C H A P T E R

14

Separation of (Phospho)Lipids byThin-Layer Chromatography

Beate Fuchs, Yulia Popkova,Rosmarie Suß, Jurgen Schiller

Medical Department, University of Leipzig, Institute of MedicalPhysics and Biophysics, Hartelstr, Leipzig, Germany

14.1 INTRODUCTION

During the last decades, it was assumed that liquid chromatographicmethods, such as high-performance liquid chromatography (HPLC),would completely replace thin-layer chromatography (TLC) for the sep-aration of lipids. However, a revival of TLC seems nowadays very like-lydparticularly regarding lipid analysis, since TLC is a frequently usedseparation technique for lipids [1e5]. Although the terms “TLC” and“high-performance thin-layer chromatography (HPTLC) will be usednearly synonymously in this review, the reader should note that HPTLC isa more sophisticated version of TLC. Differences are primarily related to(1) the different particle size of the layers (5e6 mm in comparison to10e12 mm), (2) the attention paid to sample application (manual vsautomatic), and (3) the methods employed for data processing [6].

Although there are some potential concerns (e.g., the reduced resolu-tion of TLC compared with HPLC and the potential, unwanted oxidationof lipids caused by air exposition on the layer surface), TLC is perhaps themost efficient and versatile technique for the separation of complex lipidmixtures. The following advantages are obvious [7]:

1. TLC is convenient and simple.2. The (absolutely) necessary equipment is rather inexpensive.3. The commercial availability of high quality TLC plates has strongly

increased the reproducibility of the separationdthe former weakpoint of TLC.

Instrumental Thin-Layer Chromatography

http://dx.doi.org/10.1016/B978-0-12-417223-4.00014-5 375 Copyright � 2015 Elsevier Inc. All rights reserved.

14. SEPARATION OF (PHOSPHO)LIPIDS BY THIN-LAYER CHROMATOGRAPHY376

4. TLC is rapid since dozens of samples can normally be separated inless than an hour.

5. The identification of separated lipids can be performed easily byvarious staining reactions.

6. Individual lipid fractions can be re-extracted easily and studied byother analytical methods (such as mass spectrometry (MS) or nuclearmagnetic resonance (NMR) spectroscopy) and/or isolated on apreparative scale. There are even “preparative” TLC plates with thicksilica layers that canbeused to fractionate sampleamountsofup to1 g!

7. TLC is not influenced by any “memory” effects since TLC plates areused for a single application only. This is a significant advantagecompared with HPLC and one reason why (quantitative) TLC iscertified for use in many different industrial and pharmaceuticalprocesses.

8. TLC requires much smaller solvent volumes than HPLC.

TLC is used classically for routine separations, for the identification ofindividual lipid classes (normally according to their different headgroups)and for their quantitative determination, normally by densitometry. Withthe advent of the “automated multiple development” (AMD) technique,all steps from the mixing of the required solvents, development, anddrying could be automated. In particularly, AMD supports applicationsthat require reproducible solvent gradient development. The use of gra-dients (i.e., mixtures of different solvents and/or different salt concen-trations varied in a time-dependent manner) is common in HPLC but wasnot widely used in TLC until the introduction of the AMD apparatus. Oneselected lipid-related (skin) example of this technique is available inRef. [8] and more details (not only dedicated to lipids) can be found in theexcellent book by Hahn-Deinstrop [9]. A survey of commercially availableHPTLC equipment is available at the CAMAG’s (a leading supplier ofTLC equipment) homepage (http://www.camag.com).

14.1.1 The Structure of Lipid Types Relevant to this Review

“Lipids” are most probably the biomolecules with the highest struc-tural variability. Thus, a detailed discussion of all potential lipids is clearlybeyond the scope of this chapter. However, the reader interested in more“exotic” lipids is referred to the recently published book by Claude Leray,which provides an in-depth survey of virtually all known types [10]. Inthis chapter we will focus on animal-derived lipids with physiologicaland/or medical significance. A coarse overview of these lipids is pre-sented in Figure 14.1. In particular we will highlight recent advances inthe separation of glycero(phospho)lipids and sphingolipids (SL). Thereader with little experience in the lipid field should note that lipids are

CH3

CH3

CH3

CH3

CH3

RO

O P O

O

O

X

NH2

OH

O

HO

O P O

O

ONH2

OH

O

O

OOCR

O C R

O

P O

O

O

XO

OOCR

O C

C

R

O

R

O

Fatty acids

Vitamins

Glycerolipids

Sphingosine-derived lipids

Sphingosine-derived glycolipids

Cholesteroland its derivatives

CH3COOH

CH3COOH

Saturated (e.g., stearic acid, 18:0) Unsaturated (e.g., oleic acid, 18:1)

-Tocopherol

sdipilohpsohporecylGslorecylglycairT

FIGURE 14.1 Short survey of the different classes of “lipids” relevant to this chapter,whereby only some selected examples of the most important lipid subclasses are provided.“R” indicates the presence of a fatty acyl residue, while “x” represents the (polar) headgroup.

