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2 Chromatography–mass spectrometry based
analysis of biologically active endogenous
carboxylic acids
Based on: D. Kloos, H. Lingeman, O.A. Mayboroda, A.M. Deelder, W.M.A Niessen, M. Giera Chromatography–mass spectrometry based analysis of biologically active endogenous carboxylic acids Accepted in Trends Analyt Chem.
Chapter 2
28
2.1 Abstract
Carboxylic acids, such as small carboxylic acids, fatty acids, eicosanoids and bile
acids are involved in numerous biological processes. The mapping of the participating
pathways potentially yields great insight into the physiological state of an organism.
There is an urge for more sensitive, faster and easier to handle analysis techniques.
These would enable advanced analysis and validation in larger clinical cohorts and
hopefully deliver useful disease markers. The analysis of carboxylic acids is not only
driven by the carboxylic acid group, but is largely dependent on chain length and
other functional groups present. Several LC and GC strategies, with or without
derivatisation have been developed in recent years. Here, we review the most recent
trends in endogenous carboxylic acid analysis by chromatography–mass
spectrometry. We critically evaluate sample preparation, derivatisation-techniques,
separation and mass spectrometric detection. Ultimately, the reader is guided to the
most versatile, sensitive and facile analytical methods.
2.2 Introduction
One of the most interesting developments in the biomedical sciences of the post-
genomic era is a “paradigm shift” in our understanding of the biological/regulatory
activity of the chemical entities for, which such a function was disregarded. The
compounds that traditionally were seen only as the constituents of energy
metabolism (e.g. pyruvate, succinate, fatty acids) or emulsifiers of lipids (bile acids)
are being more and more recognized as important immune-modulatory and/or
signalling molecules. In the first approximation, all those compounds appear to be far
too structurally divers for being reviewed together, but taking a broader view, one
will realize that most of those “upcoming regulatory compounds” are endogenous
Chapter 2
29
carboxylic acids (CAs). Their common functional features are the presence of a CA
function, dictating many but not all of their physicochemical properties, and an alkyl
part which can be decorated with different functional groups. One can distinguish
four important subclasses of endogenous CAs, namely: (A) small CAs, crucial to
aerobic respiration and energy metabolism1, (B) fatty acids (FAs), fundamental to
energy storage, membrane formation and involved in numerous physiological
processes such as inflammation2-4, (C) eicosanoids and docosanoids, forming a class
of highly important signalling molecules during several inflammatory and
immunological events5, and (D) bile acids (BAs), being the main metabolites of
endogenous cholesterol6 (see Figure 1 for their general structures). Recent advances
in analytical techniques for the analysis of CAs are discussed here.
Figure 1 General structures of the four relevant classes of CAs discussed in this paper: small CAs, fatty acids, eicosanoids, and bile acids
All of these analytes reside in the body and can be quantitatively analysed in one
of the body fluids or in cellular extracts. Their isolation and separation from matrix
components is fundamental to their analysis. While sample preparation can be
Chapter 2
30
distinctly different for the four CA subclasses, analyte separation is commonly
performed by either gas chromatography (GC) or liquid chromatography (LC). Both
separation techniques are nowadays frequently combined with mass spectrometric
(MS) detection for highest selectivity and sensitivity in quantitative analysis7. Both
GC–MS and LC–MS feature common requirements owing to the functional groups
present in the analytes of interest. Analysis of endogenous CAs is done with different
aims, e.g., (I) qualitative profiling of CAs present, thus involving structure elucidation
and resolving isomerism issues, (II) relative quantitation for comparison of
physiological states and/or flux determination, involving the use of isotopologues,
and (III) targeted (absolute) quantitative analysis. The aims of the study determine
the analytical strategy chosen, but in all cases general issues in the analysis of CAs are
important as well8.
2.3 General aspects
All four analyte subclasses share the presence of a CA function, i.e., they are
weak organic Brønsted-Lowry acids. However, other features shared by the CAs
might also have significant effects on the analytical process and thus demand rather
similar analytical solutions. Figure 2 gives a schematic overview of the most
prominent functional groups that may be present in an endogenous CA.
Chapter 2
31
Figure 2 Overview of relevant functional groups that may be present and influence the analysis of endogenous CAs
Chapter 2
32
2.3.1 GC–MS analysis
Intrinsically, GC–MS analysis demands the evaporation of the analyte under
investigation prior to separation and detection. With respect to CAs, several
functional groups demand attention. Firstly, the CA function has to be derivatised for
successful GC–MS analysis. From the various possible derivatisation strategies,
silylation and esterification of the CA function are the most prominent ones. Other
crucial functional groups (Figure 2) that have to be derivatised prior to GC–MS
analysis are: (A) hydroxyl groups, which can either undergo silylation or ether
formation, and (B) ketone groups, which are frequently converted into oximes,
thereby blocking possible tautomerism during subsequent derivatisation as well as
stabilizing particularly α- and β-keto CAs. After derivatisation, the CA derivatives can
be analysed in routine GC–MS systems, involving common dimethylsiloxane-coated
capillary columns and wide temperature gradients. Generally, analyte ionization is
achieved by electron ionization (EI), although electron-capture negative ionization
(ECNI) has been used as well.
Functional groups at the side chain in many cases lead to the formation of a
stereo-centre. In these cases, either chiral derivatisation yielding diastereomers or
the use of chiral GC columns form possible solutions9. The degree of unsaturation
may influence GC–MS analysis in different ways. Compounds with higher degrees of
unsaturation are more prone to autoxidation. Fragmentation of highly unsaturated
CAs in EI is mainly driven by double bonds, limiting the usefulness of the obtained
spectra10. The most important derivatisation strategies for CAs in GC–MS analysis are
summarized in Table 1.
Chapter 2
33
Table 1 Overview of important functional groups in endogenous CAs that require derivatisation in GC–MS and widely applied derivatisation strategies and reagents for this11.
