Review Review role of liquid chromatography mass...

Analytica Chimica Acta 492 (2003) 17–47

Review

Review role of liquid chromatography–mass spectrometryin the analysis of oxidation products and antioxidants

in biological systems

D.G. Watsona,∗, C. Atsrikua, E.J. Oliveiraba Department of Pharmaceutical Sciences, Strathclyde Institute of Biomedical Sciences, University of Strathclyde,

27 Taylor Street, Glasgow, Scotland G4 0NR, UKb Universidade Federal da Paraiba, LTF/UDEM, Campus I, Caixa Postal 5009, Joao Pessoa, PB, Brazil

Accepted 4 April 2003

Abstract

This review covers the applications of mass spectrometry with sample introduction in the liquid phase to the analysis ofthe oxidation products of DNA, proteins and lipids in vitro and in vivo. The analysis of antioxidant xenobiotics in biologicalsystems is also covered. The review covers the period 1997–late 2002. The most commonly used mass spectrometric modeused in this area of study is electrospray mass spectrometry that may also include collision-induced dissociation (CID). Thereview summarises for each paper the important methodological aspects of the techniques used and, where appropriate, theimportance of the observations made within the context of biology.© 2003 Elsevier B.V. All rights reserved.

Keywords: Electrospray mass spectrometry; DNA oxidation; Protein oxidation; Lipid oxidation; Determination of antioxidants

Abbreviations: 1EDG, 1,N2-Etheno-2′-deoxyguanosine; 2HDOA, 2-Hydroxy-2′-deoxyadenosine; 3-NT, 3-Nitrotyrosine; 5HMDU, 5-Hydroxymethyl-2′-deoxyuridine; 8CDDA, 8-Cyclodeoxyadenosine; 8HDDA, 8-Hydroxy-2′-deoxyadenosine; 8HDOA, 8-Hydroxy-2′-deoxyadenosine; 8HDOG, 8-Hydroxy-2′-deoxyguanosine; 8ODDA, 8-Oxo-7,8-dihydro-2′-deoxyadenosine; 8-ODDG, 8-Oxo-7,8-dihydro-2′-deoxyguanosine; AD, Alzheimer’s disease; APCI, atmospheric pressure chemical ionisation; APHDL, acute phase HDL; API, atmo-spheric pressure ionisation; BHT, butylated hydroxytoluene; C18, octadecyl; CID, collisional-induced dissociation; DA, 2′-Deoxyadenosine;DC, 2′-Deoxycytidine; DG, 2′-Deoxyguanosine; DNPH, 2,4-Dinitrophenylhydrazine; EDA, Etheno-2′-deoxyadenosine; EDC, Etheno-2′-deoxycytidine; ESI, Electrospray ionisation; GC–MS, Gas chromatography–mass spectrometry; GPC, Glycerophosphocholine; GPE, Glyc-erophosphoethanolamine; GSH, Gluthathione; GT, Gluthathione transferase; HDL, High-density lipoprotein; Hgb, Hemoglobin; HN,4-Hydroxynonenal; IS, Internal standard; LC, Liquid chromatography; LDL, Low-density lipoprotein; LOD, Limit of detection; M1-DG,Pyrimido[1,2�]purine-10(3H)-one-2′-deoxyribose; MALDI, Matrix-assisted laser desorption ionization mass spectrometry; MMLDL, Mildlyoxidised LDL; MRM, Multiple reaction monitoring; MOHCl, Methoxylamine hydrochloride; NHDL, Normal response HDL; ODS, Oc-tadecylsilane; PAPC, 1-Palmitoyl-2-arachidonylglycerophosphocholine; PC, Phosphatidylcholine; PE, Phosphatidylethanolamine; RP-HPLC,Reversed-phase high-performance liquid chromatography; SPE, Solid phase extraction; TFA, Trifluoroacetic acid; TG, Thymidine glycol;TOF, Time of flight mass spectrometry

∗ Corresponding author. Tel.:+44-1415482651; fax:+44-1415526443.E-mail address: [email protected] (D.G. Watson).

0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved.doi:10.1016/S0003-2670(03)00467-7

18 D.G. Watson et al. / Analytica Chimica Acta 492 (2003) 17–47

1. Introduction

This review covers approximately the period1997–2002. The most important recent developmentsin the application of LC–MS to the determinationof oxidation products DNA, proteins and lipids andof the antioxidants that may prevent this are dis-cussed. Research into biological oxidation has beengrowing steadily over the years and will continue tobe of interest since many of the diseases of old agemay have their origin in oxidative stress; oxidativereactions both drive living processes and ultimatelyin time weaken living systems. We have aimed tocover experimental details wherever there are somenovel aspects to them otherwise the more routineexperimental procedures have not been described indetail.

The development of atmospheric pressure ioniza-tion (API) interfaces has largely overcome the initialproblems involved in coupling two seemingly incom-patible techniques; a dynamic liquid chromatographicsystem and mass spectrometry. Undoubtedly, API isresponsible for the dramatic growth and application ofLC–MS in pharmaceutical analysis, proteomics, ge-nomics and drug metabolism[1,2]. Mass spectrometryhad relied historically on the generation of gas-phaseions under high vacuum and thus interfacing it withliquid chromatography (LC) posed obvious difficul-ties. Thus, the successful combination of these twotechniques had to await the development of an ioniza-tion mode that could generate ions directly from thecondensed phase and at atmospheric pressure. Atmo-spheric pressure ionization techniques were developedmainly from the pioneering work of Dole et al.[3], butit was commercially explored only after the contribu-tion of John Fenn and his collaborators demonstratingfor the first time the potential of electrospray ionisation(ESI) for the analysis of thermolabile macromolecules[4,5]—for which Fenn was awarded the 2002 NobelPrize in chemistry.

During the past decade, LC–MS has become themethod of choice for the determination of xenobioticsand products of metabolism in complex biologicalmatrices. The two main ionization modes used incurrent commercial instruments are electrospray ion-ization and atmospheric pressure chemical ionization(APCI). In an electrospray interface, the eluent fromthe LC column is passed through a capillary held at a

high voltage (2–5 kV) forming an aerosol of chargeddroplets. A flow of coaxial gas (usually nitrogen) helpsto desolvate the droplets resulting in a progressiveincrease in the charge density at the droplets surface,eventually leading the ions to overcome the liquid’ssurface tension and attain the gas phase in a processcommonly referred as “ion evaporation”. In APCI, theeluent of the HPLC is nebulized through a capillary ina similar way to electrospray, but instead of applyinga voltage to the capillary, a discharge pin is placedin the ion source. The electrical discharge from thepin causes solvent molecules to form a plasma, caus-ing interactions to occur between ionised moleculesof the solvent and neutral analyte molecules, in amechanism resembling chemical ionization massspectrometry.

Current benchtop LC–MS instruments are mostlybased on ion traps or quadrupole ion optics. Instru-ments can have a single mass filter or more thanone mass filter connected in tandem (LC–MS/MS).In a typical triple quadrupole instrument, threequadrupoles are operated in tandem, with the first andthird quadrupoles used as mass filters and the sec-ond quadrupole as a collision chamber, in which ionsadmitted in the first quadrupole are made to collidewith molecules of an inert gas (usually argon) result-ing in collisional-induced dissociation (CID) of theions. Excellent reviews on the mechanisms of atmo-spheric pressure ionization[6–8] and on the differentacquisition modes[9,10] possible in tandem massspectrometry are available. There are five major iondetection modes of operation in bioanalytical studiesusing a triple quadrupole mass analyser.

1.1. Full-scan mode

In full-scan mode of ion detection, the firstquadrupole (Q1) (Fig. 1) is operated in a linear (radiofrequency/direct current, RF/DC) scan mode, wherea range ofm/z values encompassing the analytes ofinterest is constantly monitored. In this configura-tion, the collision cell is evacuated while the thirdquadrupole (Q3) operates in an RF-only mode to pro-vide uninhibited transmission. The quadrupole massfilter is generally inefficient at detecting ions in thefull-scan mode and detection limits are improvedmainly by operating Q1 within a narrow scan (m/z)window.

D.G. Watson et al. / Analytica Chimica Acta 492 (2003) 17–47 19

Fig. 1. Schematic representation of a triple quadrupole MS instrument (LC–MS/MS) showing a collision cell (q2) between two quadrupolemass filters (Q1, Q3).

1.2. Selected ion monitoring

Selected ion monitoring (SIM) is a fundamentalquantitative configuration. By setting the quadrupole(Q1) to pass only ions of interest through to the de-tector, a selective detection system is created. This re-duces chemical noise leading to higher mass spectralsensitivity and selectivity. The tandem mass spectrom-etry analog to SIM experiments is known as multiplereaction monitoring (MRM). In MRM, a specific setof precursor ions is selected and their transition fromthe selected precursor ion to a specific product ion fol-lowing CID is monitored.

1.3. Product (or daughter) ion scanning

Product ion scanning is a qualitative mode that canprovide structural information of a given compound.The first quadrupole (Q1) acts as a mass filter to se-lectively isolate a single mass/charge ion from the ionsource. The selected ion undergoes fragmentation inthe collision cell and is directed to the third quadrupole(Q3), which operates in a linear scanning mode to pro-duce a mass spectrum of the fragment ions.

1.4. Parent (precursor) ion scanning

Parent ion scanning can be considered the oppo-site of product ion scanning. With Q1 set to scan in alinear mode, fragmentation is induced and the daugh-ter ions are directed to Q3, which operates in SIMmode to filter out only one specific product ion foreach precursor ion. The use of this detection strategyhelps to ascertain the origin of a particular product ionthat may be a common moiety of several parent ions.For example, given three compounds (AX, AY, AZ)with different molecular weights each of which havea common charged moiety A+ in a Q1 scan, then byconfiguring Q3 as a static mass filter for ion A+, only

Table 1Possible fragmentation pathways of a hypothetical homologousseries of three compounds

Parent ion scanninga Constant neutral lossb

Precursorion

Production

Neutral Precursorion

Production

Neutral

AX+ A+ X AX + X+ AAY+ A+ Y AY + Y+ AAZ+ A+ Z AZ+ Z+ A

a Common product ion formed from each compound.b Unique ion formed from each compound.

parent ions, which produce A+ upon fragmentation inthe collision cell will be detected (Table 1).

