Analysis of biologically-active, endogenous carboxylic acids based on chromatography-mass spectrometry

Analysis of biologically-active endogenous carboxylic acids based onchromatography-mass spectrometryD Kloos ab H Lingeman b OA Mayboroda a AM Deelder a WMA Niessen bcM Giera aa Center for Proteomics and Metabolomics Leiden University Medical Center (LUMC) Albinusdreef 2 2300RC Leiden The Netherlandsb AIMMS Division of BioAnalytical Chemistry VU University Amsterdam De Boelelaan 1083 1081 HV Amsterdam The Netherlandsc Hyphen MassSpec de Wetstraat 8 2332 XT Leiden The Netherlands

A R T I C L E I N F O

KeywordsAnalysisBile acidCarboxylic acidEicosanoidFatty acidGas chromatographyGC-MSLC-MSLiquid chromatographyMass spectrometry

A B S T R A C T

Carboxylic acids (CAs) such as small CAs fatty acids eicosanoids and bile acids are involved in numer-ous biological processes Mapping the participating pathways potentially yields great insight into the phys-iological state of an organism There is demand for analytical techniques that are more sensitive fasterand easier to handle These would enable advanced analysis and validation in larger clinical cohorts andhopefully deliver useful disease markers The analysis of CAs is not only driven by the CA group but alsolargely dependent on chain length and other functional groups present Several liquid and gas chroma-tography strategies with or without derivatization were developed in recent years Here we review themost recent trends in analysis of endogenous CAs by chromatography-mass spectrometry We criticallyevaluate sample preparation derivatization techniques separation and mass spectrometric detection Ul-timately the reader is guided to the most versatile sensitive and easy analytical methods

copy 2014 Elsevier BV All rights reserved

Contents

1 Introduction 182 General aspects 18

21 GC-MS analysis 1822 LC-MS analysis 1923 Chiral analysis using GC-MS and LC-MS 2024 Sample pretreatment 2025 Use of internal standards 20

3 Small carboxylic acids with fewer than six carbon atoms 2031 GC-MS analysis of small carboxylic acids 2132 LC-MS analysis of small carboxylic acids 22

Abbreviations 4-APC 4-(2-((4-Bromophenethyl)dimethylammonio)ethoxy)benzenaminium dibromide 9-HoDE 9-hydroxy-10E12Z-octadienoic acid AMPP N-(4-aminomethylphenyl)pyridinium APCI atmospheric-pressure chemical ionization BA bile acid BHT butylated hydroxytoluene BMP 2-Bromo-1-methylpyridinium iodideBSA bis(trimethylsilyl)acetamide BSTFA NO-Bis(trimethylsilyl) trifluoroacetamide CA carboxylic acid CACI covalent adduct chemical ionization CID collision-induceddissociation COX cyclooxygenase CSF cerebrospinal fluid DMOX 44-Dimethyloxazoline CYP cytochrome P450 DMS dimethyl sulfate ECNI electron-capture negativeionization EDC 3-(Ethyliminomethyleneamino)-NN-dimethyl-propan-1-amine hydrochloride salt EDTA ethylenediaminetetraacetic acid EI electron ionization ESI electrosprayionization FA fatty acids FAME fatty acid methyl ester f-FA free fatty acid GC gas chromatography HILIC hydrophilic interaction liquid chromatography HPAEC high-performance anion-exchange chromatography HRMS high-resolution mass spectrometry IMS-MS ion mobility spectrometry-mass spectrometry IS internal standardIT ion trap LC liquid chromatography LLE liquid-liquid extraction LOX lipoxygenase MIRACLE mass isotopomer ratio analysis of uniformly-[13Cn]-labeled extracts MPEAN-methyl-2-phenylethanamine MS mass spectrometry MSTFA N-Methyl-N-(trimethylsilyl) trifluoroacetamide MtBSTFA N-methyl-N-t-butyldimethylsilyl trifluoroacetamidePFBBr pentafluorobenzyl bromide PUFA poly-unsaturated fatty acid Q-LIT quadrupole-linear ion trap RPLC reversed-phase liquid chromatography SIL stable-isotopelabel SIM selected ion monitoring SPE solid-phase extraction SRM selected reaction monitoring TBA tetrabutylamine t-BDMS tert-Butyldimethylsilyl TCA tricarbox-ylic acid TFA trifluoracetate t-FA total fatty acids TMAE trimethylaminoethyl TMS trimethylsilyl TMSH trimethylsulfonium hydroxide TMCS trimethylchlorosilaneTQ tandem quadrupole UHPLC ultra-high-performance liquid chromatography ZIC zwitterionic chromatography

Corresponding author Tel +31 71 526 9527 fax +31 71 5266907E-mail address magieralumcnl (M Giera)

httpdxdoiorg101016jtrac2014050080165-9936copy 2014 Elsevier BV All rights reserved

Trends in Analytical Chemistry 61 (2014) 17ndash28

Contents lists available at ScienceDirect

Trends in Analytical Chemistry

journal homepage wwwelseviercom locate t rac

4 Fatty acids 2241 GC-MS analysis of fatty acids 2242 LC-MS analysis of fatty acids 23

5 Eicosanoids 2351 GC-MS analysis of eicosanoids 2452 LC-MS analysis of eicosanoids 24

6 Bile acids 2561 GC-MS analysis of bile acids 2562 LC-MS analysis of bile acids 25

7 Conclusion and perspectives 25References 26

1 Introduction

One of the most interesting developments in the biomedical sci-ences of the post-genomic era is a ldquoparadigm shiftrdquo in our under-standing of the biologicalregulatory activity of the chemical entitiesfor which such a function was disregarded The compounds that tra-ditionally were seen as only constituents of energy metabolism [egpyruvate succinate fatty acids (FAs)] or emulsifiers of lipids [bileacids (BAs)] are being more and more recognized as importantimmune-modulatory andor signaling molecules In the first ap-proximation all those compounds appear to be far too structural-ly diverse to be reviewed together but taking a broader view werealize that most of those ldquoupcoming regulatory compoundsrdquo areendogenous carboxylic acids (CAs) Their common functional fea-tures are the presence of a CA function dictating many but not allof their physicochemical properties and an alkyl part that can bedecorated with different functional groups One can distinguish fourimportant sub-classes of endogenous CAs namely

(1) small CAs crucial to aerobic respiration and energy metab-olism [1]

(2) FAs fundamental to energy storage and membrane forma-tion and involved in numerous physiological processes suchas inflammation [2ndash4]

(3) eicosanoids and docosanoids forming a class of very impor-tant signaling molecules during several inflammatory and im-munological events [5] and

(4) BAs the main metabolites of endogenous cholesterol [6] (seeFig 1 for their general structures)

In this article we discuss recent advances in analytical tech-niques for the analysis of CAs

All of these analytes reside in the body and can be quantita-tively analyzed in one of the body fluids or in cellular extracts Theirisolation and separation from matrix components is fundamentalto their analysis While sample preparation can be distinctly dif-ferent for the four CA sub-classes analyte separation is commonlyperformed by either gas chromatography (GC) or liquid chroma-tography (LC) Both separation techniques are frequently com-bined with mass spectrometric (MS) detection for the highestselectivity and sensitivity in quantitative analysis [7]

Both GC-MS and LC-MS feature common requirements due tothe functional groups present in the analytes of interest Analysisof endogenous CAs is done with different aims eg

bull (I) qualitative profiling of CAs present thus involving structureelucidation and resolving isomerism issues

bull (II) relative quantitation for comparison of physiological states andor flux determination involving the use of isotopologues and

bull (III) targeted (absolute) quantitative analysis

The aims of the study determine the analytical strategy chosenbut in all cases general issues in the analysis of CAs are also im-portant [8]

2 General aspects

All four analyte sub-classes share the presence of a CA func-tion (ie they are weak organic Broslashnsted-Lowry acids) Howeverother features shared by the CAs might also have significant effectson the analytical process and thus demand rather similar analyti-cal solutions Fig 2 shows the most prominent functional groupsthat may be present in an endogenous CA

21 GC-MS analysis

Intrinsically GC-MS analysis demands the evaporation of theanalyte under investigation prior to separation and detection Withrespect to CAs several functional groups demand attention Firstthe CA function has to be derivatized for successful GC-MS analy-sis From the various possible derivatization strategies silylation andesterification of the CA function are the most prominent as the re-action usually proceeds smoothly and particularly in the case ofsilylation requires no sample clean-up before injection Other crucialfunctional groups (Fig 2) that have to be derivatized prior to GC-MS analysis are (A) hydroxyl groups which can either undergosilylation or ether formation and (B) ketone groups which are fre-quently converted into oximes thereby blocking possible tautom-erism during subsequent derivatization and stabilizing β-ketoCAs particularly After derivatization the CA derivatives canbe analyzed in routine GC-MS systems involving commondimethylsiloxane-coated capillary columns and wide temperaturegradients Generally analyte ionization is achieved by electron