De
riva
tisa
tio
n r
eag
en
t
N-M
eth
yl-N
-(tr
imet
hyl
sily
l) t
rifl
uo
roac
etam
ide
(MST
FA)
Bis
(tri
met
hyl
sily
l)ac
etam
ide
(BSA
) N
,O-B
is(t
rim
eth
ylsi
lyl)
tri
flu
oro
acet
amid
e (B
STFA
)
Trim
eth
ylch
loro
sila
ne
(TM
CS)
N-m
eth
yl-N
-ter
t-b
uty
ldim
eth
ylsi
lyl
trif
luo
roac
etam
ide
(MtB
STFA
) Tr
imet
hyl
sulf
on
ium
hyd
roxi
de
(TM
SH)
Dim
eth
yl s
ulf
ate
(DM
S)
Met
han
ol /
ino
rgan
ic a
cid
Met
han
ol /
Acy
lch
lori
de
Met
han
ol /
Bo
ron
tri
flu
ori
de
(BF 3
) A
lco
ho
l / in
org
anic
aci
d
Alc
oh
ol /
BF 3
P
enta
flu
oro
ben
zyl b
rom
ide
(PFB
Br)
Pic
olin
yl e
ste
rs (
3-P
yrid
ylca
rbin
ol e
ster
s)
4,4
-dim
eth
ylo
xazo
line
(DM
OX
)
MST
FA, B
STFA
, BSA
, TM
CS
Dia
zom
eth
ane
Mo
sher
’s a
cid
ch
lori
de
(–S-
(–)-
α-m
eth
oxy
-α-
(tri
flu
oro
met
hyl
)ph
enyl
acet
yl c
hlo
rid
e)
Hyd
roxy
lam
ine
Met
ho
xyla
min
e
Re
acti
on
Trim
eth
ylsi
lyla
tio
n
t-b
uty
ldim
eth
ylsi
lyla
tio
n
Met
hyl
est
er
form
atio
n
Este
rifi
cati
on
DM
OX
Trim
eth
ylsi
lyla
tio
n
Met
hyl
eth
er f
orm
atio
n
Ch
iral
der
ivat
isat
ion
Oxi
mat
ion
Fun
ctio
nal
gro
up
Car
bo
xylic
aci
d
Hyd
roxy
lgro
up
R/S
iso
mer
ism
Ke
ton
e gr
ou
p
Chapter 2
34
2.3.2 LC–MS analysis
The most widely applied LC mode for CAs is ion-suppressed reversed-phase LC
(RPLC), using methanol/water or acetonitrile/water gradients, frequently using acetic
or formic acid as additives. In LC–MS, underivatised CAs must be analysed in
negative-ion mode using electrospray ionization (ESI) or atmospheric-pressure
chemical ionization (APCI). In some LC–MS instruments, negative ionization seems to
be less efficient than positive ionization. Furthermore, most CA subclasses do not
provide readily applicable intense fragment ions upon collision-induced dissociation
(CID) to be applied in the selected-reaction monitoring (SRM). Quantitative analysis
using LC–MS is mostly performed in SRM mode using tandem-quadrupole (TQ) or
quadrupole–linear-ion-trap hybrid (QTRAPTM, Q–LIT) instruments. The latter provides
enhanced full-spectrum sensitivity in MS–MS as well. Underivatised CAs upon CID
may primarily show the loss of CO2, i.e., [M-H-CO2]–, and of H2O from alcohol or
ketone functions in the molecule, if present. Other fragmentation of CAs is largely
influenced by functional groups, for instance double bonds, present in the alkyl part
of the molecule. As a rule of thumb, it can be stated that the higher the degree of
unsaturation and the higher the number of functional groups present in a CA, the
easier is its fragmentation and the easier is the formation of fragments not related to
the loss of CO2.
Although for LC–MS analysis of CAs derivatisation is not always necessary, there
are a number of reasons to perform derivatisation of CAs also in LC–MS15,16.
Derivatisation may facilitate the RPLC separation of CAs and may also direct
fragmentation and yield characteristic other neutral losses, related to the
derivatisation reagent, applicable in SRM.
Chapter 2
35
Separation of chiral CAs for LC–MS analysis can be accomplished by either the
use of chiral stationary phases, or chiral derivatisation. The usage of chiral stationary
phases has long been associated with the use of normal-phase separation systems
being less compatible with LC–MS. However, chiral RPLC separations can be achieved
using for example amylose tris(3,5-dimethylphenylcarbamate) coated ChiralPak AD-
RH columns12. Chiral derivatisation for RPLC can be successful if the chiral centres are
in close proximity, otherwise the resulting diastereomers might not be separable11.
2.3.3 Sample pre-treatment
All subclasses of CAs may be analysed in body fluids, e.g., plasma, serum, urine,
cerebrospinal fluid (CSF), while especially small CAs may also be analysed in cellular
extracts. For GC–MS, analyte extraction to an organic solvent is required prior to
derivatisation. This can be achieved by (ion-suppressed) liquid-liquid extraction (LLE),
using solvents like ethyl acetate or n-hexane. For LC–MS, protein precipitation is
performed for blood-related samples, eventually followed by a sample clean-up step
using LLE or solid-phase extraction (SPE), using RPLC or mixed-mode materials.
Removal of endogenous phospholipids is important to reduce matrix effects in LC–
MS. The combination of highly polar groups and hydrophobic alkyl chains may
present challenges to analyte recovery in LLE or SPE. For urine analysis, either dilute-
and-shoot procedures13 or clean-up using LLE or SPE is performed. In the analysis of
small CAs in cellular extracts, quenching of the cellular metabolism is important,
requiring specialized protocols14.
Chapter 2
36
2.3.4 Use of internal standards
If absolute quantitation is to be achieved, the use of stable-isotope-labelled (SIL)
internal standards (ISs) is crucial for both GC–MS and LC–MS analysis of CAs.
Although Dn-labelled ISs can be used, there is a risk of D/H-exchange during sample
pre-treatment in acidic or alkaline media15. Therefore, [13Cn]-labelled ISs are generally
preferred. SIL-ISs can be produced by organic synthesis. In the analysis of cellular
extracts, the use of mass isotopomer ratio analysis of uniformly-[13Cn]-labelled
extracts (MIRACLE)16, based on the biosynthesis of SIL-ISs in yeast-cell cultures grown
on [13C6]-glucose, is a powerful tool. Other approaches involve stable isotope coding
by derivatisation17 or quantification by standard addition.
2.4 Small carboxylic acids with less than 6 carbon atoms
The subclass of small CAs consists of short-chain FAs (≤6 carbons in the aliphatic
tail) and their hydroxylated and/or ketone containing analogues1, 8. Both mono-, di-,
and tri-CAs are among this group. Prominent examples of this subclass are the
intermediates of the Krebs’s or tricarboxylic acid (TCA) cycle and the important
clinical markers D-lactic acid18 and methylmalonic acid8 (see Figure 3). Recent interest
in small CAs has largely been boosted by translational research into metabolic
phenomena such as the Warburg effect19 and autophagy1. Therefore, the current
interest in the analysis of small CAs is likely to grow. General pitfalls include: their low
molecular weight, their high polarity, their limited stability, leading to challenges in
their extraction from aqueous matrices and, for some, volatility issues1, 20. The
importance of these issues depends on the analytical technique applied. There is a
general stability problem with keto CAs21, e.g., β-keto CAs very readily undergo
decarboxylation, as a preferred six-membered transition state can be formed22.
Chapter 2
37
Figure 3 Structures of important small CAs.
2.4.1 GC–MS analysis of small carboxylic acids
Derivatisation is crucial for successful GC–MS analysis of small CAs. Esterification
of small CAs with small alcohols is generally not successful due to the high volatility
of such derivatives. Larger alcohols could be used, but generally require the use of
rather harsh reaction conditions, involving catalysts like anhydrous sulfuric acid or
Chapter 2
38
boron trifluoride23. In this respect, silylation seems to be a better choice, as less
volatile, higher molecular-weight derivatives are formed. As modern analytical
strategies tend to move more and more towards comprehensive multi-component
analysis, the derivatisation protocols to be used have to become increasingly generic
24. A frequently applied approach consists of a combination of oximation and
silylation. Oximation using methoxylamine or hydroxylamine derivatises the ketone
groups, if present, whereas trimethylsylilation or t-butyldimethylsilylation24,25
modifies both the hydroxyl and the CA groups into trimethylsilyl (TMS) and t-
butyldimethylsilyl (tBDMS) derivatives, respectively. Typical reaction conditions
comprise oximation in a solution of the corresponding alkoxylamine hydrochloride in
pyridine at a concentration of typically 20 mg/mL at 30 °C for 90 min followed by
silylation using MtBSTFA at 70 °C or MSTFA at 37 °C for 30 min25.