14.1 INTRODUCTION 377

14. SEPARATION OF (PHOSPHO)LIPIDS BY THIN-LAYER CHROMATOGRAPHY378

not only of relevance for the storage of energy (fat tissue) and nutrition,but are also massively involved in signal transduction processes, forinstance, cell differentiation, apoptosis, and phagocytosis [11]. Thus, thedetermination of selected lipid species is of considerable scientific interestfor the characterization of metabolic processes. Therefore, lipid analysishas increasing diagnostic relevance, mainly but not exclusively, in thecontext of the lipoproteins (LP).

14.1.2 Extraction of Lipids from Biological Samples

Lipids are traditionally (and in the simplest way) defined as apolarcompounds that are insoluble in water, and consequently, can be enrichedby extraction with organic solvents, such as chloroform or hexane. Theextraction of lipids from a given body fluid, cell culture, or tissue sample,therefore, is normally the first step of lipid analysis [12]. Despite its centralrole, lipid extraction does not engender major interest! This is surprising,since the “fine-tuning” of the extraction conditions helps to improve theextraction yield and influences the accuracy in the analysis of individuallipids. There is also the risk of losing specific lipids: while bulk lipids,such as the zwitterionic phosphatidylcholines (PCs), are extracted nearlyquantitatively, major losses can be predicted if lipids with higher (e.g.,lysolipids or phosphorylated phosphoinositides) or lower polarities (suchas cholesteryl esters or triacylglycerols (TAGs)) are of interest. Someestablished methods of lipid extraction are summarized in Table 14.1,although there are more successful techniques [13e18].

Independent of the method used, the reader should keep in mind thatlipid extraction is a very important step: in all subsequent steps only thelipids extracted from the biological material are observed. In addition,solid-phase extraction can afford a useful alternative method to solventextraction for the isolation of lipids [22].

14.2 SEPARATION OF LIPIDS BY TLC

14.2.1 Stationary Phases

Silica gel, alumina, and kieselguhr are common stationary phases usedin the separation of lipid mixtures by normal-phase chromatography,with silica gel being the most widely used. Silica gel modified by chem-ically bonded ligands, such as octadecylsiloxane-bonded groups, is suit-able for reversed-phase separations.

In normal-phase TLC (most widely used for the separation of lipids),the stationary phase (often unmodified silica gel) is polar and the mobilephase is apolar (i.e., the used solvent system contains significant amounts

TABLE 14.1 Survey of Commonly Used Methods of Lipid Extraction (Note that All Methods have the Disadvantage that Some Lipids MayBe Lost during the Extraction)

Solvent System Particularly Suitable For Comments/References

CHCl3/CH3OH (1:1 v/v)“Bligh & Dyer method”

Useful for water-rich systems, particularly body fluids.Most widely used method.

Partial lipid losses may occur [13]. It is a disadvantagethat CHCl3 has a higher density than water becausethis may lead to impurities from the aqueous layer thathas to be penetrated to obtain the lipids. Forcomparison with the “Folch” method see Ref. [14].

CHCl3/CH3OH (2:1 v/v)“Folch method”

Lipids from animal, plant and bacterial tissues.Normally with lower water content in comparison toBligh & Dyer.

The tissue water is the ternary component and itsamount is very important in order to avoid lipid losses[15]. This paper is among the most cited (“top 10”)papers worldwide and has received already more than36,000 citations.

Butanol saturated with water Plant lipids, i.e., lipids entrapped in starch and ratherpolar lipids. Products of phospholipase digestion.

Provides particularly good recovery of lysolipids,i.e., of polar lipids [16].

Hexane/2-propanol (3:2 v/v) Low extraction yields of polar compounds (proteins,pigments, small molecules) as impurities since thesolvent mixture is apolar.

In contrast to CHCl3 (suspected carcinogen), hexaneand 2-propanol are solvents of low toxicity [17]. Plasticmaterial can be usedwithout the release of plasticizers.However, the method seems less effective thanchloroform extraction [18].

Chloroform/2-propanol(7:11 v/v)

Particularly suitable for erythrocytes with a high lipidcontent.

Indicated to provide higher lipid yields than otherextraction methods [19].

Chloroform/methanol/12 N HCl (2:4:0.1 v/v/v)

Acidic phospholipids, such as phosphatidylserine andphosphoinositides (that are otherwise very difficult toextract quantitatively).

Addition of HCl leads to charge screening andimproves extraction yields for acidic lipids [20]. Thismethod leads to complete hydrolysis of plasmalogens(alkenyl-acyl lipids), however, which are very sensitiveto acidic conditions [21].