Fig 1 General structures of the four relevant classes of carboxylic acids (CAs) dis-cussed in this article small CAs fatty acids eicosanoids and bile acids

18 D Kloos et alTrends in Analytical Chemistry 61 (2014) 17ndash28

ionization (EI) although electron-capture negative ionization (ECNI)has also been used

Functional groups at the side chain in many cases lead to theformation of a stereo-center In these cases either chiralderivatization yielding diastereomers or the use of chiral GC columnsform possible solutions [9] (see sub-section 23) The degree ofunsaturation may influence GC-MS analysis in different ways Com-pounds with higher degrees of unsaturation are more prone to au-toxidation Fragmentation of highly unsaturated CAs in EI is mainlydriven by double bonds limiting the usefulness of the spectra ob-tained [10] Table 1 summarizes the most important derivatizationstrategies for CAs in GC-MS analysis Silylation reagents react par-ticularly readily with water possibly affecting reaction speed andyield Hence derivatization should be carried out under nearly an-hydrous conditions [12] Other points of concern are the stabilityof the derivatives and possible artifact formation or isomerizationduring derivatization (see also sub-section 41)

22 LC-MS analysis

The most widely applied LC mode for CAs is ion-suppressedreversed-phase LC (RPLC) using methanolwater or acetonitrile(ACN)water gradients frequently using acetic or formic acid asadditives In LC-MS underivatized CAs must be analyzed in negative-ion mode using electrospray ionization (ESI) or atmospheric-pressure chemical ionization (APCI) In some LC-MS instrumentsnegative ionization seems to be less efficient than positive ioniza-tion Furthermore most CA sub-classes do not provide readily ap-plicable intense fragment ions upon collision-induced dissociation(CID) to be applied in the selected-reaction monitoring (SRM) modeQuantitative analysis using LC-MS is mostly performed in SRM modeusing tandem-quadrupole (TQ) or quadrupole-linear-ion-trap hybridinstruments (QTRAP or Q-LIT) The latter also provides enhancedfull-spectrum sensitivity in MS-MS Underivatized CAs upon CID mayprimarily show the loss of CO2 ie [M-H-CO2]- and of H2O from

alcohol or ketone functions in the molecule if present Other frag-mentation of CAs is largely influenced by functional groups (egdouble bonds present in the alkyl part of the molecule) As a rule

Fig 2 Overview of relevant functional groups that may be present and influence the analysis of endogenous CAs

Table 1Overview of important functional groups in endogenous CAs that require derivatizationin GC-MS and widely applied derivatization strategies and reagents for this [11]

Functionalgroup

Reaction Derivatization reagent

Carboxylicacid

Trimethylsilylation N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA)Bis(trimethylsilyl)acetamide (BSA)NO-Bis(trimethylsilyl)trifluoroacetamide (BSTFA)Trimethylchlorosilane (TMCS)

t-butyldimethylsilylation N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide (MtBSTFA)

Methyl ester formation Trimethylsulfonium hydroxide(TMSH)Dimethyl sulfate (DMS)Methanolinorganic acidMethanolAcyl chlorideMethanolBoron trifluoride (BF3)

Esterification Alcoholinorganic acidAlcoholBF3

Pentafluorobenzyl bromide (PFBBr)Picolinyl esters (3-Pyridylcarbinolesters)

Dimethyloxazolineformation

44-dimethyloxazoline(DMOX)

Hydroxylgroup

TrimethylsilylationMethyl ether formation

MSTFA BSTFA BSA TMCSDiazomethane

RSisomerism

Chiral derivatization Mosherrsquos acid chloride(ndashS-(ndash)-α-methoxy-α-(trifluoromethyl)phenylacetylchloride)

Ketonegroup

Oximation HydroxylamineMethoxylamine

19D Kloos et alTrends in Analytical Chemistry 61 (2014) 17ndash28

of thumb the higher the degree of unsaturation and the higher thenumber of functional groups present in a CA the easier is its frag-mentation and the easier is the formation of fragments not relatedto the loss of CO2 [13]

Although for LC-MS analysis of CAs derivatization is not alwaysnecessary there are a number of reasons also to performderivatization of CAs in LC-MS [11] Derivatization may facilitate theRPLC separation of CAs and may also direct fragmentation and yieldother characteristic neutral losses related to the derivatizationreagent applicable in SRM Two in-depth reviews aboutderivatization in LC-MS were recently presented [1415] Table 2 givesan overview of the most common LC-MS derivatization reagents forendogenous CA analysis Most derivatization reactions for LC-MStarget the CA functional group frequently with the ultimate aim offorming amide derivatives for positive-ion ESI After activation ofthe CA functional group usually with triphenylphosphine or viacarbodiimide chemistry amide formation is readily accomplishedby adding an amine-containing derivatization reagent Withcarbodiimide activation one preferably uses water-soluble re-agents such as (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)(EDC) and catalysts such as (1-hydroxybenzotriazol) (HoBt) In otherderivatization strategies the presence of water may influence theapplied chemistry In most cases a (large) excess of reagents is nec-essary somewhat depending on the sample matrix Then the excessunused reagent has to be removed prior to LC-MS analysis (eg bythe use of divert valves or on-line SPE)

23 Chiral analysis using GC-MS and LC-MS

Molecular chirality can have tremendous biological impor-tance eg involving D-lactic acid which may cause the so-calledD-lactic acidosis that is a neurological syndrome occurring in in-dividuals with short-bowel syndrome [16] Another prominentexample is the S-enantio-selectivity of mammalian LOX enzymes[17] (see also Section 4) Chiral separation of CAs using GC or LCcan be achieved by derivatization using chiral reagents resultingin the formation of diastereomers or using chiral stationary phasesSeparation of diastereomers is most efficient if the chiral centersare in close proximity [12] so in designing the derivatization it iscrucial to assess whether the CA function or another functional groupis to be targeted Several chiral amines were recently compared fortheir use in chiral CA analysis by LC-MS [18] The derivatization ofN-methyl-DL-aspartic acid using S-octanol as chiral reagent mayserve as an example for a GC-based chiral separation [19] Severalchiral stationary phases have been developed and successfullyapplied in LC-MS or GC-MS analysis of CAs The use of cyclodextrin-based columns was recently reviewed [20] The GC separation of2-bromo-substituted CA ester enantiomers may serve as an example

[21] For LC there is a wide variety of chiral columns [22] whichare used in normal-phase (NP) or RP mode An example is the chiralRPLC separation of resolvins (being essentially hydroxylated PUFA)using amylose tris(35-dimethylphenylcarbamate)-coated ChiralPakAD-RH columns [13] It is important to note that chiral separa-tions do not allow determination of absolute stereochemical con-figurations unless enantiopure standards are available the elutionorder of R- and S-enantiomers cannot usually be predicted An in-teresting approach to determination of the absolute stereochem-istry of hydroxylated CAs particularly when vibrational circulardichroism has been unsuccessful [23] involves the use of Mosherrsquosacid chloride derivatization in combination with NMR [24]Alternatively gas-phase chiral separation using ion-mobilityspectrometry-MS (IMS-MS) might have considerable impact onfuture applications [25]

24 Sample pretreatment

All sub-classes of CAs may be analyzed in body fluids [eg plasmaserum urine and cerebrospinal fluid (CSF)] while especially smallCAs may also be analyzed in cellular extracts For GC-MS analyteextraction to an organic solvent is required prior to derivatizationThis can be achieved by (ion-suppressed) liquid-liquid extraction(LLE) using solvents (eg ethyl acetate or n-hexane) For LC-MSprotein precipitation is performed for blood-related samples even-tually followed by a sample clean-up step using LLE or SPE usingRPLC or mixed-mode materials Removal of endogenous phospho-lipids is important to reduce matrix effects in LC-MS The combi-nation of highly polar groups and hydrophobic alkyl chains maypresent challenges to analyte recovery in LLE or SPE For urine anal-ysis dilute-and-shoot procedures are used [26] or clean-up usingLLE or SPE is performed In the analysis of small CAs in cellular ex-tracts quenching of the cellular metabolism is important to avoidany further biochemical reaction during the sampling period re-quiring specialized protocols [2728]

25 Use of internal standards

If absolute quantitation is to be achieved use of stable-isotope-labeled (SIL) internal standards (ISs) is crucial for both GC-MS andLC-MS analysis of CAs Although [Dn]-labeled ISs can be used thereis a risk of DH exchange during sample pretreatment in acidic oralkaline media [29] so [13Cn]-labeled ISs are generally preferredSIL-ISs can be produced by organic synthesis In the analysis of cel-lular extracts the use of mass isotopomer ratio analysis of uniformly-[13Cn]-labeled extracts (MIRACLE) [3031] based on the biosynthesisof SIL-ISs in yeast-cell cultures grown on [13C6]-glucose is a pow-erful tool Other approaches involve stable-isotope coding byderivatization [32] or quantification by standard addition