In EI-MS, TMS derivatives yield abundant M–CH3●+ ions with m/z M+●–15 as well
as relatively abundant non-specific ions with m/z 73, due to (CH3)3Si+, and m/z 75,
due to (CH3)2Si=OH+. The most prominent fragment of the tBDMS derivatives in EI-MS
usually is M–(CH3)3C●+ with m/z M+●–57, together with some low-abundance
fragments26. Whereas the derivatisation of CAs using MtBSTFA is a straightforward
reaction26, the reaction of hydroxyl groups with MtBSTFA is less favourable and thus
may lead to partial derivatisation and skewing of the results27.
Prior to derivatisation, the (highly) hydrophilic small CAs should be extracted
from the usually aqueous sample matrix into an appropriate organic solvent, e.g.,
diethyl ether, methanol or ethanol. Given the volatility of some CAs and the limited
stability of the keto CAs, temperatures should be kept as low as possible throughout
the sample pre-treatment procedure. Generic protocols have recently been
described for extraction from cellular incubations and body fluids. Extraction of small
Chapter 2
39
CAs, among other cellular metabolites, from mammalian cells involves quenching
using liquid nitrogen and extraction using a methanol/chloroform mixture28. For body
fluids such as plasma or urine, quenching of the metabolic reactions is generally less
of a concern. Protein precipitation with methanol29 has become the gold standard for
wide-range analysis of low molecular-weight analytes, including small organic CAs, in
plasma, eventually in combination with SPE if more targeted methods for a limited
number of analytes are aimed at. For urine analysis, the sample pre-treatment
usually comprises of an eventual urease step followed by freeze drying,
reconstitution in an organic solvent, and derivatisation24.
Metabolic fluxes in melanoma cell lines using oximation and tBDMS ester
formation were recently investigated by Scott et al.30. The effects of valproic acid in
children were studied by urinary analysis of small CAs31. Urine samples were directly
oximated using hydroxylamine and sodium hydroxide, the analytes extracted by LLE
and further derivatised by silylation using BSTFA, prior to GC-MS analysis.
2.4.2 LC–MS analysis of small carboxylic acids
LC seems to be the method-of-choice for the analysis of small CAs, being highly
polar compounds. At first, no derivatisation seems to be required. In practice, the
situation is somewhat more complicated. In RPLC, the small CAs generally show
insufficient retention. Therefore, the use of ion-pairing agents like tetrabutylamine
(TBA) have been proposed32. However, this leads to substantial ionization
suppression in ESI-MS and is detrimental to the equipment used33. As an alternative,
methods based on either hydrophilic interaction chromatography (HILIC)34 or anion-
exchange chromatography (HPAEC)35 have been proposed. In the latter case, post-
column electrolytic suppressors are required for the removal of high salt
concentrations applied36. Performance comparison of various column chemistries for
Chapter 2
40
HILIC, e.g., aminopropyl, amide, cyano, diol, or silica37, as well as their comparison
with RPLC, for the analysis of small CAs has been reported34, 38. Although HILIC with
aminopropyl or diol columns appear to be most successful, it seems difficult to select
an LC phase system especially directed at small CAs.
The indicated problems in both LC separation and MS detection lead to
reconsidering pre-column derivatisation of the small CAs39. The use of N-methyl-2-
phenylethanamine (MPEA) after carbodiimide activation has been applied to TCA
cycle intermediates20. The analytes were derivatised with 3-
(ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine hydrochloride salt (EDC)
and MPEA at 60 °C for 45 min in 90% ACN. After dilution with water, the sample
could be directly analysed by online SPE–LC–MS. Even though derivatisation might
advance the analysis of small CAs, the instability of particularly the keto CAs might
hamper successful analysis of these species.
For sample pre-treatments aimed at LC–MS, similar protocols are used as for GC–
MS, involving freeze drying with urine, protein precipitation with plasma, eventually
complemented by SPE24, 40. In the analysis of cellular metabolites, combined
quenching and extraction methods are needed41.
A recent example identifying succinate as an inflammatory signal in innate
immunity was reported by Tannahill et al.42. The authors applied several LC–MS
platforms with different HILIC separations; for succinate analysis the one based on a
zwitter ionic (ZIC) HILIC column was used.
Chapter 2
41
2.5 Fatty acids
FAs are mono-CAs with a long-chain aliphatic end. In mammals, straight-chain
FAs with normally an even carbon number are observed, whereas in bacteria also
branched alkyl chains and/or higher levels of odd carbon numbered FAs occur. One
distinguishes short-chain (≤6 C atoms, i.e., the small CAs in this paper, Section 3),
medium-chain (6–12 C atoms), long-chain (12–22 C atoms), and very-long-chain FAs
(>22 C atoms). The aliphatic chain may contain several double bonds. FAs with a
degree of unsaturation of two or higher are frequently called poly-unsaturated fatty
acids (PUFAs). Each double bond may be either E or Z (trans or cis); a PUFA with three
double bonds could theoretically form 8 E/Z-isomers. PUFAs formed biochemically
usually show all-Z (all-cis) configurations. The ω(n)-nomenclature is applied to
indicate the position of the first double bond relative to the aliphatic end rather than
relative to the CA end (IUPAC). Besides double bonds, FAs might also contain ketone,
hydroxyl, hydroperoxide, epoxide and other functional groups. Each of these
functionalities put specific demands to the analytical strategies, which cannot be
discussed in detail here. We focus on FAs, keto FAs, and mono-hydroxylated FAs, the
latter being the biochemical precursors of certain eicosanoids and docosanoids42.
Hydroxyl groups usually lead to a stereo-centre in the FA side chain; biochemically
formed hydroxylated FAs normally pose the S-configuration, whereas autoxidation
products are racemic mixtures. The oxidative stability is a major concern in PUFA
analysis43. Until recently, FAs were primarily analysed by GC–MS, but currently also
LC–MS methods are frequently reported.
Depending on the application, either free FA (f-FA) or total FA (t-FA) content is to
be determined. f-FA determination requires an appropriate extraction method, e.g.,
using LLE with n-hexane, i-octane or a similar solvent, without affecting the FAs
Chapter 2
42
bound in triglycerides (phosopholipids and other storage forms) or to, for instance,
proteins. For t-FA determination, a saponification step must be performed, mostly
under alkaline conditions. Care must be taken to avoid autoxidation and double-bond
isomerization. Because of the risk of D/H-exchange, Dn-SIL-ISs can only be added
after saponification15. Saponification and extraction can be combined with
esterification in a process called transesterification, which is carried out by acid-
catalysed methylation usually by the use of methanol, hexane and acetylchloride44,
thus yielding fatty acid methyl esters (FAMEs), which can be analysed by GC–MS.