1.5. Constant neutral loss scanning

In constant neutral loss scanning mode, Q1 is op-erated in a linear scan mode within a mass range thatcovers them/z ratios of the target analytes. Upon un-dergoing fragmentation in the collision cell, the ionsfrom Q1 are directed into Q3 which operates in ascan mode but offset from Q1 by a value that cor-responds to the mass of a common neutral fragment“A” ( Table 1). In this mode, all the components ofthe sample reaching the detector share the same frag-mentation pattern and this is well suited for sampleswhere several metabolites sharing the same metabolicpathway are expected.

2. Oxidation of DNA and DNA modificationmediated by oxidation

Oxidative damage to DNA may result from theaction of a number of oxidative species, which in-clude: HO•, H2O2, •O2, O3, NO•, −OONO. Thisrange of oxidants generates several types of DNAdamage including oxidation of purine and pyrimidine


bases, oligonucleotide strand breaks and DNA proteincross-links. In addition, reactive aldehydes generatedvia lipid peroxidation such as malondialdehyde[11]and 4-hydroxynonenal (HN) may add to the aminogroups of bases. About 70 products of nucleoside oxi-dation have been identified in model systems but only8 of these can be confidently said to occur withincells under normal circumstances. Assays for oxida-tive damage to DNA bases should ideally be sensitiveto the level of 1 modified base per 106 base pairs. Theuse of LC–MS in the determination of oxidative dam-age to DNA bases offers a similar level of sensitiv-ity to previously developed gas chromatography–massspectrometry (GC–MS) methods but carries less riskof promoting oxidation during the analytical proce-dure. In the case of GC–MS, a derivatisation step isrequired and it is possible that some oxidation couldoccur during this step[12]. Fig. 2 shows some of theproducts of oxidative damage to DNA and RNA bases.

3. Determination of oxidatively modified DNAbases

A method was developed for the analysis of8-ODDG (Fig. 2) in tissue and urine. Liver tissue wasextracted with buffer and DNA was purified fromthe pellet produced after centrifugation, extraction oflipids and removal of proteins using proteinase andfinally precipitation of the DNA with ethanol. Thepartially purified DNA was treated with RNAase,precipitated again and then redissolved in buffer andhydrolysed with nuclease and alkaline phosphatase.Urine samples were spiked with [15N3, 13C] 8-ODDG(IS) and were extracted with a C18 solid phase extrac-tion (SPE). RP-HPLC separation was carried out andpositive ion ESI spectra were obtained using an API365 mass spectrometer. The mass spectra in the posi-tive ion mode were composed largely of [MH]+ and afragment ion produced by the loss of the sugar moiety[B + H2]+. The [B+ H2]+ ion predominated whentandem MS was carried out. Samples were analysedin MRM mode where the transitions between [MH]+and [B+ H2]+ were monitored both for the IS andunlabeled 8-ODDG. The limit of detection (LOD) for8-ODDG was 20 fmol. The levels of 8-ODDG in liverand urine were 0.7 per 105 2′-deoxyguanosine (DG)and 20 pmol/ml of urine, respectively[13].

Nucleosides and salmon testes DNA were incu-bated with recombinant cyclooxgenase-2 (COX-2)and arachidonic acid. The reaction products wereanalysed by RP-HPLC, the DNA was hydrolysedprior to analysis. Negative ion ESI spectra wereobtained using a Quattro II instrument, quantifica-tion was carried out against stable isotope labelledstandards for 8-oxo-7,8-dihydro-2′-deoxyguanosine(8-ODDG) and 8-oxo-7,8-dihydro-2′-deoxyadenosine(8ODDA). A significant increase in 8-ODDG wasobserved which may be produced indirectly via theoxidation of arachidonic acid by COX-2[14].

Damage to DNA can arise through covalent modifi-cation of bases by reaction with oxidants and productsof lipid peroxidation. DNA from human liver sampleswas isolated and enzymatically hydrolysed. Stableisotope labelled internal standards (IS) were preparedfor 8-ODDG, pyrimido[1,2�]purine-10(3H)-one-2′-deoxyribose (M1-DG), Etheno-2′-deoxyadenosine(EDA) (Fig. 2) and Etheno-2′-deoxycytidine (EDC)(Fig. 2). The sample was loaded onto a reverse phaseextraction column with 100% ammonium acetate(pH 7) and the concentrated sample zone was thenbackflushed onto a second column. MS analysis wascarried out using a Quattro Ultima mass spectrome-ter operated in the positive ion mode. Cone voltageand collision voltage was varied depending upon theadduct. MRM was carried out for the transitions be-tween the molecular ions for the four adducts andthe ions derived via loss of the deoxyribose moiety.The limits of detection under these conditions were:8-ODDG<1 pg, M1-DG 0.3 pg, EDA 10 fg and EDC20 fg. Inter- and intraday precision for the analysisranged between 0.3 and 5.3%[15].

The formation of an 8-ODDG lesion in DNA ren-ders it susceptible to further oxidation. The oxidationof 8-ODDG containing 25 mer oligonucleotides by aCr(V) complex in phosphate buffer at room tempera-ture was studied. Separation of the reaction productswas carried out by RP-HPLC with a methanol/watergradient. The peaks eluting from the column were col-lected and lyophilised. The lyophilised fractions weredissolved in an aqueous buffer containing 2.5 mM im-idazole and 2.5 mM piperidine and 10% methanol wasadded. The samples were infused into both a QuattroII mass spectrometer and a QTOF mass spectrometer.Spectra were obtained in both the positive and nega-tive ion mode. It was found that the major oxidation


Fig. 2. Some of the more common products resulting from direct or chemically mediated oxidative damage of DNA.


products of the oligonucleotide were guanidinohydan-toin and spiroiminohydantoin. The results indicate theCr(V) could act as a co-carcinogen with xenobioticscapable of generating 8-ODDG[16].

A method was developed for the analysis of8-hydroxy-2′-deoxyadenosine (8HDDA) (Fig. 2).Calf thymus DNA in solution was treated with N2Oand �-rays. Enzyme hydrolysates of the DNA wereanalysed by RP-HPLC coupled to an Agilent MSDoperated in the positive ion mode with a fragmen-tor potential of 100 V. A mass spectrum for 8HDDAwas established using an authentic standard and wasfound to consist of [MH]+, [MNa]+ and an ion result-ing from cleavage of the glycosidic bond within themolecule. Hydrolysed DNA samples were analysedusing SIM of the three major ions in the spectrumof the 8HDDA that was quantified in comparisonwith corresponding ions for13C10–15N5 8HDDA thatdiffered by 15 amu from the ions for the unlabeledcompound. Determination of 8HDDA was also car-ried out using an established GC–MS method. Thelevels of 8HDDA determined by LC–MS and GC–MSwere 2.01 ± 0.03 and 2.12 ± 0.05 molecules/Gy ofradiation/106 base pairs, respectively. The limits ofdetection of 8HDDA determined by LC–MS andGC–MS were 10 and 2 fmol, respectively[17]. In arelated study, 8-cyclodeoxyadenosine (8CDDA) wasanalysed in irradiated calf thymus DNA. The oxi-dation product was quantified against13C10–15N5CDDA that was used as an internal standard. Thehydrolysed DNA was analysed using RP-HPLC cou-pled to an Agilent MSD operated in the positive ionmode with a fragmentor potential of 100 V. The ana-lyte gave a spectrum composed of [MH]+ and an ionresulting from the loss of most of the sugar ring. Theequivalent ions in the internal standard were shiftedby 15 and 11 amu, respectively. A detectable level ofCDDA of 1–1.5 lesions/107 base pairs could even beobserved in unirradiated DNA. The limits of detectionfor CDDA in LC–MS and GC–MS mode were 2 and1 fmol, respectively. Measurement of CDDA in livingcells may contribute to an understanding of defectivenucleotide-excision repair[18].

The ESI tandem mass spectra of the oxidised nu-cleosides 8-hydroxy-2′-deoxyguanosine (8HDOG),8-hydroxy-2′-deoxyadenosine (8HDOA), 2-hydroxy-2′-deoxyadenosine (2HDOA), thymidine glycol (TG)and 5-hydroxymethyl-2′-deoxyuridine (5HMDU)

were obtained in positive and negative ion mode usinga Quatro II tandem mass spectrometer operated usingargon as the collision gas at 25 eV. The mass spectrain the positive ion mode were composed largely of[MH]+ and a fragment ion produced by the loss ofthe sugar moiety. The negative ion spectra containedthe [M −H]− ion and ions due to the loss of the sugarmoiety and in some cases an ion due to fragmentationacross the sugar ring. Salmon testes DNA was enzy-matically hydrolysed and13C10–15N5 8HDOG wasadded as an internal standard. The sample was puri-fied by ultrafiltration and was analysed by RP-HPLCcoupled to the mass spectrometer operated in MRMmode. Fig. 3 shows MRM traces for the standardsfor the oxidised DNA; only 8HDOG was detected insalmon testes DNA. The limit of detection of 8HDOGwas determined to be 0.93 ppb[19].

A method was developed for the analysis of8HDOG and 8HDOA. The standards were analysedby RP-HPLC coupled to a Quattro Ultima tandemmass spectrometer operated in the positive ion modewith argon as the collision gas at 14 eV. The spectraof the standards were composed of [MH]+, [MNa]+and ions resulting from cleavage of the glycosidicbond within the molecule. Calibration curves for theanalytes were obtained in MRM mode monitoring thetransition between the [MH]+ ions and the fragmentions due to loss of the sugar moiety. The curves werelinear between 3.5 and 350 fmol on column withoutthe use of internal standard. Calf thymus DNA wasirradiated with �-radiation and then enzymaticallyhydrolysed. Both 8HDOG and 8HDOA were detectedeven in the untreated DNA, the 8HDOA was 15-foldlower than the 8HDOG at 7.1 per 106 bases. Theamount of the oxidised bases in the DNA increasedlinearly with radiation dose[20].

DNA and RNA nucleosides HOCl were treated withhuman neutrophils (activated with�-phorbol myris-tate), HOCl or myeloperoxidase. The resultant chlo-rinated nucleotides were isolated by RP-HPLC andinfused into a Quatro LC tandem mass spectrometer inorder to establish spectra for standard chlorinated nu-cleosides. The major products formed in calf thymusDNA was 5-chlorodeoxycytidine whereas in calf-liverRNA the major product was 8-chloroguanosine. Thetransitions used to detect chlorinated nucleosides inthe positive ionisation mode were those between themolecular cation of the chlorinated nucleosides and the


Fig. 3. Standards for DNA oxidation products run in MRM mode monitoring the transitions between the molecular ions and fragmentsderived via the loss of a deoxyribose moiety. Reproduced with permission from[19].

characteristic chlorinated daughter ion formed fromthe loss of the sugar moiety. Linear calibration curveswere obtained for all of the chlorinated nucleosidesstudied and the limit of detection was found to bearound 0.1 pmol for the different nucleosides[21].