3 Small carboxylic acids with fewer than six carbon atoms

The sub-class of small CAs consists of short-chain FAs (le6 carbonsin the aliphatic tail) and their hydroxylated andor ketone contain-ing analogues [18] Mono- di- and tri-CAs are among this groupProminent examples of this sub-class are the intermediates of theKrebsrsquos or the tricarboxylic acid (TCA) cycle and important clinicalmarkers D-lactic acid [16] and methylmalonic acid [8] (see Fig 3)Recent interest in small CAs was largely boosted by translationalresearch into metabolic phenomena such as the Warburg effect [33]and autophagy [1] so the current interest in the analysis of smallCAs is likely to grow General pitfalls include their low molecularweight their high polarity their limited stability leading to chal-lenges in their extraction from aqueous matrices and for some vol-atility issues [134] The importance of these issues depends on theanalytical technique applied There is a general stability problem

Table 2Overview of important functional groups in endogenous CAs that have been tar-geted by derivatization approaches [1415]

Functionalgroup


Carboxylicacid

AmidationEsterification

N-Methyl-2-phenylethanamide (MPEA)4-(2-((4-bromophenethyl)dimethylammonio)ethoxy)benzenaminiumdibromide (4-APEBA)N-(4-aminomethylphenyl)pyridinium(AMPP)2-Hydrazinopyridine (HP)2-Picolylamine (PA)

Esterification Three step derivatization resulting intrimethylaminoethyl ester derivatives(TMAE)

Hydroxylgroup

Sulfonic acid esterformation

Dansyl chloride


particularly for β-keto CAs [3536] which readily undergo decar-boxylation as a preferred six-membered transition state can beformed [37]

31 GC-MS analysis of small carboxylic acids

Derivatization is crucial for successful GC-MS analysis of smallCAs Esterification of small CAs with small alcohols is generally un-successful due to the high volatility of such derivatives Larger al-cohols could be used but generally require rather harsh reactionconditions involving catalysts such as anhydrous sulfuric acid orboron trifluoride [38] In this respect silylation seems to be a betterchoice as less volatile higher molecular-weight derivatives areformed

As most modern analytical strategies tend to move more andmore towards comprehensive multi-component analysis thederivatization protocols to be used have to become increasinglygeneric [39] A frequently applied approach consists of a combina-tion of oximation and silylation Oximation using methoxylamineor hydroxylamine derivatizes the ketone groups if present whereastrimethylsylilation or t-butyldimethylsilylation [3940] modifies boththe hydroxyl and the CA groups into trimethylsilyl (TMS) andt-butyldimethylsilyl (tBDMS) derivatives respectively Typical re-action conditions comprise oximation in a solution of the corre-sponding alkoxylamine hydrochloride in pyridine at a concentrationof typically 20 mgmL at 30degC for 90 min followed by silylation usingMtBSTFA at 70degC or MSTFA at 37degC for 30 min [40]

In EI-MS TMS derivatives yield abundant [M-CH3bull]+ ions with mz

M+bull-15 as well as relatively abundant non-specific ions with mz73 due to [(CH3)3Si]+ and mz 75 due to [(CH3)2Si=OH]+ The mostprominent fragment of the tBDMS derivatives in EI-MS is usually[M-(CH3)3Cbull]+ with mz M+bull-57 together with some low-abundancefragments [41] Whereas the derivatization of CAs using MtBSTFAis a straightforward reaction usually proceeding rapidly [41] thereaction of hydroxyl groups with MtBSTFA is less favorable so it maylead to partial derivatization and skewing of the results even if acatalyst such as ammonium iodide and elevated temperatures areused [42]

Prior to derivatization the (highly) hydrophilic small CAs shouldbe extracted from the usually aqueous sample matrix into an ap-propriate organic solvent (eg diethyl ether) Given the volatilityof some CAs and the limited stability of the keto CAs tempera-tures should be kept as low as possible throughout the sample-pretreatment procedure

Generic protocols were recently described for extraction fromcellular incubations and body fluids Extraction of small CAs amongother cellular metabolites from mammalian cells involves quench-ing using liquid nitrogen and extraction using a methanolchloroformmixture [43] For body fluids such as plasma or urine quenchingof the metabolic reactions is generally less of a concern Protein pre-cipitation with methanol [44] has become the gold standard forwide-range analysis of low-molecular-weight analytes includingsmall organic CAs in plasma eventually in combination with SPEif the aim is more targeted methods for a limited number of analytes

Fig 3 Structures of important small carboxylic acids (CAs)


For urine analysis the sample pretreatment usually comprises ofan eventual urease step followed by freeze drying reconstitutionin an organic solvent and derivatization [39]

Metabolic fluxes in melanoma cell lines using oximation andtBDMS ester formation were recently investigated by Scott et al [45]

The effects of valproic acid in children were studied by urinaryanalysis of small CAs [46] Urine samples were directly oximatedusing hydroxylamine and sodium hydroxide the analytes ex-tracted by LLE and further derivatized by silylation using BSTFA priorto GC-MS analysis

32 LC-MS analysis of small carboxylic acids

LC seems to be the method of choice for the analysis of smallCAs which are highly polar compounds At first no derivatizationseems to be required In practice the situation is somewhat morecomplicated In RPLC the small CAs generally show insufficient re-tention so the use of ion-pairing agents like tetrabutylamine (TBA)has been proposed [47] However this leads to substantial ioniza-tion suppression in ESI-MS and is detrimental to the equipment used[36] As an alternative methods based on hydrophilic interactionchromatography (HILIC) [48] or high-performance anion-exchangechromatography (HPAEC) [49] have been proposed In the latter casepost-column electrolytic suppressors are required for the removalof the high salt concentrations applied [50] Performance compar-ison of various column chemistries for HILIC eg aminopropylamide cyano diol or silica [51] and their comparison with RPLCfor the analysis of small CAs have been reported [4852] Al-though HILIC with aminopropyl or diol columns appears to be mostsuccessful it seems difficult to select an LC phase system especial-ly directed at small CAs

The problems indicated in both LC separation and MS detec-tion lead to reconsidering pre-column derivatization of the smallCAs [53] The use of N-methyl-2-phenylethanamine (MPEA) aftercarbodiimide activation has been applied to TCA-cycle intermedi-ates [34] The analytes were derivatized with EDC and MPEA at 60degCfor 45 min in 90 ACN After dilution with water the sample couldbe directly analyzed by on-line SPE-LC-MS Even thoughderivatization might advance the analysis of small CAs the insta-bility of the keto CAs in particular might hamper successful anal-ysis of these species

For sample pretreatment aimed at LC-MS protocols are usedsimilar to those for GC-MS involving freeze drying with urine proteinprecipitation with plasma and eventually complemented by SPE[3954] In the analysis of cellular metabolites combined quench-ing and extraction methods are needed [55]

A recent example identifying succinate as an inflammatory signalin innate immunity was reported by Tannahill et al [56] The authorsapplied several LC-MS platforms with different HILIC separationsfor succinate analysis one based on a zwitterionic (ZIC) HILIC columnwas used

4 Fatty acids

FAs are mono-CAs with a long-chain aliphatic end In mammalsstraight-chain FAs normally with an even carbon number are ob-served whereas in bacteria branched alkyl chains andor higherlevels of odd carbon-numbered FAs also occur One distinguishesshort-chain FAs (le6 C atoms (ie the small CAs in this article Section3) medium-chain FAs (6ndash12 C atoms) long-chain FAs (12ndash22 Catoms) and very-long-chain FAs (gt22 C atoms) The aliphatic chainmay contain several double bonds

FAs with a degree of unsaturation of two or higher are fre-quently called poly-unsaturated FAs (PUFAs) Each double bond maybe E or Z (trans or cis) a PUFA with three double bonds could the-oretically form eight EZ-isomers PUFAs formed biochemically

usually show all-Z (all-cis) configurations The ω(n)-nomenclatureis applied to indicate the position of the first double bond relativeto the aliphatic end rather than relative to the CA end (IUPAC)

Besides double bonds FAs might also contain ketone hydroxylhydroperoxide epoxide and other functional groups Each of thesefunctionalities puts specific demands on the analytical strategieswhich we cannot discuss in detail here We focus on FAs keto FAsand mono-hydroxylated FAs the last of these being the biochem-ical precursors of certain eicosanoids and docosanoids [4] Hy-droxyl groups usually lead to a stereo-center in the FA side chainbiochemically-formed hydroxylated FAs normally pose theS-configuration whereas autoxidation products are racemic mix-tures Oxidative stability is a major concern in PUFA analysis [57]Until recently FAs were primarily analyzed by GC-MS but cur-rently LC-MS methods are also frequently reported

Depending on the application free FA (f-FA) or total FA (t-FA)content is to be determined f-FA determination requires an appro-priate extraction method (eg using LLE with n-hexane i-octaneor a similar solvent) without affecting the FAs bound in triglycer-ides (phospholipids and other storage forms) or bound to eg pro-teins For t-FA determination a saponification step must beperformed mostly under alkaline conditions Care must be takento avoid autoxidation and double-bond isomerization Because ofthe risk of DH-exchange [Dn]-SIL-ISs can be added only after sa-ponification [29] Saponification and extraction can be combinedwith esterification in a process called transesterification which iscarried out by acid-catalyzed methylation usually by using meth-anol hexane and acetyl chloride [58] thus yielding FA methyl esters(FAMEs) which can be analyzed by GC-MS