2.5.1 GC–MS analysis of fatty acids
LLE of f-FAs from a biological matrix yields the FAs in non-polar organic solvent.
The samples can be subjected to derivatisation either directly or after drying under a
stream of nitrogen or in a SpeedVac45. Similar to small CAs, the most favourable
derivatisation methods are esterification and silylation11.
The formation of FAMEs is the most prominent derivatisation strategy for GC–
MS46. While PFBBr and silyl-ester derivatives are frequently separated on standard
phenyl-polysiloxane columns, cyanopropyl polysilphenylsiloxane columns have
become the standard GC columns for FAME analysis. A recent application involves
acetylchoride based transesterification incubating the samples overnight at room
temperature, thereby overcoming acid induced E/Z isomerization, and the separation
of positional and geometrical FAME isomers46.
However, FAMEs tend to provide excessive fragmentation in EI-MS with the ion
with m/z 74, i.e., CH2C(OH)OCH3+●, resulting from a McLafferty rearrangement, being
the most abundant ion 10. As the ion with m/z 74 is a class-specific and not a
Chapter 2
43
compound-specific fragment, it cannot be used in isotopologue analysis (13C-flux
determination), as most of the molecular information is lost.
A number of alternative derivatisation strategies have been described47,
including the formation of TMS or tBDMS derivatives26, picolinyl esters48, and
DMOX49 derivatives. The latter can also be used for double-bond localization and
branching analysis49. Derivatisation using PFBBr enables the use of ECNI in GC–MS,
which provides highly selective and mild ionization, i.e., dissociative electron capture
to generate predominantly [M–PFB]–-ions without much further fragmentation, thus
facilitating isotopologue analysis50. Recently, an overview of the use of stable
isotopes in studying lipid metabolism was published51. High-resolution GC is crucial
for the differentiation of E/Z isomers46. Another important topic is the determination
of double-bond positions, which can be achieved in different ways, e.g., specific
derivatisation agents such as picolinyl esters or DMOX derivatives52, from careful
interpretation of the fragmentation observed in EI mass spectra10, or by using
covalent adduct chemical ionization tandem MS (CACI-MS–MS) using acetonitrile in
ion-trap instruments53.
2.5.2 LC–MS analysis of fatty acids
The general interest in LC–MS and especially the introduction of ultra-high-
performance LC (UHPLC) has boosted the developments in FA analysis by LC–MS
rather than GC–MS54. Unless derivatisation is performed, FAs are analysed as M–H– in
negative-ion mode using ESI or APCI. Upon CID, little fragmentation is observed for
saturated FAs, and minor losses of CO2 for PUFAs55. Therefore, either SIM or SRM
with the same m/z for both precursor and product ion, thus attempting to at least
fragment possible co-eluting isobaric species56, is used. Post-column addition of Ba2+
Chapter 2
44
was reported, to generate [M–H+Ba]+-ions, which readily undergo charge-remote
fragmentation of the alkyl chain, providing specific fragment ions for SRM57.
In this way, f-FAs can be analysed in the low nM range, e.g., after MeOH protein
precipitation for plasma55. An interesting example is the analysis of 36 f-FAs in human
plasma, using a calibration set of known FAs to enable identification and
quantification of unknown f-FAs. The method made use of the above-described SRM
procedure56 and showed lower limits of quantification in the nM range with run
times below 10 min57. Transesterification procedures, applied to determine t-FA,
yield FAMEs, which show poor ionization characteristics in ESI and APCI. Mostly, RPLC
is used for the separation of FAs. Separation of FAs on Ag+-loaded columns provides
enhanced resolution of FAs with different E/Z isomers and double-bond positions,
eventually in combination with ozonolysis58.
Like with small CAs, quantitative performance may be enhanced by
derivatisation, either to enhance the ionization efficiency or to implement
fragmentation characteristics for SRM. A 60,000-times improved sensitivity,
compared to the analysis of underivatised FAs, has been claimed for N-(4-
aminomethylphenyl)pyridinium (AMPP) derivatives of FAs, introducing a permanent
charge59. Other derivatisation strategies involve for instance trimethylaminoethyl
(TMAE)60, 2-bromo-1-methylpyridinium iodide (BMP)61, MPEA20, and 4-(2-((4-
bromophenethyl)dimethylammonio)ethoxy)benzenaminium dibromide (4-APC)62.
Derivatisation techniques for FA analysis using LC–MS have recently been reviewed39.
Carbodiimide coupling using EDC in combination with AMMP derivatisation and
stable isotope coding was applied in the analysis of t-FAs in human serum samples61.
The derivatives were separated on a C4-column using an acetonitrile/water gradient.
Chapter 2
45
2.6 Eicosanoids
Phospholipases release mainly 20-carbon PUFAs from membrane-phospholipids.
Eicosanoids are the enzymatic oxidation products of these PUFAs, generated by
enzymes from the cyclooxygenase (COX), cytochrome P450 (CYP), and lipoxygenase
(LOX) families5. Typical eicosanoids are arachidonic acid (FA 20:4) derived
prostaglandins and leukotrienes. Isoprostanes are closely related eicosanoids,
generated by non-enzymatic oxidation of FA 20:463. Many eicosanoids mediate
critical biological effects such as for example, chemotaxis, blood clotting or broncho-
constriction. Particularly during inflammatory processes, prostaglandins and
leukotrienes, derived from FA 20:4, are important in the initial phase4, whereas
eicosapentaenoic acid derived mediators play a crucial role in the active resolution
phase of inflammation4. In addition, the family of 22-carbon PUFA derived
docosanoids comprise related highly active mediators8. The biological activity of the
eicosanoids and related compounds strongly relies on stereo-, positional- and
geometrical- isomerism4.
Artificial eicosanoids may be formed by oxidation of FA 20:4, which is present at
high levels in human body fluids such as plasma, or by activation of platelets during
venipuncture. To avoid errors in analysis, it is important to use ion chelators such as
EDTA, to freeze samples immediately at -80 °C, and to consider the use of
antioxidants such as butylated hydroxytoluene (BHT) and/or enzyme inhibitors such
as indomethacin43.
Chapter 2
46
2.6.1 GC–MS analysis of eicosanoids
Multistep derivatisation is required to achieve compatibility of eicosanoids with
GC–MS analysis. The gold standard is a combination of trimethylsilylation of hydroxyl
groups, oximation of the ketone groups (if necessary), and PFBBr derivatisation of the
CA group, thus enabling selective and sensitive analysis using ECNI in GC–MS64. A
protocol for the assessment of F2-isoprostanes as markers of oxidative stress in vivo
has been reported65. Following PFBBr ester formation, sample clean-up by thin-layer
chromatography and silylation with BSTFA, analysis is performed by ECNI in GC–MS.