The oligonucleotides d(AATGAAA), d(CGCGAT-ACGCC) and d(GAGTTGAG) were treated with 2%

OsO4. Separation by RP-HPLC was carried out usingtriethylammonium acetate buffer/CH3CN as the mo-bile phase. The fractions of the eluent were collectedand then analysed using a LCQ Deca XP ion-trap massspectrometer. The [M −H]− and the [M −2H]2− ionswere selected for MS/MS experiments. The oxidationof d(GAGTTGAG) was significantly faster than that


Fig. 4. MS–MS spectrum of the [M−2H]2− ion of an unoxidised 8 mer oligonucleotide and a thymine glycol oxidation product. Reproducedwith permission from[22].

of the other two oligonucleotides. The product-ion de-rived from the spectra [M−2H]2− for d(GAGTTGAG)and its preferentially formed 5′-end thymine glycolderivative can be seen inFig. 4. The pattern of com-mon ions and ions shifted by 34 amu in comparisonwith the unoxidised oligonucleotide indicated that theoxidation of the thymine had occurred at position 4(Fig. 4) [22].

The fragmentation mechanisms of T-rich tetra- andhexadeoxynucleotides were studied in order to de-velop a method for determining photo-oxidative dam-age in DNA. The protons on the nucleotides wereexchanged for deuterium using D2O which assistedin determining proton transfer during fragmentation.Negative ion spectra of the oligonucleotides were ob-tained using a Finnigan LCQ ion trap. Fragmenta-tion mechanisms favour the loss of 2′-deoxyadenosine(DA), DG and 2′-deoxycytidine (DC), which have ahigher proton affinity than DT, since the fragmentationmechanism involves proton transfer from an adjacentphosphate group to the base being expelled. Loss of

the base from the 3′-end is favoured over the 5′-end.When loss of the base occurs at the 5′-end this is fol-lowed by loss of the sugar moiety at that end whereaswhen loss of the 3′-base occurs this is followed byloss of D2O with the formation of a putative cyclicphosphate[23].

4. Determination of adducts resulting fromreaction of DNA with oxidation products of lipidsand xenobiotics

A tandem ESI mass spectrometry assay was de-veloped for the quantitative assay of 1,N2-Etheno-2′-deoxyguanosine (1EDG) in DNA. 1EDG can bemonitored in clinical situations where there is oxida-tive stress and it is believed to result from reactionof lipid oxidation products with DNA. DNA was ex-tracted either from rat liver or cultured fibroblasts andwas hydrolysed with nuclease P1. In order to carryout quantification15N5–1EDG was synthesised as an


Fig. 5. MRM trace for 1EDG extracted from rat liver and its labelled internal standard (1 pM). Reproduced with permission from[24].

internal standard. HPLC separation was carried outon two columns: a C18(2) Luna column for clean-upwith column switching to a C18(2) Phenosphere-Nextcolumn. The instrument used was a Quattro II massspectrometer. MRM was carried out using argon at6.7 × 10−4 mbar as the collision gas. The transitionsbetween the molecular ions for 1EDG and the inter-nal standard15N5–1EDG, respectivelym/z 292 and297 to their major fragment ions 176 and 181, re-spectively, were monitored. Linearity was achievedover the range 5–5000 fmol for the analyte withthe IS concentration being fixed at 1000 fmol. Thelimit of detection of the method was ca. 1.7 1EDGresidues/107 parent nucleoside molecules. Thus basallevels of this DNA lesion were readily detectable, e.g.in rat liver (Fig. 5) [24].

Acetaldehyde, a product of alcohol and sugarmetabolic oxidation, was reacted with calf thymusDNA. The DNA was reduced with NaBH3CN, enzy-matic hydrolysis of the product was carried out andthe hydrolysate was analysed by LC–MS. Separationwas carried out by RP-HPLC. The eluent was intro-duced into a Finnigan MAT LCQ Deca instrumentoperated in the positive ion mode with a needle volt-age of 4.5 kV and an interface temperature of 350◦C.The major adduct peak was identified as being dueto N2-ethylidene-DG. Other products included a

1,N2-adduct of DG and a product formed by linkingtwo DG residues together[25].

Reaction products between DG 3′-monophosphateand the benzene oxidation productsp-benzoquinoneand hydroquinone were characterised by MS. Reac-tion was carried out in ammonium formate buffer pH6.0 and the products were analysed by RP-HPLC. Thecollected fractions were then analysed by continuousinfusion MS into a Quattro BioQ mass spectrometerin the negative ion ESI mode. The tandem mass spec-tra of the products confirmed their structures givingprincipal ions that resulted from the loss of the sugarphosphate moiety[26].

The sugar-phosphate backbone in DNA is highlyvulnerable to oxidation and one of the products of theoxidation is 2-phosphoglycolaldehyde. The reactionof the related 2-phosphoglycolaldehyde (2-PG) with2′-deoxyguanosine and calf thymus DNA was carriedout (purine residues were removed from the DNAby heating in 0.1 M HCl). The adducts formed wereisolated by reverse phase HPLC. Mass spectra wererecorded in the ESI mode on either a Hewlett-Packard5988B instrument or a TSQ-7000 instrument. Anal-ysis by MS and NMR confirmed the formation oftwo 1,N2-cyclic adducts of DG with 2-PG. Theseadducts were also formed in calf thymus DNA[27].


5. Oxidation of proteins and protein modificationmediated by oxidation

The interaction of reactive oxygen species andmetabolites with proteins has been implicated in theetiology of a number of pathological conditions. Dis-eases associated with elevated levels of oxidised pro-teins include Alzeimer’s disease, rheumatoid arthritis,atherosclerosis, muscular dystrophy, cataractogene-sis, respiratory distress syndrome and progression ofage-related disorders[28–33]. Compared to oxida-tive modification of lipids (lipid peroxidation) andnucleic acids, the study of oxidative damage to pro-teins has not received much attention owing to thelarge number of protein targets and the relatively highnumber of different amino acid residues that haveto be investigated. Consequently, the study of oxida-tive protein modifications has necessitated the useof analytical techniques that are sensitive and havehigh throughput. Since proteomics involves analysisof proteins occurring at their natural abundances, thesensitivity of the analytical technique is of primeimportance on account of the fact that oxidativelymodified proteins may occur at low levels in vivo.Matrix-assisted laser desorption ionization coupledwith time of flight mass spectrometry (MALDI)–TOFand LC–ESI–MS/MS have emerged as the best meansfor the characterization of protein modifications andalthough they operate in entirely different ways, theygenerate complementary data and information forthe study of protein chemistry. MALDI–TOF is amatrix-dependent ionization technique and mainlyprovides peptide mass measurement, which is indis-pensable for analysis of oxidatively modified pep-tides. Although generic MALDI–TOF instruments donot provide amino acid/peptide sequence informa-tion, high-end MALDI–TOF instruments equippedwith reflectrons can be used to analyze peptide frag-mentation by a technique referred to as post-sourcedecay (PSD). In addition to providing identificationof oxidised proteins at the level of amino acid/peptidesequence, LC–ESI–MS/MS offers a flexible instru-mental approach which allows interfacing of an ESIsource with different mass analysers such as ion traps,TOF and quadrupole mass filters. A major challengein the study of oxidative modification of proteins isthe possibility of removing or inducing covalent mod-ifications of peptides during the sample work up and

peptide characterization processes, e.g. the formationof N-terminal carbamyl adducts of peptides due to useof urea in sample preparation has been reported[34].While the presence of small amounts of surfactants,buffer salts and alkylating agents used during enzy-matic protein digestion can interfere with peptide ion-ization in LC–ESI–MS, MALDI–TOF instruments aregenerally more tolerant to low levels of contaminantsin peptide samples. Consequently, sample preparationprocedures used for peptide analysis by LC–ESI–MSare crucial in the studies of protein oxidation. Addi-tionally, since modified peptides generally constituteonly a small fraction of proteins in a given cell, theMS–MS signals of unmodified peptides would be ex-pected to be more intense than for the modified ones.In order to increase the fraction of modified peptides,pre-concentration of polypeptides is commonly car-ried out by off-line preparative HPLC followed byinfusion into the ESI ion source. While this techniquereduces background interference by buffer salts of themobile phase, the process of sample pre-concentrationincreases analysis time. Typical mobile phase systemscurrently used for peptide analysis consist of acetoni-trile, water and 0.1% trifluoroacetic acid (TFA) withgradient rates of 1% min−1 from 0 to 80% acetonitrile.The identification of oxidatively modified proteins isfurther facilitated by MS–MS data mining softwaresuch as the Sequest program and the “Scoring Algo-rithm for Spectral Analysis” (SALSA). Some of themodifications that occur at amino acid residues inproteins as a result of oxidation are shown inFig. 6.

5.1. In vitro

Free radical–protein interactions are believed tobe closely related to the physiology of ageing andpathologies such as atheroslerosis. The occurrenceof hydroxyleucines in normal human plasma andlow-density lipoprotein (LDL), and their elevation incataractous as against normal human lens proteinswas demonstrated. Hydroxy derivatives of leucinewere generated in vitro by�-radiolysis of leucineprior to their determination in biological samples.Plasma and LDL proteins were purified by TCA pre-cipitation following acetone and diethylether washingbefore acid hydrolysis in 6N HCl, whereas frozenlens samples were powdered and freeze-dried beforehydrolysis. Analysis was performed by ESI–MS on a


Fig. 6. Some reactions of amino acids within proteins promoted by oxidation.

VG Platform mass spectrometer and electron impact(EI) mass spectra were recorded on a Finnigan MATTSQ-46 quadrupole analyser with ionization potentialof 70 eV and source temperature of 140◦C. Followingprotein hydrolysis, analytes were separated and pre-concentrated on a semi-preparative LC-NH2 columnwith 81% acetonitrile in sodium phosphate buffer.The fractions were infused into the ESI source wasvia a syringe pump at a flow rate of 5�l/min [34].