41 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 derivatizationdirectly or after drying under a stream of nitrogen or in a SpeedVac[59] As for small CAs the most favorable derivatization methodsare esterification and silylation [11]

The formation of FAMEs is the most prominent derivatizationstrategy for GC-MS [60] While PFBBr and silyl-ester derivatives arefrequently separated on standard phenyl-polysiloxane columnscyanopropyl polysilphenylsiloxane columns have become the stan-dard GC columns for FAME analysis A recent application involvedacetylchoride-based transesterification incubating the samples over-night at room temperature thereby overcoming acid-induced EZisomerization and separation of positional and geometrical FAMEisomers [60]

However FAMEs tend to provide excessive fragmentation in EI-MS with the ion with mz 74 ie [CH2C(OH)OCH3]+bull resulting froma McLafferty rearrangement being the most abundant ion [10] Asthe ion with mz 74 is a class-specific and not a compound-specificfragment it cannot be used in isotopologue analysis (13C-flux de-termination) as most of the molecular information is lost Fig 4shows a comparison of three different FA derivatives and their be-havior during GC-EI-MS analysis [62]

A number of derivatization strategies have been described [61]such as formation of TMS or tBDMS derivatives [41] including a re-cently introduced sequential one-pot application of both reagentsfor the comprehensive analysis of FAs and sterols [62] picolinyl esters[63] and DMOX [64] derivatives The latter can also be used fordouble-bond localization and branching analysis [64] Derivatizationusing PFBBr enables the use of ECNI in GC-MS which provides highlyselective and mild ionization ie dissociative electron capture togenerate predominantly [M-PFB]ndashions without much further frag-mentation thus facilitating isotopologue analysis [65]

Recently an overview of the use of stable isotopes in studyinglipid metabolism was published [66] High-resolution GC is crucial


for the differentiation of EZ isomers [67] Another important topicis the determination of double-bond positions which can be achievedin different ways eg specific derivatization agents such as picolinylesters or DMOX derivatives [68] from careful interpretation of thefragmentation observed in EI mass spectra [10] or using covalent-adduct chemical-ionization tandem MS (CACI-MS-MS) using ACNin IT instruments [69]

42 LC-MS analysis of fatty acids

General interest in LC-MS especially the introduction of ultra-high-performance LC (UHPLC) has boosted developments in FA anal-ysis by LC-MS rather than GC-MS [70] Unless derivatization isperformed FAs are analyzed as [M-H]- in negative-ion mode usingESI or APCI Upon CID little fragmentation is observed for satu-rated FAs and minor losses of CO2 for PUFAs [13] SIM or SRM istherefore used with the same mz for both precursor and production thus attempting to at least fragment possible co-eluting iso-baric species [71] Post-column addition of Ba2+ was reported to gen-erate [M-H + Ba]+-ions which readily undergo charge-remotefragmentation of the alkyl chain providing specific fragment ionsfor SRM [72]

In this way f-FAs can be analyzed in the low-nM range eg afterMeOH protein precipitation for plasma [13] An interesting exampleis the analysis of 36 f-FAs in human plasma using a calibration setof known FAs to enable identification and quantification of unknownf-FAs The method made use of the SRM procedure described above[71] and expanded the concept even further measuring not only aquantification trace but also what the authors called a differentialenergy qualifier ion ratio for the identification of co-eluting

impurities The method showed lower limits of quantification in thenM range with run times below 10 min [13] Transesterification pro-cedures applied to determine t-FA yield FAMEs which show poorionization characteristics in ESI and APCI Mostly RPLC is used forthe separation of FAs Separation of FAs on Ag+-loaded columns pro-vides enhanced resolution of FAs with different EZ isomers anddouble-bond positions eventually in combination with ozonoly-sis [73] An application by Joacutenasdoacutettir et al combined capillary LCand IT-MS3 to characterize hydroxylated FAs in intact phospholip-ids without previous hydrolysis [74]

As with small CAs quantitative performance may be enhanced byderivatization to enhance the ionization efficiency or to implement frag-mentation characteristics for SRM An improvement in sensitivity bya factor of 60000 compared to the analysis of underivatized FAs hasbeen claimed for N-(4-aminomethylphenyl)pyridinium (AMPP) de-rivatives of FAs introducing a permanent charge [75] Otherderivatization strategies involve eg trimethylaminoethyl (TMAE) [76]2-bromo-1-methylpyridinium iodide (BMP) [77] MPEA [34] and 4-(2-((4-bromophenethyl)dimethylammonio)ethoxy)benzenaminiumdibromide (4-APC) [78]

Derivatization techniques for FA analysis using LC-MS were re-cently reviewed [53] Carbodiimide coupling using EDC in combina-tion with AMMP derivatization and stable-isotope coding was appliedin the analysis of t-FAs in human-serum samples [77] The derivativeswere separated on a C4 column using an ACNwater gradient

5 Eicosanoids

Phospholipases release mainly 20-carbon PUFAs from membrane-phospholipids Eicosanoids are the enzymatic oxidation products

Fig 4 Overlaid extracted ion chromatograms of the characteristic fragments of the derivatives of FA181(Z) with (A) mz 2642 for the methyl ester (B) mz 3392 for theTMS derivative and (C) mz 3392 for the tBDMS derivative The respective EI spectra (70 eV) obtained are shown on the right hand side AndashC Reprinted with permissionfrom [61] copy2014 John Wiley amp Sons Ltd


of these PUFAs generated by enzymes from the cyclooxygenase(COX) cytochrome P450 (CYP) and lipoxygenase (LOX) families [5]Typical eicosanoids are arachidonic acid (FA 204)-derived prosta-glandins and leukotrienes Isoprostanes are closely relatedeicosanoids generated by non-enzymatic oxidation of FA 204 [79]Many eicosanoids mediate critical biological effects (eg chemo-taxis blood clotting or broncho-constriction) Particularly during in-flammatory processes prostaglandins and leukotrienes derivedfrom FA 204 are important in the initial phase [4] whereaseicosapentaenoic acid-derived mediators play a crucial role in theactive resolution phase of inflammation [4] In addition the familyof 22-carbon PUFA-derived docosanoids comprise related highlyactive mediators [8] The biological activity of the eicosanoids andrelated compounds strongly relies on stereo positional and geo-metrical isomerism [4]

Artificial eicosanoids may be formed by oxidation of FA 204which is present at high levels in human body fluids such as plasmaor by activation of platelets during venipuncture To avoid errorsin analysis it is important to use ion chelators such as EDTA to freezesamples immediately at -80degC and to consider the use of antioxi-dants such as butylated hydroxytoluene (BHT) andor enzyme in-hibitors such as indomethacine [57]

51 GC-MS analysis of eicosanoids

Multistep derivatization is required to achieve compatibility ofeicosanoids with GC-MS analysis The gold standard is a combinationof trimethylsilylation of hydroxyl groups oximation of the ketone groups(if necessary) and PFBBr derivatization of the CA group thus en-abling selective and sensitive analysis using ECNI in GC-MS [80] A pro-tocol for the assessment of F2-isoprostanes as markers of oxidative stressin vivo has been reported [81] Following PFBBr-ester formation sampleclean-up by thin-layer chromatography and silylation with BSTFA anal-ysis is performed by ECNI in GC-MS Particularly for structural confir-mation purposes GC-MS with EI fragmentation after diazomethanederivatization is still an important tool [82]

52 LC-MS analysis of eicosanoids

The sample pretreatment protocol for GC-MS involving a two(three)-step derivatization is obviously quite laborious and that

explains why LC-MS analysis is frequently applied instead [67]LC-MS allows the analysis of underivatized compounds greatlyfacilitating sample pretreatment and minimizing possible analytelosses

An important challenge in eicosanoid analysis is resolution ofthe large number of possible stereoisomers and EZ isomers (egleukotriene B4 contains four double bonds and two stereo-centersso it can thus theoretically exist as 64 different isomers) The highseparation efficiency achievable using UHPLC with columns packedwith small porous or solid-core particles (lt2 μm) and excellentretention-time stability are of the utmost importance especiallybecause differentiation based on fragmentation in MS-MS is notalways possible [83] This is illustrated for the detection of 9-hydroxy-10E12Z-octadienoic acid (9-HoDE) in human plasma in Fig 5 Thealmost co-eluting unknown peak 2 most probably represents the10E12E-isomer [85]

As eluent systems in RP separations MeOHwater ACNwaterand mixtures thereof have been described Given the impact of ste-reoisomerism and EZ isomerism on their biological activity chiralseparation of eicosanoids can be of considerable concern [84] Asthe elution order of enantiomers cannot be predicted only com-parison with standards or with published results obtained underidentical conditions allows the deduction of absolute stereochem-istry [46] IMS-MS andor Mosherrsquos acid chloride derivatization incombination with NMR techniques as described in sub-section 23might offer alternatives in future applications