Particularly for structural confirmation purposes, GC–MS with EI fragmentation after
diazomethane derivatisation is still an important tool66.
2.6.2 LC–MS analysis of eicosanoids
The sample pre-treatment protocol for GC–MS, involving a two (three)-step
derivatisation, is obviously quite laborious, which explains why nowadays LC–MS
analysis is frequently applied instead46. LC–MS allows the analysis of underivatised
compounds, greatly facilitating sample pre-treatment and minimizing possible
analyte losses.
An important challenge in eicosanoid analysis is the resolution of the high
number of possible stereo- and E/Z-isomers. Leukotriene B4, for example, contains 4
double bonds and 2 stereo-centres and can thus theoretically exist as64 different
isomers. The high separation efficiency achievable by the use of UHPLC with columns
packed with small porous or solid-core particles (<2 μm) and excellent retention time
stability are of utmost importance, especially because differentiation based on
fragmentation in MS–MS is not always possible67. As eluent systems in RP-
separations MeOH/water, ACN/water as well as mixtures thereof have been
Chapter 2
47
described. Given the impact of stereo- and E/Z isomerism on their biological activity,
chiral separation of eicosanoids can be of considerable concern 68. As the elution
order of enantiomers cannot be predicted, only comparison with standards or with
published results obtained under identical conditions allows the deduction of
absolute stereochemistry31. Sample pre-treatments of plasma samples is mainly
based on protein precipitation followed by sample clean-up using C18-SPE with or
without the involvement of a hexane wash step68. While sample preparation of blood
derived samples is rather straightforward, the analysis of urinary samples does
involve more tedious sample preparation protocols which is mainly due to the
occurrence of strong matrix effects. A protocol using a mixed mode SPE (Oasis HLB)
in combination with APCI LC–MS was described69. Another protocol involves the use
of a weak anion-exchange material70. Compared to C18 based SPE, very clean extracts
were obtained by methanol elution of the eicosanoids; most matrix components
remained on the SPE cartridge under these conditions.
Given the low endogenous levels of eicosanoids, ultimate sensitivity must be
achieved using SRM in TQ or Q–LIT instruments68. Upon CID, the presence of hydroxyl
and ketone groups in the alkyl side-chain induces specific cleavages leading to
analyte-specific fragment ions71. Current trends in the implementation of high-
resolution mass spectrometry (HRMS) in quantitative bio analysis may be beneficial
in eicosanoid analysis, as (almost) co-eluting isobaric compounds can be resolved by
HRMS72 In this respect, combined ion-mobility spectrometry and MS (IMS–MS)
should be explored as well73.
LC–MS analysis of eicosanoids has recently been reviewed74. Recent applications
involving AMPP labelling of oxidized fatty acids and LC–MS using either a TQ
instrument75 or an LTQ-Orbitrap mass spectrometer72 were reported. Mouse serum
Chapter 2
48
samples were derivatised after SPE clean-up and analysed by a generic RPLC
separation using an acetonitrile/water gradient.
2.7 Bile Acids
BAs, in particular cholic and chenodeoxycholic acids, are the major CYP-mediated
catabolic-metabolites of cholesterol 6. Just recently, BAs have emerged as signalling
molecules with systemic endocrine function76. Particularly in the context of metabolic
diseases such as obesity or type-2 diabetes, BA signalling might possibly be exploited
as novel therapeutic intervention strategy76, 77. As a result, analysis and profiling of
BAs has recently received considerable attention. In this respect, a comprehensive
sample pre-treatments protocol allowing the analysis of neutral, acidic and basic
sterol-derivatives is needed78. All aspects of the analysis of BAs have been extensively
reviewed recently79.
2.7.1 GC–MS analysis of bile acids
The subclass of BAs is not a favourable compound class for GC–MS. Apart from
the CA and hydroxyl groups, which already require derivatisation, BAs may contain
several other polar and labile conjugates with groups like sulfate, phosphate, amide
and glucuronate, that are not readily derivatised towards GC–MS 8. Thus, BA analysis
by GC–MS is limited to deconjugated compounds, which can be analysed as
TMS/methyl ester derivatives. The fragmentation of BAs in EI can be highly useful
and complementary in structure elucidation to product-ion mass spectra obtained by
ESI-MS and CID79.
Chapter 2
49
2.7.2 LC–MS analysis of bile acids
LC–MS can be readily used for the analysis of BAs as well as their conjugated
analogues80, 81. In all instances, sample pre-treatment is less complicated than for
GC–MS. The presence of multiple isomeric BAs put high demands on efficient
separation, especially because CID provides little compound-specific fragmentation.
Reversed-phase UHPLC is generally applied81, 82. For SRM in negative-ion mode,
mostly group-specific product ions are applied, e.g., m/z 74 (C2H4NO2–) for glyco-BAs,
m/z 80 (SO3–●) for tauro-BAs, and m/z 97 (HSO4
–) (or neutral loss of 80 Da, SO3) for
sulfate-conjugated BAs, whereas unconjugated BAs do not show significant
fragmentation83. As such, CID readily enables the identification of the conjugates, but
provides little structural information on the BAs themselves80.
An interesting recent study provided evidence that dietary fats can result in
changes of the host BA composition, thus altering conditions for gut microbial
assemblage perturbing immune homeostasis84.
2.8 Conclusion and perspectives
Analytical strategies for the analysis of four subclasses of CAs by GC–MS and LC–
MS are reviewed. Attention is paid to typical protocols for the analysis of these
compound classes. The biological importance of the compounds discussed clarifies
the analytical relevance. Despite the progress in the development of useful analytical
protocols, many challenges remain to be (further) addressed, including
differentiation between various isomeric species, both in terms of separation and
MS–MS fragmentation, and low-level analysis for both absolute quantification and
flux analysis. Significant progress in this respect may be expected in the years to
come.
Chapter 2
50
2.9 References
1 J.D. Rabinowitz, E. White, Autophagy and Metabolism, Science 330 (2010) 1344-1348.
2 P.C. Calder, n-3 Fatty acids, inflammation and immunity: new mechanisms to explain old actions, Proc. Nutr. Soc. 72 (2013) 326-336.
3 I.R. Klein-Wieringa, S.N. Andersen, J.C. Kwekkeboom, M. Giera, B.J.E. de Lange-Brokaar, G.J.V.M. van Osch, A.-M. Zuurmond, V. Stojanovic-Susulic, R.G.H.H. Nelissen, H. Pijl, T.W.J. Huizinga, M. Kloppenburg, R.E.M. Toes, A. Ioan-Facsinay, Adipocytes Modulate the Phenotype of Human Macrophages through Secreted Lipids, The Journal of Immunology 191 (2013) 1356-1363.
