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

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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

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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

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Bo

ron

tri

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(BF 3

) A

lco

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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

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ch

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de

(–S-

(–)-

α-m

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(tri

flu

oro

met

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enyl

acet

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hlo

rid

e)

Hyd

roxy

lam

ine

Met

ho

xyla

min

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Re

acti

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Trim

eth

ylsi

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tio

n

t-b

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ldim

eth

ylsi

lyla

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Met

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est

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form

atio

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Este

rifi

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on

DM

OX

Trim

eth

ylsi

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n

Met

hyl

eth

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orm

atio

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Ch

iral

der

ivat

isat

ion

Oxi

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Fun

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gro

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Car

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Ke

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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

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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

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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.

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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.

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50

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