Pretreatment of plasma samples is mainly based on protein pre-cipitation followed by sample clean-up using C18-SPE with or withoutthe involvement of a hexane wash step [84] While preparation ofblood-derived samples is rather straightforward the analysis ofurinary samples does involve more tedious sample-preparation pro-tocols mainly due to the occurrence of strong matrix effects A pro-tocol using mixed mode SPE (Oasis HLB) in combination with APCILC-MS was described [86] Another protocol involves the use of aweak anion-exchange material [87] Compared to C18-based SPE veryclean extracts were obtained by methanol elution of the eicosanoidsmost matrix components remained on the SPE cartridge under theseconditions

Given the low endogenous levels of eicosanoids ultimate sen-sitivity must be achieved using SRM in TQ or Q-LIT instruments [84]Upon CID the presence of hydroxyl and ketone groups in the alkyl

Fig 5 Analysis of 9-HoDE in human plasma by LCndashMSMS Above SRM transition mz 295 rarr 171 left standard (A) right plasma sample (B) Below enhanced product ionspectra of 9-HoDE (C) and the unknown peak (D) Reprinted with permission from [84]


side-chain induces specific cleavages leading to analyte-specific frag-ment ions [88] Current trends in the implementation of high-resolution MS (HRMS) in quantitative bioanalysis may be beneficialin eicosanoid analysis as (almost) co-eluting isobaric compoundscan be resolved by HRMS [89] In this respect IMS-MS should alsobe explored [25]

LC-MS analysis of eicosanoids was recently reviewed [90] Recentapplications involving AMPP labeling of oxidized FAs and LC-MSusing either a TQ instrument [91] or an LTQ-Orbitrap mass spec-trometer [89] were reported Mouse-serum samples were derivatizedafter SPE clean-up and analyzed by a generic RPLC separation usingan ACNwater gradient

6 Bile acids

BAs particularly cholic and chenodeoxycholic acids are the majorCYP-mediated catabolic metabolites of cholesterol [6] Just re-cently BAs emerged as signaling molecules with systemic endo-crine function [92] Particularly in the context of metabolic diseasessuch as obesity or type-2 diabetes BA signaling might be ex-ploited as a novel therapeutic intervention strategy [9293] As aresult analysis and profiling of BAs recently received considerableattention In this respect a comprehensive sample-pretreatment pro-tocol is needed to allow the analysis of neutral acidic and basic sterolderivatives [94] All aspects of the analysis of BAs were exten-sively reviewed recently [95]

61 GC-MS analysis of bile acids

The sub-class of BAs is not a favorable compound class for GC-MS Apart from the CA and hydroxyl groups which already requirederivatization BAs may contain several other polar and labile con-jugates with groups (eg sulfate phosphate amide and glucuronate)that are not readily derivatized towards GC-MS [8] Thus BA anal-ysis by GC-MS is limited to deconjugated compounds which canbe analyzed as TMSmethyl-ester derivatives The fragmentationof BAs in EI can be very useful and complementary in structure

elucidation to product-ion mass spectra obtained by ESI-MS andCID [95]

62 LC-MS analysis of bile acids

LC-MS can be readily used for the analysis of BAs and their con-jugated analogues [96] In all instances sample pretreatment is lesscomplicated than for GC-MS The presence of multiple isomeric BAsputs high demands on efficient separation especially because CIDprovides little compound-specific fragmentation RP-UHPLC isgenerally applied [9697] For SRM in negative-ion mode mostlygroup-specific product ions are applied [eg mz 74 (C2H4NO2

minus) forglyco-BAs mz 80 (SO3

minus bull) for tauro-BAs and mz 97 (HSO4minus) (or neutral

loss of 80 Da SO3) for sulfate-conjugated BAs] whereas unconjugatedBAs do not show significant fragmentation [98] As such CID readilyenables the identification of the conjugates but provides little struc-tural information on the BAs themselves [99]

An interesting recent study provided evidence that dietary fatscan result in changes of host BA composition thus altering condi-tions for gut microbial assemblage perturbing immune homeosta-sis [100] A very recent study compared GC-MS LC-MS and a novelLC-UV analysis platform based on the formation of BA phenacyl esterderivatives for the analysis of BAs in human feces Protocols for ex-traction deconjugation and derivatization were provided for all threeapproaches [101] An interesting LC-MSMS platform has been re-ported for the analysis of conjugated and unconjugated BAs in humanurine employing RPLC and TQ-MS [102]

7 Conclusion and perspectives

As a summary of the approaches that we have discussed for the anal-ysis of small CAs FAs eicosanoids and BAs Table 3 presents a selec-tion of timely and comprehensive applications for each analyte classFuture perspectives clearly include increasing demand for the analy-sis of CAs as more and more biological functions of CAs are beingunraveled Most probably LC-MSMS platforms will further replaceGC-MS analysis systems The main reasons for this trend are

Table 3Applications for the analysis of four classes of carboxylic acids (CAs) ndash small CAs fatty acids (FAs) eicosanoids and bile acids (BAs)

Analyte class Sub-class (if applicable) Matrixsample preparation Analyticaltechnique

Comment Ref

Small CA TCA cycle intermediates Cultured cellsproteinprecipitation

LC-MSMS Full description of MIRACLE approach for intracellularmetabolite analysis using U-13C-labelled cell extracts

[31]

TCA cycle intermediates Cultured cellsproteinprecipitation

LC-MSMS Comparison study of different separation strategies forhighly hydrophilic cellular metabolites

[48]

Global metabolic profiling ofurine

Urineurease treatmentprotein precipitation

GC-MS Global metabolic profiling of urine using GC-MS andmethoxymationtrimethylsilylation

[39]

FA f-FA Plasmaprotein precipitation LC-MSMS Development of prediction models for theidentification of unknown FA based on a calibration setand introduction of the concept called differentialqualifier ion ratio

[13]

f-FA SeveralLLE GC-ECNI-MS PFBBr derivative analysis highly selective andsensitive

[65]

t-FA Plasma cellstrans-esterification

GC-EI-MS Analysis of FAMEs Modified trans-esterification foraccurate double bond isomer determination

[60]

Eicosanoids Eicosanoids and PUFA Plasmaon-line SPE LC-MSMS On-line sample preparation combined with highresolution separation and high sensitivity QTrapanalysis

[85]

F2-isoprostanes SeveralSPE thin layerchromatography

GC-ECNI-MS Analysis of F2-isoprostanes as markers of oxidativestress using GC-ECNI-MS

[80]

Oxidized FAs TissueSPE LC-Orbitrap MS HRMS analysis after derivatization allowing resolutionof (almost) co-eluting substances

[89]

BAs FecesEthanol extractionfollowed by deconjugationderivatization and SPE

LC-UV Analysis of phenacyl ester derivatives - cost effectiveroutine platform

[101]

Urinedilution with ethanolfiltration

LC-MSMS Analysis of 39 conjugated and unconjugated bile acidsin urine samples

[102]


(1) less elaborate sample-preparation protocols(2) (usually) no need for derivatization(3) greater versatility of LC-MS platforms especially for non-

volatile and fragile analytes(4) increasing sensitivity particularly of TQ-MS systems and(5) recent advances in LC-column technology enabling rapid high-

resolution separations

Although many pitfalls in the field of CA analysis have been over-come during the past decade three major tasks remain

bull make the comprehensive separation and analysis of isomersapplicable to routine analysis platforms

bull enable the comprehensive separation and analysis of enanti-omers and

bull facilitate absolute quantification

IMS-MS possibly offers analytical solutions for the separation andunambiguous identification of isomers and enantiomers without theneed for highly sophisticated LC or GC separation systems Howeverregarding absolute quantification major analytical issues still need tobe resolved The MIRACLE approach has overcome some pitfalls in cel-lular metabolite analysis but other issues continue to attract our at-tention Matrix effects are of considerable concern and in particularthey are a major pitfall in the development of comprehensive multi-component platforms To eliminate matrix effects and to enable abso-lute quantification SIL-ISs are needed ideally for each analyte underinvestigation Furthermore standardized protocols have to be furtherdeveloped for sample collection and storage so as to allow leak-freequenching and to avoid autoxidation during sample handling

It will be interesting to see if the future will bring us more uni-versal MS tools for absolute quantification and how far novel ion-ization techniques might possibly overcome the current limitationsof ESI and APCI ionization Also IMS-MS possibly allowing themapping of metabolic fine structures so that isomers andor en-antiomers can further be resolved will most probably become a tech-nique to be applied in the analysis of the CAs

References

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[2] PC Calder n-3 Fatty acids inflammation and immunity new mechanismsto explain old actions Proc Nutr Soc 72 (2013) 326ndash336

[3] IR Klein-Wieringa SN Andersen JC Kwekkeboom M Giera BJEde Lange-Brokaar GJVM van Osch et al Adipocytes modulate the phenotypeof human macrophages through secreted lipids J Immunol 191 (2013)1356ndash1363