4 C.N. Serhan, N.A. Petasis, Resolvins and Protectins in Inflammation Resolution, Chem. Rev. 111 (2011) 5922-5943.
5 C.D. Funk, Prostaglandins and Leukotrienes: Advances in Eicosanoid Biology, Science 294 (2001) 1871-1875.
6 J.Y.L. Chiang, Bile Acid Metabolism and Signalling, John Wiley & Sons, Inc., 2013.
7 J. Acimovic, A. Lövgren-Sandblom, K. Monostory, D. Rozman, M. Golicnik, D. Lutjohann, I. Björkhem, Combined gas chromatographic/mass spectrometric analysis of cholesterol precursors and plant sterols in cultured cells, J. Chromatogr. B 877 (2009) 2081-2086.
8 D.W. Johnson, Contemporary clinical usage of LC/MS: Analysis of biologically important carboxylic acids, Clin. Biochem. 38 (2005) 351-361.
9 A.M. Stalcup, Chiral Separations, Annual Review of Analytical Chemistry 3 (2010) 341-363.
10 S.A. Mjøs, The prediction of fatty acid structure from selected ions in electron impact mass spectra of fatty acid methyl esters, Eur. J. Lipid Sci. Technol. 106 (2004) 550-560.
11 V.Zaikin and J. Halket, A Handbook of derivatives for mass spectrometry, IM Publications, Chisester, UK, 2009.
12 S.F. Oh, T.W. Vickery, C.N. Serhan, Chiral lipidomics of E-series resolvins: Aspirin and the biosynthesis of novel mediators, Biochim. Biophys. Acta 1811 (2011) 737-747.
13 H.G. Gika, C. Ji, G.A. Theodoridis, F. Michopoulos, N. Kaplowitz, I.D. Wilson, Investigation of chronic alcohol consumption in rodents via ultra-high-performance liquid chromatography–mass spectrometry based metabolite profiling, J. Chromatogr. A 1259 (2012) 128-137.
14 T. Damme, M. Lachová, F. Lynen, R. Szucs, P. Sandra, Solid-phase extraction based on hydrophilic interaction liquid chromatography with acetone as
Chapter 2
51
eluent for eliminating matrix effects in the analysis of biological fluids by LC-MS, Anal. Bioanal. Chem. 406 (2014) 401-407.
15 J. Lee, E.-S. Jang, B. Kim, Development of isotope dilution-liquid chromatography/mass spectrometry combined with standard addition techniques for the accurate determination of tocopherols in infant formula, Anal. Chim. Acta 787 (2013) 132-139.
16 M.R. Mashego, L. Wu, J.C. Van Dam, C. Ras, J.L. Vinke, W.A. Van Winden, W.M. Van Gulik, J.J. Heijnen, MIRACLE: mass isotopomer ratio analysis of U-13C-labelled extracts. A new method for accurate quantification of changes in concentrations of intracellular metabolites, Biotechnol. Bioeng. 85 (2004) 620-628.
17 P. Bruheim, H.F.N. Kvitvang, S.G. Villas-Boas, Stable isotope coded derivatizing reagents as internal standards in metabolite profiling, J. Chromatogr. A 1296 (2013) 196-203.
18 C. Petersen, D-Lactic Acidosis, Nutr. Clin. Pract. 20 (2005) 634-645. 19 M.G. Vander Heiden, L.C. Cantley, C.B. Thompson, Understanding the
Warburg Effect: The Metabolic Requirements of Cell Proliferation, Science 324 (2009) 1029-1033.
20 D. Kloos, R.J.E. Derks, M. Wijtmans, H. Lingeman, O.A. Mayboroda, A.M. Deelder, W.M.A. Niessen, M. Giera, Derivatisation of the tricarboxylic acid cycle intermediates and analysis by online solid-phase extraction-liquid chromatography–mass spectrometry with positive-ion electrospray ionization, J. Chromatogr. A 1232 (2012) 19-26.
21 M. Fuchs, J. Engel, M. Campos, R. Matejec, M. Henrich, H. Harbach, M. Wolff, K. Weismüller, T. Menges, M.C. Heidt, I.D. Welters, M. Krüll, G. Hempelmann, J. Mühling, Intracellular alpha-keto acid quantification by fluorescence-HPLC, Amino Acids 36 (2009) 1-11.
22 K.J. Pedersen, The Decomposition of α-Nitrocarboxylic Acids. With Some Remarks on the Decomposition of β-Ketocarboxylic Acids, The Journal of Physical Chemistry 38 (1933) 559-571.
23 C. Hallmann, B.G.K. van Aarssen, K. Grice, Relative efficiency of free fatty acid butyl esterification: Choice of catalyst and derivatisation procedure, J. Chromatogr. A 1198–1199 (2008) 14-20.
24 E.C. Chan, K.K. Pasikanti, J.K. Nicholson, Global urinary metabolic profiling procedures using gas chromatography-mass spectrometry, Nat. Protoc. 6 (2011) 1483-1499.
25 O. Fiehn, J. Kopka, R.N. Trethewey, L. Willmitzer, Identification of Uncommon Plant Metabolites Based on Calculation of Elemental Compositions Using Gas
Chapter 2
52
Chromatography and Quadrupole Mass Spectrometry, Anal. Chem. 72 (2000) 3573-3580.
26 K.R. Kim, M.K. Hahn, A. Zlatkis, E.C. Horning, B.S. Middleditch, Simultaneous gas chromatography of volatile and non-volatile carboxylic acids as tert.-Butyldimethylsilyl derivatives, J. Chromatogr. A 468 (1989) 289-301.
27 D. Saraiva, R. Semedo, M.d.C. Castilho, J.M. Silva, F. Ramos, Selection of the derivatisation reagent—The case of human blood cholesterol, its precursors and phytosterols GC–MS analyses, J. Chromatogr. B 879 (2011) 3806-3811.
28 M.A. Lorenz, C.F. Burant, R.T. Kennedy, Reducing Time and Increasing Sensitivity in Sample Preparation for Adherent Mammalian Cell Metabolomics, Anal. Chem. 83 (2011) 3406-3414.
29 J. A, J. Trygg, J. Gullberg, A.I. Johansson, P. Jonsson, H. Antti, S.L. Marklund, T. Moritz, Extraction and GC/MS Analysis of the Human Blood Plasma Metabolome, Anal. Chem. 77 (2005) 8086-8094.
30 D.A. Scott, A.D. Richardson, F.V. Filipp, C.A. Knutzen, G.G. Chiang, Z.e.A. Ronai, A.L. Osterman, J.W. Smith, Comparative Metabolic Flux Profiling of Melanoma Cell Lines: Beyond the Warburg Effect, J. Biol. Chem. 286 (2011) 42626-42634.
31 K.E. Price, R.E. Pearce, U.C. Garg, B.A. Heese, L.D. Smith, J.E. Sullivan, M.J. Kennedy, J.F. Bale, R.M. Ward, T.K.H. Chang, F.S. Abbott, J.S. Leeder, Effects of Valproic Acid on Organic Acid Metabolism in Children: A Metabolic Profiling Study, Clin. Pharmacol. Ther. 89 (2011) 867-874.
32 U. Hofmann, K. Maier, A. Niebel, G. Vacun, M. Reuss, K. Mauch, Identification of metabolic fluxes in hepatic cells from transient 13C-labelling experiments: Part I. Experimental observations, Biotechnol. Bioeng. 100 (2008) 344-354.