Cytochrome c was treated with H2O2 and di-bromonitrosobenzenesulfonic (DNBS) was used totrap the free radicals that were formed. ESI–MS wasused to monitor adduct formation where shifts in themass spectrum indicated the addition of 1 or 2 DNBSmoieties. The experiment was repeated to determine

where or not cytochromec in the presence of H2O2could cause free radicals to be generated in other pro-teins/peptides. A nonapeptide containing two tyrosineand one tryptophan (Trp) residue was found, follow-ing analysis by tandem MS to form an adduct withDNBS at the tryptophan residue in the presence of cy-tochrome C/H2O2. Ascorbic acid was found to inhibitthe formation of radical species from the peptide[35].

Numerous side chains of amino acids found inextracellular proteins are known to be modified byhypochlorite which is produced as a result of antiba-cterial and inflammatory response of neutrophils andmacrophages. Hypochlorite initiated modificationof low-density lipoproteins is believed to induce arth-erosclerotic plaque formation[36]. The modification/


relative reactivity of amino acid residues (His, Arg,Tyr, Lys, Trp and Met) in short N-blocked peptidesfollowing treatment with hypochlorite was investi-gated. Isolated peptides were digested with 6 M HCland subjected to analysis following derivatizationwith 2,4-dinitrophenylhydrazine (DNPH). Detectionand analysis of modified peptides was achieved byRP-HPLC using gradients between 0.1% aqueous TFAand acetonitrile. Peptides were evaluated before andafter modification using electrospray mass spectrome-try and by high-resolution nuclear magnetic resonance(NMR). The relative reactivity of lysine and other pep-tide bound amino acids to oxidation by hypochloritewas investigated[37]. The role of protein modificationin atherosclerosis has been recently reviewed[38].

In order to characterize amino acid residues thatare highly susceptible to carbonyl formation duringhypochlorite attack, bovine insulin was treated withhypochlorite at pH 4. This was followed by derivatiza-tion with DNPH (which is used as a label for carbonylreactive compounds) and tryptic digestion of bovine

Fig. 7. Tandem MS of the B-chain of (A) native insulin (B) insulin after treatment with HOCl and derivatisation with DNPH. Reproducedwith permission from[19].

insulin. RP-HPLC analysis of native insulin, modi-fied insulin and tryptic peptides was achieved usinga Vydac wide pore C4 column at room temperature.Fractions of eluent containing DNPH-labelled peakswere collected, evaporated to dryness and reconsti-tuted in 50:50 MeOH/H2O, with 1% acetic acid. Elec-trospray MS was carried out using a Hewlett-Packard5988A quadrupole mass spectrometer equipped with10 kV high-energy dynode electron multiplier. Sam-ples were analysed by flow injection and for CID massspectrometry, the quadrupole was scanned from 14 to500 Da. The electrospray CID–MS analysis of nativeinsulin B-chain and that from DNPH-labelled insulinis shown inFig. 7. In the CID mass spectra, the B-chainobtained from native insulin showed a fragment ofm/z 120, consistent with the intact N-terminal Phe ofthe B-chain (Fig. 7A) whereas in the spectrum of theDNPH-modified B-chain (Fig. 7B), the fragment atm/z 120 is absent and replaced with ions atm/z 398 and426 which represent the major and minor phenyalaninemodification observed in DNPH-labelled insulin[39].


The oxidation of bovine�-casein by hypochlo-rite (HOCl) was investigated. Following exposure toHOCl at 4◦C for 15 min, derivatization with DNPHand digestion with trypsin, the resultant peptides wereseparated by RP-HPLC using a gradient betweenaqueous TFA and acetonitrile. Samples were collectedin the eluent and infused through a silica capillary intothe mass spectrometer. Electrospray tandem MS wascarried out with a Sciex API III+ triple quadrupolemass spectrometer with collision energy settings of20–30 eV. One peptide isolated from a peak absorbingat 365 nm was identified as AVP(Y∗)PQR, corre-sponding to amino acids 177–183 of bovine�-casein.Analysis of the peptide by both electrospray andMALDI mass spectrometry identified a molecular ion[MH]+ of 1008.5 Da, which represented an increaseof 178 Da from the calculated monoisotopic [MH]+of the unmodified peptide of 830.45 Da. Daughterion spectra of the doubly charged parent ion of thepeptide further revealed that oxidation converted tyro-sine to the quinone methide which was subsequentlyconverted to the corresponding DNPH. It was demon-strated that methionine (M) and tyrosine (Y) residueswere most susceptible to oxidation upon exposure ofbovine�-casein to HOCl[40].

LC–ESI–MS/MS and the SALSA algorithm havebeen applied to the identification and characterizationof products formed from the reaction of epoxides withhuman hemoglobin (Hgb) in vitro. Hgb adducts maybe formed by reaction with epoxides formed fromstyrene, ethylene and butadiene and thus constitutebiomarkers for exposure to these chemicals. Follow-ing the synthesis of Hgb adducts and tryptic digestionof the precipitated globins, the digested globin sam-ples were diluted with acetonitrile/water and analysedusing a Thermofinnigan LCQ ion trap mass spectrom-eter equipped with an electrospray ionization source.Chromatography was carried out on a Vydac proteinand peptide C18 column (250 mm× 1 mm) at a flowrate of 15�l min−1 with a mobile phase gradient of3–95% acetonitrile: (0.5% formic acid/0.01% TFA).Data-dependent scanning was used to achieve auto-mated acquisition of MS–MS spectra of peptide ions,with a dynamic exclusion mass set to±1.5 m/z andions residing on the exclusion list for 5 min. By usingsequence motif-based data analysis of MS–MS spec-tra, the mass and position of adducts were established.The adduct sites were identified as the N-terminal va-

lines of both Hb� and Hb�, glutamic acid 7, cysteine93 and histidines 77, 97 and 143 of the�-chain andhistidine 45 of the� chain[41].

Oxidative modifications of low-density lipopro-teins are believed to contribute to the pathogene-sis of atherosclerosis. The oxidation of LDL “invitro” is known to be accelerated significantly bymetal ions and inhibited by chelating agents. Fol-lowing Cu2+-catalyzed oxidation of LDL isolatedfrom human plasma, nine peptides from oxidisedLDL in which the tryptophan residues had been con-verted to the respective kynurenines were identified.DNPH-modified and unmodified delipidated LDLwere digested (trypsin), and the digested apolipopro-tein B-100/peptide mixtures were analysed usingRP-HPLC eluted with a TFA/acetonitrile gradient.The major peptide peaks were collected manuallyand were subsequently analysed with a FinniganMAT TSQ-700 mass spectrometer on which wasmounted a modified Vestec electrospray source. Thesamples infused through a silica capillary into themass spectrometer[42]. The oxidation of apolipopro-tein B-100 by myeloperoxidase was also studied.ESI tandem mass spectrometry was employed for theidentification of myeloperoxidase-catalysed oxida-tion of N-terminal amine of apolipoprotein B-100 inlow-density lipoproteins isolated from human plasma.Following derivatization of oxidised proteins with2,4-dinitrophenylhydrazine, delipidation and trypticdigestion, peptides were separated by RP-HPLC usinggradient elution with TFA–acetonitrile. Mass spec-trometric analysis was carried out using a modifiedSciex QqTOF instrument which could be operatedboth in the ESI and the MALDI modes. A net gain of179 amu was observed for the protein after oxidationand reaction with DNPH and this combined with thepattern of B and Y ions in the spectrum were consis-tent with preferential oxidation of the N-terminus ofthe protein by MPO[43].

The nitration of tyrosine to 3-nitrotyrosine (3-NT)has been associated with the onset of diseases likeAlzheimer’s (AD) and Parkinson’s diseases, 3-NTmight thus used as a biomarker for such pathologies.The determination of 3-NT and tyrosine in biologicalsamples has been performed by LC–MS/MS. Fol-lowing the isolation and digestion of protein fromrat brain tissue, the digested samples were extractedusing SPE. LC–MS/MS assay was carried out with


a Micromass Quattro mass spectrometer operated inthe positive ESI mode. A collision energy setting of13 eV was used and the transition between the molec-ular ion m/z 227.1 and major fragment ion atm/z181.1 of 3-NT was monitored. Separation was carriedout using RP-HPLC. The assay of 3-NT was linearbelow 100 ng/ml and the limit of detection was below100 pg/ml[44].

5.2. In clinical samples

Oxidative alterations of proteins by reactive oxy-gen species (ROS) have been implicated in the pro-gression of ageing and age-related neurodegenerativedisorders such as Alzheimer’s disease. Protein car-bonyls, which constitute biomarkers of protein ox-idation, are increased in AD brain, indicating thatoxidative protein modification is involved in thegenesis of AD. Detection and isolation of proteinscontaining reactive carbonyl groups in post-mortembrain tissue of five AD and control subjects was car-ried out by two-dimensional gel protein mapping andimmunological techniques. The specific identity ofoxidized proteins was investigated using MicromassMALDI–TOF instrument operated with a 337 nm N2laser at 20–35% power in the positive ion reflectronmode. Peptide mass fingerprinting was used for identi-fication of tryptic protein fragments by using the Mas-cot search engine (http://www.matrixscience.com)querying the entire theoretical human peptide massesin the NCBI and SwissProt databases. The three tar-gets of protein oxidation in AD were identified asglutamine synthase, creatine kinase BB and Ubiquitincarboxy-terminal hydrolase L-1[45].

6. LC–MS methods for the analysis of theproducts of lipid oxidation

Lipid peroxidation has been implicated in thepathogenesis of a number of human diseases includ-ing atherosclerosis, cancer and neurodegenerativediseases. Polyunsaturated fatty acids are easily trans-formed into hydroperoxides (LOOHs) either by enzy-matic or free radical action and such processes occurduring ageing injury or disease. LOOHs are unsta-ble and the O–O bond is readily cleaved to yield analkoxy radical. These radicals can then go on to react

with other lipid molecules or undergo rearrangementto produce a variety of products. The oxidised prod-ucts may be formed as low molecular weight cleavageproducts or may remain attached to the remainderof the lipid structure. Some examples of lipid oxi-dation products are shown inFig. 8. Peroxides andconsequently hydroxyacids can form at a number ofpoints in the chain of a polyunsaturated lipid. Directdetermination of lipid peroxides and determinationof hydroxylated lipids by LC–MS is one of the mostcommon measurements. The short chain aldehydesand hydroxyaldehydes are usually determined by us-ing GC–MS[46]. The ability of living organisms toprevent and repair damage due to lipid peroxidationmay have an important impact on the prevention ofdisease and slowing down the ageing process. Thecharacterization and biological testing of the productsof phospholipid oxidation has been recently reviewed[47,48].