[4] CN Serhan NA Petasis Resolvins and protectins in inflammation resolutionChem Rev 111 (2011) 5922ndash5943

[5] CD Funk Prostaglandins and leukotrienes advances in eicosanoid biologyScience 294 (2001) 1871ndash1875

[6] JYL Chiang Bile Acid Metabolism and Signaling John Wiley amp Sons Inc 2013[7] J Acimovic A Loumlvgren-Sandblom K Monostory D Rozman M Golicnik D

Lutjohann et al Combined gas chromatographicmass spectrometric analysisof cholesterol precursors and plant sterols in cultured cells J Chromatogr B877 (2009) 2081ndash2086

[8] DW Johnson Contemporary clinical usage of LCMS analysis of biologicallyimportant carboxylic acids Clin Biochem 38 (2005) 351ndash361

[9] AM Stalcup Chiral sepazrations Ann Rev Anal Chem 3 (2010) 341ndash363[10] SA Mjoslashs 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) 550ndash560

[11] VZAJ Halket A Handbook of Derivatives for Mass Spectrometry IMPublications Chisester UK 2009

[12] CF Poole Alkylsilyl derivatives for gas chromatography J Chromatogr A 1296(2013) 2ndash14

[13] C Hellmuth M Weber B Koletzko W Peissner Nonesterified fatty aciddetermination for functional lipidomics comprehensive ultrahigh performanceliquid chromatography-tandem mass spectrometry quantitation qualificationand parameter prediction Anal Chem 84 (2012) 1483ndash1490

[14] T Santa Derivatization reagents in liquid chromatographyelectrosprayionization tandem mass spectrometry Biomed Chromatogr 25 (2011) 1ndash10

[15] T Santa Derivatization in liquid chromatography for mass spectrometricdetection Drug Discov Ther 7 (2013) 9ndash17

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as reagents for HPLC-MS enantioseparation of chiral carboxylic acids J SepSci 35 (2012) 1551ndash1559

[19] D-T Nguyen K-R Kim G Lee M-J Paik Chiral separation of N-methyl-dl-aspartic acid in rat brain tissue as N-ethoxycarbonylated (S)-(+)-2-octyl esterderivatives by GC-MS Biomed Chromatogr 26 (2012) 1353ndash1356

[20] Y Xiao S-C Ng TTY Tan Y Wang Recent development of cyclodextrin chiralstationary phases and their applications in chromatography J ChromatogrA 1269 (2012) 52ndash68

[21] I Špaacutenik D Kaceriakovaacute J Krupciacutek DW Armstrong GC separation ofenantiomers of alkyl esters of 2-bromo substituted carboxylic acidsenantiomers on 6-TBDMS-23-di-alkyl- β- and γ-cyclodextrin stationary phasesChirality (2014)

[22] DW Armstrong B Zhang Product review chiral stationary phases for HPLCAnal Chem 73 (2001) 557Andash561A

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[24] TR Hoye CS Jeffrey F Shao Mosher ester analysis for the determination ofabsolute configuration of stereogenic (chiral) carbinol carbons Nat Protocols2 (2007) 2451ndash2458

[25] P Dwivedi C Wu LM Matz BH Clowers WF Siems HH Hill Gas-phasechiral separations by ion mobility spectrometry Anal Chem 78 (2006)8200ndash8206

[26] HG Gika C Ji GA Theodoridis F Michopoulos N Kaplowitz ID WilsonInvestigation of chronic alcohol consumption in rodents via ultra-high-performance liquid chromatography-mass spectrometry based metaboliteprofiling J Chromatogr A 1259 (2012) 128ndash137

[27] T Damme M Lachovaacute F Lynen R Szucs P Sandra Solid-phase extractionbased on hydrophilic interaction liquid chromatography with acetone as eluentfor eliminating matrix effects in the analysis of biological fluids by LC-MSAnal Bioanal Chem 406 (2014) 401ndash407

[28] S Noack W Wiechert Quantitative metabolomics a phantom TrendsBiotechnol 32 (2014) 238ndash244

[29] J Lee E-S Jang B Kim Development of isotope dilution-liquidchromatographymass spectrometry combined with standard additiontechniques for the accurate determination of tocopherols in infant formulaAnal Chim Acta 787 (2013) 132ndash139

[30] MR Mashego L Wu JC Van Dam C Ras JL Vinke WA Van Winden et alMIRACLE mass isotopomer ratio analysis of U-13C-labeled extracts A newmethod for accurate quantification of changes in concentrations of intracellularmetabolites Biotechnol Bioeng 85 (2004) 620ndash628

[31] BD Bennett J Yuan EH Kimball JD Rabinowitz Absolute quantitation ofintracellular metabolite concentrations by an isotope ratio-based approachNat Protocols 3 (2008) 1299ndash1311

[32] P Bruheim HFN Kvitvang SG Villas-Boas Stable isotope coded derivatizingreagents as internal standards in metabolite profiling J Chromatogr A 1296(2013) 196ndash203

[33] MG Vander Heiden LC Cantley CB Thompson Understanding the warburgeffect the metabolic requirements of cell proliferation Science 324 (2009)1029ndash1033

[34] D Kloos RJE Derks M Wijtmans H Lingeman OA Mayboroda AM Deelderet al Derivatization of the tricarboxylic acid cycle intermediates and analysisby online solid-phase extraction-liquid chromatography-mass spectrometrywith positive-ion electrospray ionization J Chromatogr A 1232 (2012) 19ndash26

[35] M Fuchs J Engel M Campos R Matejec M Henrich H Harbach et alIntracellular alpha-keto acid quantification by fluorescence-HPLC Amino Acids36 (2009) 1ndash11

[36] D Siegel H Permentier D-J Reijngoud R Bischoff Chemical and technicalchallenges in the analysis of central carbon metabolites by liquid-chromatography mass spectrometry J Chromatogr B 966 (2014) 21ndash33

[37] KJ Pedersen The decomposition of α-nitrocarboxylic acids With someremarks on the decomposition of β-ketocarboxylic acids J Phys Chem 38(1933) 559ndash571

[38] C Hallmann BGK van Aarssen K Grice Relative efficiency of free fatty acidbutyl esterification choice of catalyst and derivatisation procedure JChromatogr A 1198ndash1199 (2008) 14ndash20

[39] C Eric Chun Yong P Kishore Kumar KN Jeremy Global urinary metabolicprofiling procedures using gas chromatography-mass spectrometry NatProtoc 6 (2011) 1483ndash1499

[40] O Fiehn J Kopka RN Trethewey L Willmitzer Identification of uncommonplant metabolites based on calculation of elemental compositions using gaschromatography and quadrupole mass spectrometry Anal Chem 72 (2000)3573ndash3580

[41] KR Kim MK Hahn A Zlatkis EC Horning BS Middleditch Simultaneousgas chromatography of volatile and non-volatile carboxylic acids as tert-butyldimethylsilyl derivatives J Chromatogr A 468 (1989) 289ndash301

[42] D Saraiva R Semedo MDC Castilho JM Silva F Ramos Selection of thederivatization reagent ndash the case of human blood cholesterol its precursorsand phytosterols GC-MS analyses J Chromatogr B 879 (2011) 3806ndash3811


[43] MA Lorenz CF Burant RT Kennedy Reducing time and increasing sensitivityin sample preparation for adherent mammalian cell metabolomics AnalChem 83 (2011) 3406ndash3414

[44] J A J Trygg J Gullberg AI Johansson P Jonsson H Antti et al Extractionand GCMS analysis of the human blood plasma metabolome Anal Chem77 (2005) 8086ndash8094

[45] DA Scott AD Richardson FV Filipp CA Knutzen GG Chiang ZEA Ronaiet al Comparative metabolic flux profiling of melanoma cell lines beyondthe warburg effect J Biol Chem 286 (2011) 42626ndash42634

[46] KE Price RE Pearce UC Garg BA Heese LD Smith JE Sullivan et al Effectsof valproic acid on organic acid metabolism in children a metabolic profilingstudy Clin Pharmacol Ther 89 (2011) 867ndash874

[47] U Hofmann K Maier A Niebel G Vacun M Reuss K Mauch Identificationof metabolic fluxes in hepatic cells from transient 13C-labeling experimentspart I Experimental observations Biotechnol Bioeng 100 (2008) 344ndash354

[48] SU Bajad W Lu EH Kimball J Yuan C Peterson JD Rabinowitz Separationand quantitation of water soluble cellular metabolites by hydrophilicinteraction chromatography-tandem mass spectrometry J Chromatogr A 1125(2006) 76ndash88

[49] JC van Dam MR Eman J Frank HC Lange GWK van Dedem SJ HeijnenAnalysis of glycolytic intermediates in Saccharomyces cerevisiae using anionexchange chromatography and electrospray ionization with tandem massspectrometric detection Anal Chim Acta 460 (2002) 209ndash218

