However, d3-MCF is expensive toever, the analytical precision was much better than those

substrates are also expensive. An alternative way is to use acombination of d0-MCF and d0/d3-methanol. This will not

Isotope-coded fatty acid transmethylation (IFAT)

perform ICD correction for analytes lacking carboxylictransmethylation (IFAT) as a novel ICD approach . IFAT
integrates extraction, transmethylation, and isotopic label-75)

obtained using external standards only .

buy and even its synthesis is not cost-eﬀective as the used

Recently, Li et al. reported the isotope-coded fatty acid76)

groups; however, it is still feasible to use this strategy forcreating internal standards for amino acids as their carbox-

ing in one-step using ultrasonic irradiation (Fig. 3a). In thisylic groups will be labeled with the d3-methanol . Further60)

technique, the samples were mixed simultaneously with29

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Medical Mass Spectrometry Vol. 4 No. 1

sodium hydroxide in d0/d3-methanol for ICD transmethylation and n-hexane solution for extraction. The wave shocks

tion. Polyunsaturated fatty acids were found to decrease in
response to DDT exposure. On the other hand, saturated
long-chain fatty acids were found to be increased inand cavitation produced by the ultrasonic irradiation
speeded up the alkaline-catalyzed transmethylation andresponse to DDT in a concentration-dependent way. From
all these reported methods, we can conclude the outstand-facilitated the mass transfer of the produced fatty acidmethyl esters into the top n-hexane layer, which was thening performance of IFAT in the ﬁeld of lipidomic and evenseparated and injected into the GC-MS system. The

79)

in proteomics .

obtained sensitivity were at the picogram level with thisapproach. This technique has many advantages including

Oximes formation using aminooxyethyl propionates

carrying out heterogeneous reactions eﬃciently where solid

Recent reports showed that the ﬁrst multiplex ICD
reagent was synthesized based on three isotopic 2-aminoo-

samples can be easily derivatized and analyzed with the aid

xyethyl propionates (AEP) and applied to chemo-selectiveof ultrasonic irradiation. In addition, it provides accuratequantiﬁcation of fatty acids where ICD can be used for pro-compounds for the determination of aldehydes and ketones
80)
using GC-MS . The three derivatives were as follow:

viding the internal standards for a perfect comparison of thecomposition of fatty acids in diﬀerent samples. Further-13

C0-d3-AEP,

C1-d3-AEP,

13

C2-d3-AEP. Upon reaction of

13

more, the IFAT strategy volatilizes the labeled analytes,

the aforementioned three AEPs with carbonyl compounds,

thus, it isolates them from the background matrices andminimizes the interferences from the biological compo-

the formed oximes yielded three mass products ions (m/znents. This approach was applied successfully for quantita-

cleavage (Fig. 4a). The reagents were designed speciﬁcallyto produce these reporter ions as they lie in the zone of min-32, 33, and 34), upon their electron ionization (EI) induced

tive determination of the fatty acid composition of plant oiland budding yeast cell lysates . The same research group

imal interference (ZMI; 31‒37 m/z) explored by theused the IFAT approach for quantitative analysis of fatty
acids in diﬀerent microorganisms aiming at classifying andauthors. Through this triplex ICD strategy, simultaneous
GC-MS analysis of multiple samples that allows identiﬁca-

identifying unknown microorganisms based on their fattytion and quantiﬁcation of multiple analytes became possi-

acids identity and quantity. This method is best suited fortyping fungal species due to their distinguished lipid bio-

ble with accuracy reached about 95%. It is noteworthy that76)

this type of oximes formation reactions is performed

77）

synthetic pathways compared with bacteria and animals .

Furthermore, IFAT was applied for qualitative analysis of
S-fatty acylation of proteins. Cellular proteins wereextracted from diﬀerent cell states and then separated bysodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein bands are excised followed byin-gel IFAT. Acyl moieties can be directly released from
S-fatty acylated proteins by IFAT alkaline-catalyzed transmethylation (Fig. 3b). The resulted fatty acid methyl esters
after the ICD with d0/d3-methanol were identiﬁed by

GC-MS. This approach was successfully applied to monitor
the changes of S-fatty acid acylation of zebraﬁsh liver protein upon its exposure to the insecticide, dichlorodiphenyltrichloroethane (DDT). This approach was found to be simpler, more eﬀective, and by far less hazardous than thecommonly used radioactive fatty acid analogs metabolicFig. 4.

78)

incorporation approach . Furthermore, the IFAT approach
was used again for monitoring changes that occur to zebrafish metabolomics upon exposure to DDT, but this timethrough monitoring of the aberrant hepatic lipid composi-

Aminooxyethyl propionates reagents and theirreaction scheme where (a) the reaction scheme of
AEP with carbonyls and (b) the structure of MAP.

The isotope-coded parts of the reagents are marked withasterisks (*).

30

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Medical Mass Spectrometry Vol. 4 No. 1

need to remove excess reagents as discussed previously inoxyethyl propionates oximation ICD reagents. The reported
ICD GC-MS methods showed great success in metabolic

the introduction part. This simultaneously multiple samples

proﬁling and sometimes in absolute quantiﬁcation, how-analysis approach can signiﬁcantly increase the analyticalthroughput cost-eﬀectively and improve the accuracy of theever, the reported methods are not so much. The reasons forthe non-wide use of ICD in GC-MS analysis can be the highquantiﬁcation at the same time. The method was applied

price and limited commercial availability of ICD reagents.
Moreover, GC-MS methods targeted for metabolomics arethrough in situ aqueous derivatization, thus, there is no

successfully for the absolute quantiﬁcations of the two
methyl ketones namely 2-nonanone and 2-undecanone inmostly performed in scan mode, leading to the generation80)

the turmeric root extract .

of very complex chromatograms, with many fragment ions

Despite the success of AEP triplex ICD, there is still aconnected to each analyte. Therefore, the introduction of
isotope-coded internal standards can signiﬁcantly further

concern about the susceptibility of the ester linkage towardhydrolytic cleavage, in addition to the possibility of transac-

increase the chromatogram and mass spectra complexity,
compromising the data analysis process. Thus, cost-eﬀec-

ylation reactions with nucleophiles, by either the AEP itself
or its by-products. These reactions could perform ICD fortive ICD such as IFAT was introduced to overcome such ametabolites that are not originally targeted by the aminooxy

problem. Moreover, new techniques such as ZMI reporter

functionality. Furthermore, the EI induced cleavage process

ICD were introduced and improved recently. With thisof AEP derivatization products needs to be enhanced inemerging technique, we expect great future development in
the ICD-GC-MS ﬁeld either through the design of neworder to improve that mass spectral tag detection in the
ZMI through minimizing uninformative fragmentations.
Thus, the same research group of Nantz and coworkers pur-

reagents which is either commercially available or can besued the synthesis and evaluation of an alternative N-me-

reagents in diﬀerent analytical areas, especially in the

thoxy amide analog, N-methoxy-N-(2-aminooxyethyl)-pro-

metabolomics ﬁeld.

pionate (MAP). In MAP, the ester group of AEP is replacedwith an N-methoxy amide group (Fig. 4b). This design for

Declarations of Interest Statement

easily synthesized to satisfy the increasing needs for such

All the authors declare that there is no conﬂict of interest.

the reagent increased the its stability towards hydrolysis
and increased adduct stability towards uninformative frag-References

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the ZMI reporter ion. Moreover, the N-methoxy amide

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