6.1. Analysis of oxidised phospholipids whichpromote monocyte adhesion

LDL was isolated from the plasma of normal blooddonors and the formation of mildly oxidised LDL(MMLDL) was catalysed by Fe2+ and the modellipid 1-palmitoyl-2-arachidonylglycerophosphocho-line (PAPC) was oxidised by exposure to air for24–48 h. Oxidised lipids were fractionated by normalphase chromatography followed by further separationof the isolated fractions by reverse phase chromatog-raphy. Lipids were introduced into the API III+ESI–MS instrument either via HPLC or via flow in-jection in acetonitrile/aqueous formic acid for positiveion spectra or methanol containing 1 mM ammoniumacetate for negative ion. The oxidised phospholipidswere quantified against a dimyristoylphosphatidyl-choline internal standard. Analysis of PAPC byESI–MS revealed a range of oxidation products, theidentities of these as such were confirmed by in-creasing the orifice voltage to promote formation ofcommon fragments such as the ion atm/z 183 due tothe phosphocholine head group. Increase in the com-mon fragment ions corresponded with a decrease inthe molecular ions of the oxidation products. The bi-ological activity of the phospholipid fractions derivedfrom PAPC was assessed in a monocyte adhesionassay and biological activity was found for fractions

http://www.matrixscience.com


Fig. 8. Some examples of the products of lipid peroxidation of the an unsaturated fatty acid within a phosphatidylcholine lipid.

yielding ions atm/z 594–595, 610–611 and 828–829.Phospholipid fractions isolated from MMLDL alsocontained species with these ions. Reduction withsodium borohydride and reaction with methoxylaminehydrochloride (MOHCl) and pentafluorobenzyl bro-mide followed by LC–ESI–MS were used to charac-terise the biologically active oxidation products. Twoof the lipids in the biologically active fractions wereidentified as 1-palmitoyl-2-(5-oxovaleryl)-GPC and1-palmitoyl-2-glutaryl-GPC. The third active lipidwith m/z 828–829 was not completely characterised

[49]. In an extension of this study, the structure ofthe unknown active component was elucidated usinga combination of techniques including: derivatisationwith MOHCl, which reacted with epoxide and ketonegroups in the unknown producing shifts in the ESIspectra of the indicating the number of such groupsin the molecule. Reduction with sodium borohydrideor borodeuteride indicated the number of ketone andhydroperoxide groups. Treatment with phospholi-pase A2 was used to release free fatty acids. Thefree fatty acids liberated from the oxidation products


Fig. 9. MS–MS spectrum of a biologically active lipid isolated from oxidised PAPC (A) before and (B) after exchange in H218O.

Reproduced with permission from[50].

were analysed using tandem MS in the negative ionmode. Exchange of oxygen atoms in the analyte andH2

18O was used in the elucidation of the fragmen-tation pathways of the biologically active oxidationproduct.Fig. 9 shows the fragmentation scheme forthe fatty acid liberated from the dehydration productof the biologically active fraction by phospholipaseA2 before and after exchange with H2

18O. Thus, thebiologically active compound could be identified as1-palmitoyl-2-(5,6-epoxyisoprostane E2)-GPC[50].

The presence of 5-hydroxy-8-oxoct-6-enoylGPC(HOOAPC) in oxidised PAPC was determined usingRP-HPLC coupled to ESI–MS by comparison with asynthetic standard for this oxidation product. Furtherconfirmation of identity was carried out using tan-dem MS to produce characteristic fragments of themolecule and derivatisation with MOHCl. HOOAPCwas found to be active in a monocyte adhesion assayand increased the biosynthesis of monocyte chemo-tactic protein and interleukin-8[51].

6.2. Analysis of oxidised plasmalogen lipids

Phospholipids were extracted from the membranesof red blood cells and fractionated into their sub-classes using normal phase HPLC. MS–MS wascarried out using a Sciex API III+ triple quadrupolemass spectrometer in the negative ion mode withCID at 11 eV using argon gas. Initially, the massspectrometer was used in MS mode to identify thoselipids specifically containing arachidonic acid. Thiswas done by elevating the orifice potential to−110 Vto produce an ion atm/z 303 due to the carboxylateion of the arachidonate chain. In order to further char-acterize the arachidonate–containing lipids tandemmass spectrometry was carried out. Predicted precur-sor ions for the arachidonic acid–containing lipidswere selected and the transition yieldingm/z 303 wasmonitored. It was found that the most abundant lipidscontaining arachidonate at position 2 were 16:0p/20:4glycerophosphoethanolamine (GPE) (Fig. 10) and


Fig. 10. A plasmalogen lipid.

16:0a:20:4 where position 1 is acylated rather thanalkylated. Direct oxidation of red blood cells was alsocarried out usingt-butylhydroperoxide (tBuOOH) andthis was followed by tandem MS with monitoring ofmono-oxygenated carboxylate ion derived from thearachidonic chain atm/z 319. Similar experimentswere carried out on lipids derived from bovine brain. Inorder to fully characterize the mono-oxygenated fattyacid species, saponification of the oxidised lipid frac-tions was carried out. The liberated mono-oxygenatedfatty acids could then be characterised by their typicalfragmentation patterns obtained following separationby reverse phase HPLC. The mono-oxygenated fattyacids derived from arachindonate were a mixtureof epoxides (ETEs) and hydroxides (HETEs). Tenoxygenated products were identified on the basis oftheir fragments promoted by� cleavage adjacent tothe hydroxyl or epoxy groups in the fatty acid chain.Fig. 11 shows ion chromatograms derived via dif-ferent transitions fromm/z 319 [52]. In an extensionof the work described above, 1-O-hexadecen-1′-enyl-arachidonylglycerophosphocholine was oxidised us-ing 2,2′-azobis-(2-amidopropane)hydrochloride. Thesample was separated by RP-HPLC. The column ef-fluent was split to yield a flow of 25�l/min. to themass spectrometer and 25�l/min into a fraction col-lector. MS was carried out using a Sciex API III+triple quadrupole mass spectrometer in the positive

ion mode with CID at 30 eV using argon gas. CIDwas used to identify chromatographic peaks thatyielded m/z 184 typical of the phosphocholine headgroup. Analysis in the negative ion mode was used toidentify the oxidation products. A high negative ori-fice potential (−100 V) was used to promote demethy-lation of the choline head group yielding [M − 15]−ions from acetate adduct ions formed with the dif-ferent molecular species. The peaks observed in thenegative ion mode were then subjected to tandem MStypically yielding fragments characteristic of the acylgroup on position 2 and characteristic of the rest ofthe molecule derived via loss of the acyl group at po-sition 2 as a neutral ketene fragment. Saponificationof the lipids was carried out and the products derivedfrom oxidation of the arachidonic acid group werethen analysed using multiple reaction monitoring of,predominantly� cleavage fragments, derived from theions m/z 319 and 335 which are derived via additionof one or two oxygen atoms to arachidonic acid. Theresults indicated that the plasmenyl lipid underwentunique and complex oxidation that may be related toits ability to protect cells from oxidative stress[53].In a closely related study, oxidation of 16:0p/20:4GPC was carried out using Cu2+ and H2O2 used as asource of hydroxyl radicals. The LC–MS conditionswere similar to those described above. The oxidationproducts of HAGP differed from those produced using


Fig. 11. LC–MS–MS of monohydroxy and monoepoxy arachidonic acids derived via saponification of oxidised lipids extracted from redblood cell membranes. The chromatograms are normalised relative to added [18O2] 12-HETE internal standard. Reproduced with permissionfrom [52].

AAPH in that mono- and deoxygenated oxidationproducts of arachidonic acid were produced as thefree acids rather than remaining esterified as part ofthe phospholipid[54]. The findings of the results fromthis and related work has recently been reviewed[55].

Post-mortem brain tissue samples were obtainedfrom patients who had suffered from Alzheimer’s dis-ease of varying severity. Dipentadecanoylglycero-pho-sphoethanolamine was added as an internal standardto samples prior to extraction. Desalted extracts weredirectly infused in chloroform/methanol-containingLiOH into a TSQ-7000 mass spectrometer operatedin the negative ion mode. The molecular ions of thepredominant phosphoethanolamine lipids could beclearly seen (Fig. 12) and marked differences wereobserved between white and gray matter and betweenthe white matter of normal brains and brains withearly Alzheimer’s disease. The enrichment of do-cosahexanoic acid (m/z 791 and 775) and arachidonicacid (m/z 751) containing lipids in gray matter wasimmediately obvious. The identity of these lipids wasconfirmed by tandem MS yielding the product ions atm/z 327 and 303 for docosahexanoic acid and arachi-donic acid, respectively. The white matter from thebrains of AD subjects was markedly deficient in theplasmalogen lipids (m/z 775, 751, 727, 701 comparedwith IS m/z 663; Fig. 12) when compared with the

white matter from normal brains. The main depletionwas in the 18:1–18:1 plasmalogen lipid (m/z 727)[56].

6.3. Oxidation of HDL and LDL

In the acute phase normal response high-densitylipoprotein (NHDL) is converted into acute phaseHDL (APHDL) and becomes proinflammatory andunable to protect LDL against oxidative modification.Blood samples were obtained from healthy volun-teers and from patients who had undergone bypasssurgical procedures 34–38 h after surgery. The HDLfractions were isolated and then extracted with chlo-roform/methanol. Normal phase HPLC separationswere carried and the column effluent was fed into aHewlett-Packard 5988B mass spectrometer with anESI interface operated in both the positive and neg-ative ion mode. The oxygenated acids were specifi-cally identified by LC–ESI–MS and in comparisonwith standards. As well as other variations in lipidcomposition, the isoprostane containing phosphatidyl-cholines (PCs) of NHDL were present in much largeramounts than those in APHDL[57].