33 D. Siegel, H. Permentier, D.-J. Reijngoud, R. Bischoff, Chemical and technical challenges in the analysis of central carbon metabolites by liquid-chromatography mass spectrometry, J. Chromatogr. B. doi: 10.1016/j.jchromb.2013.11.022
34 S.U. Bajad, W. Lu, E.H. Kimball, J. Yuan, C. Peterson, J.D. Rabinowitz, Separation and quantitation of water soluble cellular metabolites by hydrophilic interaction chromatography-tandem mass spectrometry, J. Chromatogr. A 1125 (2006) 76-88.
35 J.C. van Dam, M.R. Eman, J. Frank, H.C. Lange, G.W.K. van Dedem, S.J. Heijnen, Analysis of glycolytic intermediates in Saccharomyces cerevisiae using anion exchange chromatography and electrospray ionization with tandem mass spectrometric detection, Anal. Chim. Acta 460 (2002) 209-218.
36 V. Ruiz-Calero, M.T. Galceran, Ion chromatographic separations of phosphorus species: a review, Talanta 66 (2005) 376-410.
Chapter 2
53
37 B. Buszewski, S. Noga, Hydrophilic interaction liquid chromatography (HILIC)—a powerful separation technique, Anal. Bioanal. Chem. 402 (2012) 231-247.
38 D.-P. Kloos, H. Lingeman, W.M.A. Niessen, A.M. Deelder, M. Giera, O.A. Mayboroda, Evaluation of different column chemistries for fast urinary metabolic profiling, J. Chromatogr. B 927 (2013) 90-96.
39 P. Deng, Y. Zhan, X. Chen, D. Zhong, Derivatisation methods for quantitative bioanalysis by LC–MS/MS, Bioanalysis 4 (2011) 49-69.
40 S. Becker, L. Kortz, C. Helmschrodt, J. Thiery, U. Ceglarek, LC–MS-based metabolomics in the clinical laboratory, J. Chromatogr. B 883–884 (2012) 68-75.
41 S. Dietmair, N.E. Timmins, P.P. Gray, L.K. Nielsen, J.O. Krömer, Towards quantitative metabolomics of mammalian cells: Development of a metabolite extraction protocol, Anal. Biochem. 404 (2010) 155-164.
42 G.M. Tannahill, A.M. Curtis, J. Adamik, E.M. Palsson-McDermott, A.F. McGettrick, G. Goel, C. Frezza, N.J. Bernard, B. Kelly, N.H. Foley, L. Zheng, A. Gardet, Z. Tong, S.S. Jany, S.C. Corr, M. Haneklaus, B.E. Caffrey, K. Pierce, S. Walmsley, F.C. Beasley, E. Cummins, V. Nizet, M. Whyte, C.T. Taylor, H. Lin, S.L. Masters, E. Gottlieb, V.P. Kelly, C. Clish, P.E. Auron, R.J. Xavier, L.A.J. O’Neill, Succinate is an inflammatory signal that induces IL-1bgr through HIF-1agr, Nature 496 (2013) 238-242.
43 A.E. Barden, E. Mas, K.D. Croft, M. Phillips, T.A. Mori, Minimizing artifactual elevation of lipid peroxidation products (F2-isoprostanes) in plasma during collection and storage, Anal. Biochem. 449 (2014) 129-131.
44 G. Lepage, C.C. Roy, Direct transesterification of all classes of lipids in a one-step reaction, J. Lipid Res. 27 (1986) 114-120.
45 G.-L. Wei, E.Y. Zeng, Gas chromatography-mass spectrometry and high-performance liquid chromatography-tandem mass spectrometry in quantifying fatty acids, TrAC, Trends Anal. Chem. 30 (2011) 1429-1436.
46 J. Ecker, Profiling eicosanoids and phospholipids using LC-MS/MS: Principles and recent applications, J. Sep. Sci. 35 (2012) 1227-1235.
47 W.W. Christie, Preparation of ester derivatives of fatty acids for chromatographic analysis, in: W.W. Christie (Ed.), Advances in lipid methodoligy - two, Oily Press, Dundee, UK, 1993, pp. 69-111.
48 W. Christie, E. Brechany, S. Johnson, R. Holman, A comparison of pyrrolidide and picolinyl ester derivatives for the identification of fatty acids in natural samples by gas chromatography-mass spectrometry, Lipids 21 (1986) 657-661.
Chapter 2
54
49 V. Svetashev, Mild Method for Preparation of 4,4-Dimethyloxazoline Derivatives of Polyunsaturated Fatty Acids for GC–MS, Lipids 46 (2011) 463-467.
50 O. Quehenberger, A.M. Armando, E.A. Dennis, High sensitivity quantitative lipidomics analysis of fatty acids in biological samples by gas chromatography–mass spectrometry, Biochim. Biophys. Acta 1811 (2011) 648-656.
51 J. Ecker, G. Liebisch, Application of stable isotopes to investigate the metabolism of fatty acids, glycerophospholipid and sphingolipid species, Prog. Lipid Res. 54 (2014) 14-31.
52 P. Gómez-Cortés, C. Tyburczy, J.T. Brenna, M. Juárez, M.A. de la Fuente, Characterization of cis-9 trans-11 trans-15 C18:3 in milk fat by GC and covalent adduct chemical ionization tandem MS, J. Lipid Res. 50 (2009) 2412-2420.
53 C. Pelt, B. Carpenter, J.T. Brenna, Studies of structure and mechanism in acetonitrile chemical ionization tandem mass spectrometry of polyunsaturated fatty acid methyl esters, J. Am. Soc. Mass. Spectrom. 10 (1999) 1253-1262.
54 A. Latorre, A. Rigol, S. Lacorte, D. Barceló, Comparison of gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry for the determination of fatty and resin acids in paper mill process waters, J. Chromatogr. A 991 (2003) 205-215.
55 C. Hellmuth, M. Weber, B. Koletzko, W. Peissner, Nonesterified Fatty Acid Determination for Functional Lipidomics: Comprehensive Ultrahigh Performance Liquid Chromatography–Tandem Mass Spectrometry Quantitation, Qualification, and Parameter Prediction, Anal. Chem. 84 (2012) 1483-1490.
56 S. Schiesel, M. Lämmerhofer, W. Lindner, Quantitative LC-ESI-MS/MS metabolic profiling method for fatty acids and lipophilic metabolites in fermentation broths from β-lactam antibiotics production, Anal. Bioanal. Chem. 397 (2010) 147-160.
57 N. Zehethofer, D.M. Pinto, D.A. Volmer, Plasma free fatty acid profiling in a fish oil human intervention study using ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry, Rapid Commun. Mass Spectrom. 22 (2008) 2125-2133.
58 B. Nikolova-Damyanova, Retention of lipids in silver ion high-performance liquid chromatography: Facts and assumptions, J. Chromatogr. A 1216 (2009) 1815-1824.