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[51] B Buszewski S Noga Hydrophilic interaction liquid chromatography (HILIC)ndash a powerful separation technique Anal Bioanal Chem 402 (2012) 231ndash247

[52] D-P Kloos H Lingeman WMA Niessen AM Deelder M Giera OAMayboroda Evaluation of different column chemistries for fast urinarymetabolic profiling J Chromatogr B 927 (2013) 90ndash96

[53] P Deng Y Zhan X Chen D Zhong Derivatization methods for quantitativebioanalysis by LC-MSMS Bioanalysis 4 (2011) 49ndash69

[54] S Becker L Kortz C Helmschrodt J Thiery U Ceglarek LC-MS-basedmetabolomics in the clinical laboratory J Chromatogr B 883ndash884 (2012)68ndash75

[55] S Dietmair NE Timmins PP Gray LK Nielsen JO Kroumlmer Towardsquantitative metabolomics of mammalian cells development of a metaboliteextraction protocol Anal Biochem 404 (2010) 155ndash164

[56] GM Tannahill AM Curtis J Adamik EM Palsson-McDermott AFMcGettrick G Goel et al Succinate is an inflammatory signal that inducesIL-1[bgr] through HIF-1[agr] Nature 496 (2013) 238ndash242

[57] AE Barden E Mas KD Croft M Phillips TA Mori Minimizing artifactualelevation of lipid peroxidation products (F2-isoprostanes) in plasma duringcollection and storage Anal Biochem 449 (2014) 129ndash131

[58] G Lepage CC Roy Direct transesterification of all classes of lipids in aone-step reaction J Lipid Res 27 (1986) 114ndash120

[59] G-L Wei EY Zeng Gas chromatography-mass spectrometry and high-performance liquid chromatography-tandem mass spectrometry in quantifyingfatty acids Trends Analyt Chem 30 (2011) 1429ndash1436

[60] J Ecker M Scherer G Schmitz G Liebisch A rapid GC-MS method forquantification of positional and geometric isomers of fatty acid methyl estersJ Chromatogr B 897 (2012) 98ndash104

[61] WW Christie WW Christie (Editor) Preparation of Ester Derivatives of FattyAcids for Chromatographic Analysis Oily Press Dundee UK 1993 pp 69ndash111

[62] D Kloos E Gay H Lingeman F Bracher C Muumlller OA Mayboroda et alComprehensive GC-MS analysis of fatty acids and sterols using sequentialone-pot silylation quantification and isotopologue analysis Rapid CommunMass Spectrom (2014)

[63] W Christie E Brechany S Johnson R Holman A comparison of pyrrolidideand picolinyl ester derivatives for the identification of fatty acids in naturalsamples by gas chromatography-mass spectrometry Lipids 21 (1986) 657ndash661

[64] V Svetashev Mild method for preparation of 44-dimethyloxazoline derivativesof polyunsaturated fatty acids for GC-MS Lipids 46 (2011) 463ndash467

[65] O Quehenberger AM Armando EA Dennis High sensitivity quantitativelipidomics analysis of fatty acids in biological samples by gaschromatography-mass spectrometry Biochim Biophys Acta 1811 (2011)648ndash656

[66] J Ecker G Liebisch Application of stable isotopes to investigate themetabolism of fatty acids glycerophospholipid and sphingolipid species ProgLipid Res 54 (2014) 14ndash31

[67] J Ecker Profiling eicosanoids and phospholipids using LC-MSMS principlesand recent applications J Sep Sci 35 (2012) 1227ndash1235

[68] P Goacutemez-Corteacutes C Tyburczy JT Brenna M Juaacuterez MA de la FuenteCharacterization of cis-9 trans-11 trans-15 C183 in milk fat by GC andcovalent adduct chemical ionization tandem MS J Lipid Res 50 (2009)2412ndash2420

[69] C Pelt B Carpenter JT Brenna Studies of structure and mechanism inacetonitrile chemical ionization tandem mass spectrometry of polyunsaturatedfatty acid methyl esters J Am Soc Mass Spectrom 10 (1999) 1253ndash1262

[70] A Latorre A Rigol S Lacorte D Barceloacute Comparison of gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry for thedetermination of fatty and resin acids in paper mill process waters JChromatogr A 991 (2003) 205ndash215

[71] S Schiesel M Laumlmmerhofer W Lindner Quantitative LC-ESI-MSMS metabolicprofiling method for fatty acids and lipophilic metabolites in fermentation

broths from β-lactam antibiotics production Anal Bioanal Chem 397 (2010)147ndash160

[72] N Zehethofer DM Pinto DA Volmer Plasma free fatty acid profiling in afish oil human intervention study using ultra-performance liquidchromatographyelectrospray ionization tandem mass spectrometry RapidCommun Mass Spectrom 22 (2008) 2125ndash2133

[73] B Nikolova-Damyanova Retention of lipids in silver ion high-performanceliquid chromatography facts and assumptions J Chromatogr A 1216 (2009)1815ndash1824

[74] HS Joacutenasdoacutettir S Nicolardi W Jonker R Derks M Palmblad A Ioan-Facsinayet al Detection and structural elucidation of esterified oxylipids in humansynovial fluid by electrospray ionization-fourier transform ion-cyclotron massspectrometry and liquid chromatography-ion trap-MS3 detection of esterifiedhydroxylated docosapentaenoic acid containing phospholipids Anal Chem85 (2013) 6003ndash6010

[75] JG Bollinger G Rohan M Sadilek MH Gelb LCESI-MSMS detection of FAsby charge reversal derivatization with more than four orders of magnitudeimprovement in sensitivity J Lipid Res 54 (2013) 3523ndash3530

[76] C Pettinella SH Lee F Cipollone IA Blair Targeted quantitative analysisof fatty acids in atherosclerotic plaques by high sensitivity liquidchromatographytandem mass spectrometry J Chromatogr B 850 (2007)168ndash176

[77] W-C Yang J Adamec FE Regnier Enhancement of the LCMS analysis of fattyacids through derivatization and stable isotope coding Anal Chem 79 (2007)5150ndash5157

[78] M Eggink M Wijtmans A Kretschmer J Kool H Lingeman IP Esch et alTargeted LC-MS derivatization for aldehydes and carboxylic acids with a newderivatization agent 4-APEBA Anal Bioanal Chem 397 (2010) 665ndash675

[79] U Jahn J-M Galano T Durand Beyond prostaglandins ndash chemistry andbiology of cyclic oxygenated metabolites formed by free-radical pathwaysfrom polyunsaturated fatty acids Angew Chem Int Ed 47 (2008) 5894ndash5955

[80] GL Milne SC Sanchez ES Musiek JD Morrow Quantification of F2-isoprostanes as a biomarker of oxidative stress Nat Protoc 2 (2007) 221ndash226

[81] W Liu JD Morrow H Yin Quantification of F2-isoprostanes as a reliable indexof oxidative stress in vivo using gas chromatography-mass spectrometry(GC-MS) method Free Radical Biol Med 47 (2009) 1101ndash1107

[82] K Kasuga R Yang TF Porter N Agrawal NA Petasis D Irimia et al Rapidappearance of resolvin precursors in inflammatory exudates novelmechanisms in resolution J Immunol 181 (2008) 8677ndash8687

[83] M Giera A Ioan-Facsinay R Toes F Gao J Dalli AM Deelder et al Lipidand lipid mediator profiling of human synovial fluid in rheumatoid arthritispatients by means of LC-MSMS Biochim Biophys Acta 2012 (1821) 1415ndash1424

[84] R Yang N Chiang SF Oh CN Serhan Metabolomics-Lipidomics ofEicosanoids and Docosanoids Generated by Phagocytes John Wiley amp SonsInc 2001

[85] L Kortz J Dorow S Becker J Thiery U Ceglarek Fast liquid chromatography-quadrupole linear ion trap-mass spectrometry analysis of polyunsaturatedfatty acids and eicosanoids in human plasma J Chromatogr B 927 (2013)209ndash213

[86] 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 947ndash948 (2014) 173ndash178

[87] A Taylor R Bruno M Traber Women and smokers have elevated urinaryF2-isoprostane metabolites a novel extraction and LC-MS methodology Lipids43 (2008) 925ndash936

[88] RC Murphy RM Barkley K Zemski Berry J Hankin K Harrison C Johnsonet al Electrospray ionization and tandem mass spectrometry of eicosanoidsAnal Biochem 346 (2005) 1ndash42

[89] X Liu SH Moon DJ Mancuso CM Jenkins S Guan HF Sims et al Oxidizedfatty acid analysis by charge-switch derivatization selected reactionmonitoring and accurate mass quantitation Anal Biochem 442 (2013) 40ndash50

[90] L Kortz J Dorow U Ceglarek Liquid chromatography-tandem massspectrometry for the analysis of eicosanoids and related lipids in humanbiological matrices A review J Chromatogr B 964 (2014) 1ndash11