The ability of apolipoprotein A-1 (A1) and apeptidomimetic analogue of this protein to preventoxidation of the lipids within human low-densitylipoprotein was studied. The lipids were isolated


Fig. 12. Negative ion ESI spectra of phosphatidylethanolamine lipids extracted from temporal gray and white matter from normal humanbrain (a and b); (c) temporal white matter from the brain of a subject with mild AD.

from A1 and LDL by solvent extraction followed bysolid phase extraction and were then fractionated byRP-HPLC. ESI–MS was used to confirm the transferof fatty acid hydroperoxides and lipid hydroxidesfrom LDL to A1 by monitoring the molecular ionsof these species in the negative ion mode. Removalof oxidised ‘seeding lipids’ from LDL removed theability of human arterial walls to oxidize LDL andNHDL was observed to exert a similar effect. In pre-vious studies, it had been found that three oxidisedphospholipids having molecular ions ofm/z 594, 610and 828 were associated with the ability of LDL topromote monocyte adhesion to artery walls. The oxi-dised free fatty acids 13(S)-hydroperoxyoctadecenoic

acid (HPODE) and 15(S)-hydroperoxyeicosatetrae-noic acid were found to act as ‘seeding lipids’ in pro-moting the formation of the oxidised phosplipids asjudged by monitoring of their molecular ions in lipidsextracted from LDL[58,59].

The hydrolysis of lipids in normal and acute phaseHDL by the acute phase enzyme phospholipase A2was studied. HDL was isolated from the plasma ofhealthy volunteers and from the plasma of patientsundergoing cardiac surgery. Total lipids were sep-arated by normal phase HPLC that was connectedto a Hewlett-Packard 5998B MS system. Both pos-itive and negative ion spectra were obtained. Anal-ysis by LC–MS revealed that after 4 h ca. 30% of


phosphatidylcholine lipid NHDL was hydrolysedby phospholipase A2 in comparison with ca. 80%of APHDL. During hydrolysis levels of lyso phos-phatidylcholine increased more rapidly in the case ofAPHDL lipids compared with NHDL lipids. ESI–MSprofiles of the fatty acids released by hydrolysisof the lipid fractions revealed that a much largeramount of oxidised fatty acid material was present inAPHDL compared with NHDL[60]. The methods foranalysing HDL and oxidatively modified LDL havebeen recently reviewed[61,62].

6.4. Miscellaneous analyses of oxidised lipidsamples in clinical and biological samples

Cytochrome P450 derived linoleate arachidonatemetabolites were quantified in urine by tandem MSin healthy human subjects, Sprague–Dawley ratsand spontaneously hypertensive rats. Oxylipid stan-dards were chemically synthesised and purified us-ing HPLC. Odd chain length monounsaturated fattyacids were used to prepare internal standards usedto adjust for losses during extraction. Urine sampleswere spiked with internal standard, saturated sodiumchloride solution was then added and the sampleswere extracted and the extracts were analysed byRP-HPLC and the column eluent was passed into aQuattro Ultima mass spectrometer. ESI was carriedout in the negative ion mode argon was used as thecollision gas with collision voltages in the range−15to −26 V depending on the analyte. Changing thecone voltage had a marked effect on the intensityof [M − 1]− ions. Having optimised the molecularion intensity, CID was carried out and SRM wascarried out on each of the target analytes. Cleavageof the chains of the oxidised lipids was typicallypromoted at a site adjacent to added oxygen atom.Limits of quantification were typically 0.01–0.5 nMin urine with a precision of ca.±15%. In rat urine,octadecanoid oxidation products were present in therange 10–80 ng/ml and ecosanoid oxidation productsat <1.5 ng/ml. In human urine, octadecanoid oxida-tion products were in the range 0.005–497 pmol/mgcreatinine and ecosanoid oxidation products werein the range 0–1.13 pmol/mg creatinine. Thus, a ro-bust method was established for the quantification ofthese potentially important markers of disease states[63].

Aldehydes formed from phosphatidylcholine lipidspresent in human artheromas were examined in aor-tic plaques removed from patients undergoing en-darterectomy or aortic reconstruction. The atheromasamples were homogenised in phosphate buffer andthe aldehydes were converted to DNPH derivatives.The derivatives were then extracted, treated with phos-pholipase C and then analysed by reverse phase HPLCin combination with thermospray mass spectrometryusing a Hewlett-Packard 5985B mass spectrometer.Negative ion spectra were obtained for the DNPHderivatives which gave predominately [M − 1]−ions. Underivatised phospholipid extracts were alsoanalysed using a Hewlett-Packard 5988B instrumentwith an electrospray interface. Chromatography wascarried out using a normal phase column. Both neg-ative and positive ion spectra were obtained. Themajor core aldehydes in atheromas were identified as1-C18-alkyl,1-C16-alkyl, 1-C16-acyl and 1-C18-acylderivatives with 2-oxovaleroyl or 2-oxononanolylgroups. The 1-C16-alkyl-2-(5-oxovaleroyl)-phosphati-dylcholine lipid was found to have strong plateletaggregating properties[64].

Phospholipids from rat liver were analysed directlyby ESI–MS, analysed following off-line fractionationby HPLC or by TLC. ESI–MS was carried out us-ing a Quattro II triple quadrupole instrument. Analy-sis was carried out in the positive ion mode and CIDwith argon being carried out at 15–40 V. The study re-vealed that there was a linear response to variation inphospholipid concentration below 2 nmol/ml. If sam-ples were pre-fractionated using TLC or HPLC therewas a selective loss of molecular species with lessthan three double bonds. Oxidation of the phospho-lipids occurred within 30 min of their application toTLC plates[65].

6.5. Miscellaneous in vitro model systems andmethodological variations

Lipids were extracted from human platelets and thedifferent phospholipid classes were fractionated us-ing TLC. The reaction of the lipid peroxidation prod-uct 4-hydroxynonenal were studied by incubating withthe phosphatidylethanolamine (PE) lipids in buffer atpH 8.5. HPLC separation was carried out on a sil-ica gel column and fractions were collected and anal-ysed by ESI–MS in the negative ion mode using a


Finnigan TSQ-7000 instrument. HN reacted with PElipids to produce a Schiff base adduct, a Michael ad-dition adduct and a pyrrole adduct[66].

The oxidation of linoleic acid by soybean lipoxy-genase was studied. The enzymatic mixture was ex-tracted and the extract was evaporated and dissolvedin methanol for analysis by LC–MS. ESI–MS anal-ysis was carried out using a TSQ-7000 instrument.The most prominent ions were due to NH4

+ adductsin the positive ion mode andM − 1 ions could beobserved in the negative ion mode. CID spectra wereobtained with argon as the collision gas and a col-lision energy of 7 eV. The product ion spectrum for13-hydroperoxy-9-Z-11-octadienoic acid gave ionsresulting from the loss of H2O2 and NH4

+ and aprominent ion atm/z 195 located at the position ofthe peroxide group which directs cleavage betweenC12 and C13 indicating the presence of OOH at C13.Similarly, a OOH group at C9 produced an ion atm/z155 due to cleavage between C9 and C10 [67].

Phospholipid vesicles containing arachidonate wereoxidised with H2O2 and Cu(II) with aim of testingthe action of oxidised phospholipids on prothrombi-nase activity. Oxidised lipids were infused into a VGQuattro II mass spectrometer. ESI–MS revealed thepresence of mono-, di-, tri- and tetrahydroperoxidederivatives of the arachidonate containing lipid. It wasobserved that up a certain level the oxidised lipidsaccelerated prothrombinase activity[68].

Lipid oxidation products were studied in commer-cially available phospholipids. The samples were ei-ther analysed without pre-treatment or treated withpentamethyl-6-chromanol in order to produce oxidisedlipids. Analysis of oxidation products was carried outusing a TSQ-7000 instrument. RP-HPLC separationwas carried out with 0.5 mM AgBF4 being mixedpost-column with the eluent from the HPLC columnor AgBF4 was added to the mobile phase to producea 0.15 mM solution in order to carry out silver ionco-ordination ion spray mass spectrometry (CIS-MS)of lipid peroxidation products either CIS-MS allowedunambiguous identification of the oxidation products.Although ESI–MS allows identification of the molec-ular ions of oxidation products when tandem MS ex-periments are carried out the spectra produced aredominated by the ion for the phosphocholine headgroup. CIS-MS results in formation of Ag+ adductsof the fragment ions which result in spectra which al-

low the identification of the position in the lipid chainat which oxidation has occurred[69].

Intact oxidised phospholipids in oxidatively stressedmammalian cell lines and phospholipid vesicles weredetermined by ESI–MS. Phospholipid vesicles wereincubated witht-butylhydroperoxide and FeSO4 or inthe presence of NaOCl in order to study the formationof chlorohydrins. A human monocytic cell line wassubjected to similar oxidative stress. The phospholipidsamples were extracted with methanol/hexane and celllipids were extracted with methanol/chloroform. Sam-ples were analysed by positive ion ES–MS using aVG Platform mass spectrometer. The more extensivelyoxidised lipids eluted earliest and the chlorohydrinseluted over a similar time range[70].

The conjugation product formed between glutha-thione (GSH) and 15-A2t-isoprostane (AIP) was anal-ysed following a reaction catalysed by gluthathionetransferase (GT). AIP was incubated with GT andGHS and the products were isolated by SPE. Theproducts were analysed using reverse phase HPLCconnected to a TSQ-7000 MS. CID was carried outusing argon gas at 0.033 Pa with a collision energyof −20 eV. The parent ion for the GSH conjugate ofAIP gave a characteristic fragment arising from theloss of GSH. The rate of conjugation of GSH to AIPproved to be very rapid[71].

A solution containing calf lung surfactant extract(CLSE) was treated with ozone and the sample was ex-tracted into chloroform/methanol. The products wereseparated by straight phase HPLC. Mass spectrometrywas carried out in the positive ion and negative ionmode using a PE Sciex API III+ triple quadrupole in-strument. In negative ion mode, the (M − 15) ion wascollisionally activated as described previously[19].The major oxidation product of the phospholipidsin CLSE was found to be 1-palmitoyl-(9′-oxono-nanoyl)-glycerophosphocholine which yielded a char-acteristicm/z 171 ion in its tandem mass spectrum(Fig. 13). This lipid was synthesised and was foundto be significantly cytotoxic when cultures of humanlung epithelial-like cells were treated with it[72].