Chapter 2
55
59 J.G. Bollinger, G. Rohan, M. Sadilek, M.H. Gelb, LC/ESI-MS/MS detection of FAs by charge reversal derivatisation with more than four orders of magnitude improvement in sensitivity, J. Lipid Res. 54 (2013) 3523-3530.
60 C. Pettinella, S.H. Lee, F. Cipollone, I.A. Blair, Targeted quantitative analysis of fatty acids in atherosclerotic plaques by high sensitivity liquid chromatography/tandem mass spectrometry, J. Chromatogr. B 850 (2007) 168-176.
61 W.-C. Yang, J. Adamec, F.E. Regnier, Enhancement of the LC/MS Analysis of Fatty Acids through Derivatisation and Stable Isotope Coding, Anal. Chem. 79 (2007) 5150-5157.
62 M. Eggink, M. Wijtmans, A. Kretschmer, J. Kool, H. Lingeman, I.P. Esch, W.A. Niessen, H. Irth, Targeted LC–MS derivatisation for aldehydes and carboxylic acids with a new derivatisation agent 4-APEBA, Anal. Bioanal. Chem. 397 (2010) 665-675.
63 U. Jahn, J.-M. Galano, T. Durand, Beyond Prostaglandins—Chemistry and Biology of Cyclic Oxygenated Metabolites Formed by Free-Radical Pathways from Polyunsaturated Fatty Acids, Angew. Chem. Int. Ed. 47 (2008) 5894-5955.
64 G.L. Milne, Sanchez, Stephanie C, Musiek, Erik S, Morrow, Jason D, Quantification of F2-isoprostanes as a biomarker of oxidative stress, Nat. Protoc. 2 (2007) 221-226.
65 W. Liu, J.D. Morrow, H. Yin, Quantification of F2-isoprostanes as a reliable index of oxidative stress in vivo using gas chromatography–mass spectrometry (GC-MS) method, Free Radical Biol. Med. 47 (2009) 1101-1107.
66 K. Kasuga, R. Yang, T.F. Porter, N. Agrawal, N.A. Petasis, D. Irimia, M. Toner, C.N. Serhan, Rapid Appearance of Resolvin Precursors in Inflammatory Exudates: Novel Mechanisms in Resolution, The Journal of Immunology 181 (2008) 8677-8687.
67 M. Giera, A. Ioan-Facsinay, R. Toes, F. Gao, J. Dalli, A.M. Deelder, C.N. Serhan, O.A. Mayboroda, Lipid and lipid mediator profiling of human synovial fluid in rheumatoid arthritis patients by means of LC–MS/MS, Biochim. Biophys. Acta 1821 (2012) 1415-1424.
68 R. Yang, N. Chiang, S.F. Oh, C.N. Serhan, Metabolomics-Lipidomics of Eicosanoids and Docosanoids Generated by Phagocytes, John Wiley & Sons, Inc., 2001.
69 S. Noble, D. Neville, R. Houghton, Determination of 8-iso-prostaglandin F2α (8-iso-PGF2α) in human urine by ultra-performance liquid chromatography–tandem mass spectrometry, J. Chromatogr. B 947–948 (2014) 173-178.
Chapter 2
56
70 A. Taylor, R. Bruno, M. Traber, Women and Smokers Have Elevated Urinary F2-Isoprostane Metabolites: A Novel Extraction and LC–MS Methodology, Lipids 43 (2008) 925-936.
71 R.C. Murphy, R.M. Barkley, K. Zemski Berry, J. Hankin, K. Harrison, C. Johnson, J. Krank, A. McAnoy, C. Uhlson, S. Zarini, Electrospray ionization and tandem mass spectrometry of eicosanoids, Anal. Biochem. 346 (2005) 1-42.
72 X. Liu, S.H. Moon, D.J. Mancuso, C.M. Jenkins, S. Guan, H.F. Sims, R.W. Gross, Oxidized fatty acid analysis by charge-switch derivatisation, selected reaction monitoring, and accurate mass quantitation, Anal. Biochem. 442 (2013) 40-50.
73 P. Dwivedi, C. Wu, L.M. Matz, B.H. Clowers, W.F. Siems, H.H. Hill, Gas-Phase Chiral Separations by Ion Mobility Spectrometry, Anal. Chem. 78 (2006) 8200-8206.
74 L. Kortz, J. Dorow, U. Ceglarek, Liquid chromatography-tandem mass spectrometry for the analysis of eicosanoids and related lipids in human biological matrices. A review, J. Chromatogr. B. doi: 10.1016/j.jchromb.2014.01.046
75 J.G. Bollinger, W. Thompson, Y. Lai, R.C. Oslund, T.S. Hallstrand, M. Sadilek, F. Turecek, M.H. Gelb, Improved Sensitivity Mass Spectrometric Detection of Eicosanoids by Charge Reversal Derivatisation, Anal. Chem. 82 (2010) 6790-6796.
76 C. Thomas, Pellicciari, Roberto, Pruzanski, Mark, Auwerx, Johan, S. , Kristina, Targeting bile-acid signalling for metabolic diseases, Nat. Rev. Drug Discov. 7 (2008).
77 G. Porez, J. Prawitt, B. Gross, B. Staels, Bile acid receptors as targets for the treatment of dyslipidemia and cardiovascular disease: Thematic Review Series: New Lipid and Lipoprotein Targets for the Treatment of Cardiometabolic Diseases, J. Lipid Res. 53 (2012) 1723-1737.
78 W.J. Griffiths, J. Sjövall, Analytical strategies for characterization of bile acid and oxysterol metabolomes, Biochem. Biophys. Res. Commun. 396 (2010) 80-84.
79 W.J. Griffiths, J. Sjövall, Bile acids: analysis in biological fluids and tissues, J. Lipid Res. 51 (2010) 23-41.
80 M. Maekawa, M. Shimada, T. Iida, J. Goto, N. Mano, Tandem mass spectrometric characterization of bile acids and steroid conjugates based on low-energy collision-induced dissociation, Steroids 80 (2014) 80-91.
81 J. Ding, E.T. Lund, J. Zulkoski, J.P. Lindsay, D.L. McKenzie, High-throughput bioanalysis of bile acids and their conjugates using UHPLC coupled to HRMS, Bioanalysis 5 (2013) 2481-2494.
Chapter 2
57
82 S.P.R. Bathena, S. Mukherjee, M. Olivera, Y. Alnouti, The profile of bile acids and their sulfate metabolites in human urine and serum, J. Chromatogr. B 942–943 (2013) 53-62.
83 J. Huang, S.P.R. Bathena, I.L. Csanaky, Y. Alnouti, Simultaneous characterization of bile acids and their sulfate metabolites in mouse liver, plasma, bile, and urine using LC–MS/MS, J. Pharm. Biomed. Anal. 55 (2011) 1111-1119.
84 S. Devkota, Y. Wang, M.W. Musch, V. Leone, H. Fehlner-Peach, N. A., D.A. Antonopoulos, B. Jabri, E.B. Chang, Dietary-fat-induced taurocholic acid promotes pathobiont expansion and colitis in Il10-/- mice, Nature 487 (2012) 104-108.