[91] JG Bollinger W Thompson Y Lai RC Oslund TS Hallstrand M Sadileket al Improved sensitivity mass spectrometric detection of eicosanoids bycharge reversal derivatization Anal Chem 82 (2010) 6790ndash6796

[92] C Thomas R Pellicciari M Pruzanski J Auwerx K Schoonjans Targetingbile-acid signalling for metabolic diseases Nat Rev Drug Discov 7 (2008)

[93] G Porez J Prawitt B Gross B Staels Bile acid receptors as targets for thetreatment of dyslipidemia and cardiovascular disease thematic review seriesnew lipid and lipoprotein targets for the treatment of cardiometabolic diseasesJ Lipid Res 53 (2012) 1723ndash1737

[94] WJ Griffiths J Sjoumlvall Analytical strategies for characterization of bile acidand oxysterol metabolomes Biochem Biophys Res Commun 396 (2010)80ndash84

[95] WJ Griffiths J Sjoumlvall Bile acids analysis in biological fluids and tissues JLipid Res 51 (2010) 23ndash41

[96] J Ding ET Lund J Zulkoski JP Lindsay DL McKenzie High-throughputbioanalysis of bile acids and their conjugates using UHPLC coupled to HRMSBioanalysis 5 (2013) 2481ndash2494

[97] SPR Bathena S Mukherjee M Olivera Y Alnouti The profile of bile acidsand their sulfate metabolites in human urine and serum J Chromatogr B942ndash943 (2013) 53ndash62


[98] J Huang SPR Bathena IL Csanaky Y Alnouti Simultaneous characterizationof bile acids and their sulfate metabolites in mouse liver plasma bile andurine using LC-MSMS J Pharm Biomed Anal 55 (2011) 1111ndash1119

[99] M Maekawa M Shimada T Iida J Goto N Mano Tandem mass spectrometriccharacterization of bile acids and steroid conjugates based on low-energycollision-induced dissociation Steroids 80 (2014) 80ndash91

[100] S Devkota Y Wang MW Musch V Leone H Fehlner-Peach A Nadimpalliet al Dietary-fat-induced taurocholic acid promotes pathobiont expansionand colitis in Il10-- mice Nature 487 (2012) 104ndash108

[101] G Kakiyama A Muto H Takei H Nittono T Murai T Kurosawa et al Asimple and accurate HPLC method for fecal bile acid profile in healthy andcirrhotic subjects validation by GC-MS and LC-MS J Lipid Res 55 (2014)978ndash990

[102] A Muto H Takei A Unno T Murai T Kurosawa S Ogawa et al Detectionof Δ4-3-oxo-steroid 5β-reductase deficiency by LC-ESI-MSMS measurementof urinary bile acids J Chromatogr B 900 (2012) 24ndash31


4 Fatty acids 2241 GC-MS analysis of fatty acids 2242 LC-MS analysis of fatty acids 23

5 Eicosanoids 2351 GC-MS analysis of eicosanoids 2452 LC-MS analysis of eicosanoids 24

6 Bile acids 2561 GC-MS analysis of bile acids 2562 LC-MS analysis of bile acids 25

7 Conclusion and perspectives 25References 26

1 Introduction

One of the most interesting developments in the biomedical sci-ences of the post-genomic era is a ldquoparadigm shiftrdquo in our under-standing of the biologicalregulatory activity of the chemical entitiesfor which such a function was disregarded The compounds that tra-ditionally were seen as only constituents of energy metabolism [egpyruvate succinate fatty acids (FAs)] or emulsifiers of lipids [bileacids (BAs)] are being more and more recognized as importantimmune-modulatory andor signaling molecules In the first ap-proximation all those compounds appear to be far too structural-ly diverse to be reviewed together but taking a broader view werealize that most of those ldquoupcoming regulatory compoundsrdquo areendogenous carboxylic acids (CAs) Their common functional fea-tures are the presence of a CA function dictating many but not allof their physicochemical properties and an alkyl part that can bedecorated with different functional groups One can distinguish fourimportant sub-classes of endogenous CAs namely

(1) small CAs crucial to aerobic respiration and energy metab-olism [1]

(2) FAs fundamental to energy storage and membrane forma-tion and involved in numerous physiological processes suchas inflammation [2ndash4]

(3) eicosanoids and docosanoids forming a class of very impor-tant signaling molecules during several inflammatory and im-munological events [5] and

(4) BAs the main metabolites of endogenous cholesterol [6] (seeFig 1 for their general structures)

In this article we discuss recent advances in analytical tech-niques for the analysis of CAs

All of these analytes reside in the body and can be quantita-tively analyzed in one of the body fluids or in cellular extracts Theirisolation and separation from matrix components is fundamentalto their analysis While sample preparation can be distinctly dif-ferent for the four CA sub-classes analyte separation is commonlyperformed by either gas chromatography (GC) or liquid chroma-tography (LC) Both separation techniques are frequently com-bined with mass spectrometric (MS) detection for the highestselectivity and sensitivity in quantitative analysis [7]

Both GC-MS and LC-MS feature common requirements due tothe functional groups present in the analytes of interest Analysisof endogenous CAs is done with different aims eg

bull (I) qualitative profiling of CAs present thus involving structureelucidation and resolving isomerism issues

bull (II) relative quantitation for comparison of physiological states andor flux determination involving the use of isotopologues and

bull (III) targeted (absolute) quantitative analysis

The aims of the study determine the analytical strategy chosenbut in all cases general issues in the analysis of CAs are also im-portant [8]

2 General aspects

All four analyte sub-classes share the presence of a CA func-tion (ie they are weak organic Broslashnsted-Lowry acids) Howeverother features shared by the CAs might also have significant effectson the analytical process and thus demand rather similar analyti-cal solutions Fig 2 shows the most prominent functional groupsthat may be present in an endogenous CA

21 GC-MS analysis

Intrinsically GC-MS analysis demands the evaporation of theanalyte under investigation prior to separation and detection Withrespect to CAs several functional groups demand attention Firstthe CA function has to be derivatized for successful GC-MS analy-sis From the various possible derivatization strategies silylation andesterification of the CA function are the most prominent as the re-action usually proceeds smoothly and particularly in the case ofsilylation requires no sample clean-up before injection Other crucialfunctional groups (Fig 2) that have to be derivatized prior to GC-MS analysis are (A) hydroxyl groups which can either undergosilylation or ether formation and (B) ketone groups which are fre-quently converted into oximes thereby blocking possible tautom-erism during subsequent derivatization and stabilizing β-ketoCAs particularly After derivatization the CA derivatives canbe analyzed in routine GC-MS systems involving commondimethylsiloxane-coated capillary columns and wide temperaturegradients Generally analyte ionization is achieved by electron

Fig 1 General structures of the four relevant classes of carboxylic acids (CAs) dis-cussed in this article small CAs fatty acids eicosanoids and bile acids




22 LC-MS analysis





Functionalgroup


Carboxylicacid








Hydroxylgroup



RSisomerism


Ketonegroup















Functionalgroup


Carboxylicacid




Hydroxylgroup


Dansyl chloride




















4 Fatty acids




















5 Eicosanoids



















6 Bile acids















Comment Ref



[31]



[48]




[39]


[13]


[65]



[60]


[85]



[80]


[89]



[101]



[102]











References













































































































22 LC-MS analysis





Functionalgroup


Carboxylicacid








Hydroxylgroup



RSisomerism


Ketonegroup















Functionalgroup


Carboxylicacid




Hydroxylgroup


Dansyl chloride




















4 Fatty acids




















5 Eicosanoids



















6 Bile acids















Comment Ref



[31]



[48]




[39]


[13]


[65]



[60]


[85]



[80]


[89]



[101]



[102]











References























































































































Functionalgroup


Carboxylicacid




Hydroxylgroup


Dansyl chloride




















4 Fatty acids




















5 Eicosanoids



















6 Bile acids















Comment Ref



[31]



[48]




[39]


[13]


[65]



[60]


[85]



[80]


[89]



[101]



[102]











References





























































































































4 Fatty acids




















5 Eicosanoids



















6 Bile acids















Comment Ref



[31]



[48]




[39]


[13]


[65]



[60]


[85]



[80]


[89]



[101]



[102]











References



















































































































4 Fatty acids




















5 Eicosanoids



















6 Bile acids















Comment Ref



[31]



[48]




[39]


[13]


[65]



[60]


[85]



[80]


[89]



[101]



[102]











References


















































































































5 Eicosanoids



















6 Bile acids















Comment Ref



[31]



[48]




[39]


[13]


[65]



[60]


[85]



[80]


[89]



[101]



[102]











References


























































































































6 Bile acids















Comment Ref



[31]



[48]




[39]


[13]


[65]



[60]


[85]



[80]


[89]



[101]



[102]











References













































































































6 Bile acids















Comment Ref



[31]



[48]




[39]


[13]


[65]



[60]


[85]



[80]


[89]



[101]



[102]











References




















































































































References
















































































































































































Documents

Analysis of biologically-active, endogenous carboxylic acids based on chromatography-mass spectrometry