Lithiated adducts were used to facilitate the identi-fication of phospholipids extracted from tissue. Whilstoxidation products were not analysed in this study, thismodification of technique might prove useful whenapplied to the analysis of lipid oxidation products.Standard lipids and lipid extracts from rat brain


Fig. 13. Tandem MS spectrum of a cytotoxic aldehyde isolatedfrom CLSE treated with ozone. Reproduced with permission from[72].

were dissolved in chloroform/methanol and LiOHwas added to give a concentration of 2 nmol/�l. Thesamples were infused into a TSQ-7000 instrumentand tandem MS was carried out using argon as thecollision gas at 32–36 eV in the positive ion mode.Without the addition of lithium, MS–MS spectra weredominated by the head group ion atm/z 184. Lithi-ated adducts exhibited more complex fragmentationyielding ions resulting from losses of the phospho-choline head group, fatty acid ester groups and alsoions due to acylium ions derived from fatty acids.There was evidence that the ions derived via lossesof fatty acids were more abundant for the loss of thesubstituent at sn-1 reflecting the position of the fattyacid substituent[73].

7. Analysis of antioxidant xenobiotics inbiological systems

7.1. Phenolic compounds

Polyphenols (Fig. 14) are of widespread dietaryoccurrence and have well reported “in vitro” antiox-

idant activity [74]. Relevant dietary sources includeonions, apples, wine, green tea and citrus juice[75].There has been an association between flavonoidintake and inverse risk of coronary heart diseaseand certain types of cancer[76,77], two diseases inwhich oxidation plays a pivotal role. In addition,soy isoflavones have estrogenic activity that mightbe therapeutically relevant[78]. Despite the interestraised by the different pharmacological activities ofpolyphenols there are still certain aspects related totheir metabolism and bioavailability that need furtherstudy, including whether or not they can be absorbedas glycosides or need previous hydrolysis for absorp-tion as aglycones. The determination of polyphenolsand their metabolites in biological fluids is importantin this context. Liquid chromatography coupled withmass spectrometric analysis has become the methodof choice for the determination of xenobiotics incomplex biological matrices. Polyphenols are usu-ally analysed by liquid chromatography–electrospraymass spectrometry in negative ion mode using an acidas a modifier to improve peak shape. Trifluoroaceticacid gives usually the best peak shape, but stronglysuppress ionization in negative ion mode electrospray.A recent study[79], demonstrated that substitution ofTFA by acetic acid caused a 230-fold increase in thesensitivity of negative electrospray for the detectionof 3′,4′,5′-trimethoxyflavone with only a marginal in-crease in peak width. In a recent study on the effectof eluent composition on the ionisation efficiency offlavonoids in LC–MS, Rauha et al.[80] found that thebest sensitivity (LOD between 0.8 and 13�M for aninjection volume of 20�l) for these analytes is foundwith ionspray (pneumatically-assisted electrospray)operated in the negative ion mode using ammoniumacetate buffer pH 4.0. The authors compared dif-ferent ionization techniques (ionspray, atmosphericpressure chemical ionization, and atmospheric pres-sure photoionization) for flavonoid analysis and thebest conditions for positive ionspray and APCI werefound using 0.4% formic acid (pH 2.3) as a buffer,and in negative ionspray and APCI using ammoniumacetate buffer adjusted to pH 4.0. The usefulness ofa new strategy for the electrospray ionization massspectrometric detection of flavonoids using metalcomplexation with a neutral ligand was demonstratedby Satterfield and Brodbelt[81]. The authors used2,2′-bipyridine (bpy) as a ligand in the presence of


Fig. 14. Structure of biologically important flavonoids from different classes.

divalent metal cations to enhance sensitivity. Underthese circumstances, ternary complexes are formedbetween the metal, the flavonoid and the ligand:[MII (flavonoid-H)bpy]+. The signal intensity of theternary complexes were 2 orders of magnitude greaterthan the corresponding protonated flavonoids and 1.5orders greater than the deprotonated molecules. Thework also compared negative and positive electro-spray for the analysis of several flavonoids, show-ing that sensitivity is greatly superior when nega-

tive ion mode is used, reflecting the acidity of theflavonoids.

7.2. In vitro analyses

In a study aimed at determining the regioselec-tivity of flavonoid glucuronidation, Boersma et al.[82] analysed luteolin (5,7,3′,4′-tetrahydroxyflavone)and quercetin (3,5,7,3′,4′-pentahydroxyflavone) con-jugates using negative ion electrospray LC–MS.


Samples from incubation of flavonoids with liver andintestine microsomes or with recombinant UGT iso-forms were directly injected into a RP-HPLC systemand separated with a flow rate of 1 ml/min and a gra-dient of acetonitrile and water containing 0.1% aceticacid.

The comparative importance of oxidative and con-jugative pathways for the metabolism of the flavonoidgalangin was studied by Otake et al.[83] using hu-man liver microsomes and liver S9 fractions. Sampleswere prepared by precipitating proteins from the in-cubation media with an equal volume of methanol(100�l medium + 100�l methanol) and injectingthe supernatant (150�l) collected after centrifuga-tion. Glucuronides were separated using a octade-cyl (C18) YMC octadecylsilane (ODS)-AQ column(50 × 2.0 mm i.d.) with a flow rate of 0.2 ml/minand a mobile phase consisting of a gradient of wa-ter and acetonitrile both with 0.1% acetic acid. Themass spectrometer (API 300, Applied Biosystem)with a turbo ionspray ionization interface was oper-ated in positive ionization mode. Glucuronides weredetected by using the mass spectrometer in constantneutral loss mode with an offset of 176 amu (loss ofthe glucuronic acid moiety). Ylmazer et al. studiedthe glucuronidation[84] and oxidative metabolism[85] of another flavonoid, the prenylated chalconexanthohumol, isolated from Hop.

Xanthohumol glucuronides formed by incubationwith rat or human liver microsomes were analysedby LC–MS using a PE Sciex III+ triple quadrupoleoperated in positive ion mode APCI. The conjugateswere separated using RP-HPLC with a gradient of40–100% acetonitrile in 0.1% formic acid in 30 minand a flow rate of 0.8 ml/min. Samples were pre-pared by precipitating proteins from the incubationmedia with methanol, and collecting the supernatantafter centrifugation. The oxidative metabolism offlavonoids was also studied by Nielsen et al.[86]using rat liver microsomes, who showed that mosthydroxylated metabolites were formed only whenAroclor 1254-induced microsomes were used. Theformation of GSH conjugates from quercetin and lu-teolin oxidation products was investigated by positiveelectrospray mass spectrometry by Galati et al.[87].Quercetin or luteolin (1 mM) were first incubated withhorseradish peroxidase, GSH and H2O2. Aliquotsof the incubation medium (5�l) were directly intro-

duced into the mass spectrometer (PE Sciex III+ triplequadrupole) using an HPLC pump to deliver a flowrate of 30�l/min. The metabolism of the flavone tan-geretin (5,6,7,8,4′-pentamethoxyflavone) in rats[88]was investigated by Nielsen et al. Tangeretin was me-tabolized into several demethylated and hydroxylatedmetabolites which were identified by LC–MS and1HNMR. LC–MS was performed on individual fractionsisolated from chromatographic analysis of incubationmedium. Analysis was carried out using RP-HPLCinterfaced to a VG Platform II single quadrupole massspectrometer operated in positive ion APCI mode.The glucuronidation of quercetin and kaempferol bythe recombinant UGT isoform UGT1A9[89] and byfreshly isolated rat hepatocytes[90] was investigatedby Oliveira et al., who identified four monoglu-curonides of quercetin and two monoglucuronides ofkaempferol upon incubation of these flavonoids withUGT1A9-expressing microsomes and rat hepatocytes.A Thermoquest Automass intstrument operated innegative ion electrospray mode was used. A similarstudy by Galijatovic et al.[91] identified glucuronideand sulphate metabolites of the flavonoid chrysin afterincubation with CACO-2 and HepG2 cell lines. Themetabolites as well as unmetabolized chrysin wereextracted by SPE using an C18 cartridge and analysedby RP-HPLC interfaced to a Finnigan LCQ ion trapoperated in positive ion mode with a mobile phaseconsisting of MeOH/0.3% aqueous TFA (55:45).

7.3. In vivo studies

In vitro studies of flavonoid metabolism and dis-position are important in order to establish generali-sations which might be difficult to make in “in vivo”studies due to the interaction of different enzyme sys-tems and factors beyond experimental control. How-ever, physiological relevance of these generalizationscan only be assessed by “in vivo” studies.

An interesting method for the determination ofcatechins (Fig. 15) in human urine after green teaconsumption was developed by Li et al.[92]. Urinesamples (300�l) were extracted with 300�l ofdichloromethane, centrifuged, and then the aqueousphase (200�l) was filtered (0.2�m centrifugal filter)and directly injected into the HPLC. To compensatefor the low content of organic solvent in the mobilephase during analyte elution, a second pump was used


Fig. 15. LC–MS/MS analysis of catechins in human urine. S2 and U2 are product ion chromatograms and their respective product ionspectra of standard (S2) and urine-extracted samples (U2) containing epigallocatechin (EGC). S3 and U3 are product ion chromatogramsand their respective product ion spectra of standard (S3) and urine-extracted samples (U3) containing (−)-epicatechin. Urine samples werepre-treated with�-glucuronidase. Reproduced with permission from[92].

to deliver a 0.3 ml/min flow of MeOH/H2O (8:2 v/v)into the post-column eluent in order to increase the ef-ficiency of ionisation. The eluent was delivered with-out splitting to a Finnigan LCQ ion trap mass spec-trometer operated in negative ion mode. Cremin et al.[93], investigated the fate of hydroxycinnamates afterprune consumption by healthy volunteers. Plasma andurine samples were either pre-treated with a mixtureof �-glucuronidase+ sulphatase or analysed withoutenzymatic hydrolysis. Aliquots of plasma were ex-tracted using a liquid extraction method using ethylacetate. The method showed good recovery for caf-feic acid (98.0 ± 6.1% in plasma and 99.1 ± 4.3% inurine) but recoveries for chlorogenic acid and ferrulicacid were lower than 50% for the levels commonlyfound in plasma and urine, reflecting the higher polar-ity of these metabolites. The hydroxycinnamates weredetected by mass spectrometry using electrosprayionization with negative ion detection.

A method to detect naringin in human plasma wasdeveloped by Ishi at al.[94]. The authors studied thefate of naringin administered to human volunteers af-ter a 500 mg dose of the glycoside. Urine samples werecollected at predetermined time intervals from 0 to24 h after administration of naringin. Untreated urine

samples or samples pre-treated with�-glucuronidasewere analysed. Hesperidin was added as internal stan-dard and the samples were extracted according to apreviously published SPE method[95]: The cartridges(Sep-Pak Accell QMA 5 g) were conditioned with5 ml of methanol followed by 5 ml of distilled water,the samples were applied and the cartridges washedwith 2 ml of distilled water, purged and finally elutedwith 80% methanol. The samples were analysed byRP-HPLC interfaced to a LCQ quadrupole ion trapinstrument operated in positive ion mode. This papershowed evidence of naringin absorption without theneed for prior hydrolysis of the sugar moiety, althoughthe dose of 500 mg did not reflect dietary intake andthus might not be physiologically relevant.

7.4. Carotenoids, tocopherols and retinoids

Carotenoids (Fig. 16) are conjugated hydrocarbonsformed by the condensation of eight isoprenoid units(carotenes) and their hydroxylated derivatives (xantho-phylls). As antioxidants, carotenoids are implicated inthe protection against certain types of cancer[96,97],although it has not yet been conclusively demonstratedwhether or not carotenoids have intrinsic antitumoural


Fig. 16. Structure of some biologically relevant carotenoids, tocopherols and tocotrienols.


activity or are just markers of a diet rich in fruitsand vegetables. Retinoids are structurally related tocarotenoids and are formed of retinoic acid and struc-turally related compounds. Retinoids are involved inthe regulation of cell cycle and differentiation[98].Vitamin E is composed of�, �, �, and�-tocopherolsand of tocotrienols which have a triunsaturated iso-prenoid side chain (Fig. 14). Vitamin E (especially as�-tocopherol) plays a major role in preventing oxida-tion of lipids in cell membranes.

The analysis of carotenoids and tocopherols in bi-ological fluids is challenging, especially because oftheir instability to heat, direct light, oxygen and ex-tremes of pH as well as difficulties in the separation ofgeometric isomers. Due care must thus be exercisedto ensure the stability of analytes during both sampleprocessing and analysis. This includes the addition ofantioxidants: butylated hydroxytoluene (BHT) is usu-ally used, but ascorbic acid and pyrogallol have alsobeen used. Processing samples in subdued light or inthe dark is also common practice. Chromatography ofcarotenoids and related compounds is usually carriedout using reversed-phase C18 columns or triacontyl(C30) columns. Triacontyl columns are commonly re-ferred as carotenoid columns and usually provide im-proved resolution for isomeric carotenoids[99]. MostLC–MS methods for the analysis of carotenoids in bio-logical fluids make use of atmospheric pressure ionisa-tion techniques (API), in special atmospheric pressurechemical ionisation, since due to the absence of proto-nation sites, electrospray is less efficient for the ionisa-tion of carotenoids. Examples of techniques other thanAPI for carotenoid analysis include a study of the ef-fect of eluent composition on the ionization efficiencyof a particle–beam interface with electron-capturenegative ion chemical ionisation (PB–NCI–MS) forthe determination of carotenes (lycopene,�-carotene)and xanthophylls (astaxanthin, lutein, zeaxanthin,�-cryptoxanthin)[100]. In this case, the authors usedtwo narrow bore reversed-phase columns in seriesfor separation. Detection limits between 0.2 and 3 ngfor carotenes and 0.02–20 ng for xanthophylls wereachieved. Another study using particle–beam inter-face with negative ion chemical ionization was thatby Pawlosky et al.[101] who developed a method forthe quantification of alltrans-�-carotene-d8 in hu-man plasma usingtrans-�-carotene-13C40 as internalstandard. Samples of plasma (750�l) were extracted

with 750�l of BHT in ethanol (0.005% w/v) and1.5 ml of hexane. The hexane layer was filtered andthe aqueous layer was further extracted with hexane,and the combined hexane fractions evaporated to dry-ness under nitrogen and dissolved in mobile phaseprior to HPLC analysis. Separation was achieved us-ing a C18 column and a mobile phase consisting ofMeCN/CH2Cl2/MeOH (65:25:10) with 0.1% diiso-propylethylamine. Nebulization was achieved withhelium (405 kPa) and methane was used as reagentgas. The precision of the method as determinedfrom replicate analysis of a pooled plasma samplewas 2.4 and 3.9% for alltrans-�-carotene-d8 andtrans-�-carotene, respectively. Linearity was in therange of 3–300 ng/ml plasma.

Although atmospheric pressure chemical ionisationis the most commonly used ionisation method formass spectrometry of carotenoids, Rentel et al.[102],describes the analysis by electrospray of severalcarotenoids and tocopherols by formation of adductswith silver. The method consists of post-column ad-dition of a solution of AgClO4 in acetone. Detectionlimits (using flow injection) of as low as 500 fmolfor canthaxanthin and 300 fmol for�-carotene arereported. This technique is sometimes referred asco-ordination ion spray. An application of the methodfor the identification of tocopherols and tocotrienolsin a palm oil extract is described by Strohschein et al.[103].

van Breemen et al.[104] investigated the pres-ence of lycopene and its isomers in human serumand prostate tissue after dietary supplementation witha tomato-based sauce. Human resected prostate tis-sue (100–200 mg wet weight) was homogenised inMeOH/H2O (1:1 v/v+ 0.1% pyrogallol) and saponi-fied by heating with 100�l of 70% potassium hy-droxide. Samples were extracted with hexane and theextracts were evaporated to dryness and redissolved in50�l diethylether and 150�l of MeOH/MeCN/THF(50:45:5 v/v/v). Serum samples were processed simi-larly omitting the saponification step. Separation wasachieved using a YMC C30 column and elution wascarried out using methanol/methyl-tert-butyl ether(55:45 v/v) at 1.0 ml/min for 15 min followed by agradient to methanol/methyl-tert-butyl ether (45:55v/v). An Agilent G1946A LCMSD mass spectrome-ter was operated using SIM in the positive ion APCImode for detection of lycopene atm/z 537.


An interesting method for monitoring the bio-conversion of �-carotene to retinol in humanswas developed by Wang et al.[105]. The methodused APCI LC–MS in positive ion mode to detect[13C10]-�-carotene and [13C10]retinyl palmitate inserum of children after administration of physiologicaldoses of the13C-labelled compounds (80�g of each).The advantage of using13C-labelled compounds isthat carotenoids and retinoids can be determined evenin the presence of high endogenous concentrationsof these analytes, a situation commonly found inintervention studies (seeFig. 17). Another advan-tage is that the retention behavior in reversed-phasechromatography of13C-labelled�-carotene and theunlabeled compound is the same, in contrast with[2H8]-�-carotene which has a different retention timecompared with the unlabeled�-carotene[101]. Serumsamples were mixed with 1 ml of a solution contain-ing 1 ml of 30% NaCl (aq.) and 1 ml of 70% EtOHand extracted three times with hexane under subdued

Fig. 17. Selected ion chromatograms using APCI LC–MS (positive ion mode) of carotenoids (traces at left) and retinoids (traces at right)in a serum extract before (A) and after (B) administration of labelled [13C10]-�-carotene and [13C10]retinyl palmitate. Note in both casesthe appearance of labelled compounds in serum after administration. Reproduced with permission from[105].

light. The hexane extracts were combined, evaporatedto dryness and the residue redissolved in 200�l ofMeOH/methyl-tert-butyl ether (1:1 v/v) and 60�l wasinjected into the HPLC. Separation of analytes wasachieved using an YMC C30 column and a mobilephase consisting of methanol containing 1 mM am-monium acetate (A) and methyl-tert-butyl ether (B).A HP G1946A LCMSD quadrupole mass spectrom-eter was operated in positive ion APCI. The methodhad detection limits of less than 1 pmol on column forboth�-carotene and retinol and linearity was demon-strated between 0 and 2000 pmol on column. Thismethod was later applied to determine the bioavail-ability of �-carotene in children[106]. Anotherapplication of APCI mass spectrometry for the deter-mination of carotenoids was developed by Hagiwaraet al. [107] who used squalene as internal standardfor the determination of lycopene,�-carotene and�-carotene in serum samples. Although the methodhad a limited dynamic range, it had good detection


limits for carotenoids (3 ng/ml plasma) and also goodrecoveries for all analytes (89–102%).

A method for the quantification of labelled andunlabeled Vitamin E in plasma was described by Lau-ridsen et al.[108] using APCI in positive ion modeand selected reaction monitoring for highly selectivedetection of labelled and unlabeled�-tocopherols.Standards were prepared from unlabeled and labelledtocopheryl acetates (RRR-�-5-(CD3)-tocopheryl ac-etate and all rac-�-5,7-(CD3)2-tocopheryl acetate) bysaponification using alcoholic KOH. Plasma samples(100�l) were mixed with BHT (1 mg/ml, ethanol),ethanol, SDS, and a known amount of internal stan-dard (d9-ambo-�-tocopherol). The samples wereextracted with hexane, the hexane extracts were evap-orated to dryness under nitrogen and dissolved in200�l of MeOH/EtOH. LC–MS/MS was performedon a Sciex API III+ triple quadrupole mass spec-trometer using APCI in positive ion mode. Multiplereaction monitoring was used for quantitative LC–MS(transitions: d0-�-tocopherol,m/z 430.4 → 165.2;d3-�-tocopherolm/z 433.4 → 168.2; d6-�-tocopherolm/z 436.4 → 171.2; d9-�-tocopherolm/z 439.4 →174.2). Chithalen et al[109] investigated the oxida-tive metabolism of alltrans-retinoic acid by CYP26A.A Quattro Ultima triple quadrupole mass spectrom-eter was used off-line for the LC–MS analysis offractions isolated from incubation media of retinoicacid with mammalian cells expressing hCYP26. Themass spectrometer was operated using the nega-tive ion ESI mode. Several metabolites, including4-OH-all-trans-retinoic acid, 4-oxo-all-trans-retinoicacid, and 18-OH-all-trans-retinoic acid were detected.

A problem usually associated with the use of elec-trospray ionisation for the analysis of retinoids is thenon-linear response over a wide concentration range,as described in a paper by van Breemen et al.[110] onthe analysis of retinol and retinyl palmitate in serum.The authors used APCI in positive ion mode insteadof electrospray. Linearity was demonstrated over therange 0.0524–3400 pmol/�l for all trans-retinol and0.0249–28.3 pmol/�l for all trans-retinyl palmitate.

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