14.2

SEPARATIO

NOFLIPID

SBYTLC

379

14. SEPARATION OF (PHOSPHO)LIPIDS BY THIN-LAYER CHROMATOGRAPHY380

of apolar solvents, such as hexane or chloroform). Layers prepared fromsmaller particles (HPTLC layers) result in higher separation quality andlower detection limits, i.e., smaller lipid amounts can be applied. Normal-phase chromatography is the standard method of (phospho)lipid sepa-rations based on polarity differences due to the different headgroups.

Silver nitrate and boric acid are common silica gel modifiers: silvernitrate is, in particular, useful for the separation of glycerolipids (or freefatty acids (FFA)) with different degrees of unsaturation [23]. The Agþ ionis able to form complexes with the p electrons of the lipid double bondsleading to the selective retardation of unsaturated compounds. Even thedetermination of the position of double bonds within an alkyl chain (thatotherwise requires oxidation (for instance by ozone) and careful analysisof the oxidation products [24]) is possible by this approach [25].

In contrast, the impregnation of silica with boric acid is the methodof choice to differentiate isomeric lipids, such as 1,2- and 1,3-monoacylglycerols [26] or lipids with carbohydrate moieties. Boric acidforms complexes with lipids containing vicinal hydroxyl groups resultingin the slower migration of these compounds. In addition, boric acid doesalso bind to acidic phospholipids (such as phosphatidylserine (PS)) andmodifies their migration properties as well. Ethylene-diamine-tetraaceticacid (EDTA) acts in a similar manner and improves the detection ofPS by forming more compact spots [27]. Ammonium sulfate can be usedto improve the separation of phosphatidylinositol (PI) and PS.Using silica gel plates impregnated with 0.4% (NH4)2SO4 andchloroformemethanoleacetic acid acetoneewater (40:25:7:4:2 v/v/v/v/v)as mobile phase, five different phospholipids (PS, phosphatidylethanol-amine (PE), PI, PC, and sphingomyelin (SM)) and three lysophospholipids(LPS (lysophosphatidylserine), LPE (lysophosphatidylethanolamine), andLPC (lysophosphatidylcholine)) can be successfully separated [28]. Someselected Rf values obtained by using boric acid-impregnated silica gel asthe stationary phase and chloroformeethanolewateretriethylamine (30:35:6:35 v/v/v/v) as the mobile phase are summarized in Table 14.2 [29].

14.2.2 Detection Systems

The majority of TLC-separated lipids can be easily identified by char-acteristic color reactions. A large number of spray agents are commer-cially available for this purpose [30]. These are normally sorted accordingto their specificity and if they are destructive or nondestructive reagents.

14.2.2.1 Nondestructive and Nonspecific Reagents

This is the preferred staining method because the lipid structure is notpermanently modified and prior knowledge of the identity of the lipid isnot required. One of the most frequently used (and oldest) methods is the

TABLE 14.2 Survey of Selected Rf Values for Important Lipids Using BoricAcid-Impregnated Silica Gel Layers and ChloroformeEthanoleWatereTriethylamine (30:35:6:35 v/v/v/v) as Mobile Phase for theSeparation. The Difference between Cerebroside Type I and II is fromthe Presence of Either a Glucose or a Galactose Residue, Respectively

Rf Compound Rf Compound

0.08 Lysophosphatidylcholine (LPS) 0.47 Phosphatidylglycerol (PG)

0.11 Sphingomyelin (SM) 0.51 Phosphatidylethanolamine (PE)

0.21 Phosphatidylcholine (PC) 0.58 Phosphatidic acid (PA)

0.22 Cerebroside type I 0.68 Cardiolipin (CL)

0.25 Cerebroside type II 0.70 Ceramide (CER)

0.26 Phosphatidylinositol (PI) 0.74 Free fatty acids

0.32 Sulfatides 0.81 N-acylphosphatidylethanolamine

0.32 Lysophosphatidylethanolamine (LPE) 0.96 Cholesterol

0.38 Phosphatidylserine (PS) 0.98 Mono-, di-, and triacylglycerols

Reproduced with permission from Ref. [29].

14.2 SEPARATION OF LIPIDS BY TLC 381

exposure of the developed TLC plate to iodine vapors [31]. This leads to a(reversible) brown complex between iodine and the double bonds of thelipids; afterward the iodine can be removed by exposing the TLC plate tovacuum. There are differences in dependence on the double bond content,however, and fully saturated lipids (not very abundant in biologicalsystems) are difficult to visualize. In addition, iodine can be difficult toremove from highly unsaturated lipids. “Mild” staining can be achievedalso by using 2,7-dichlorofluorescein or rhodamine 6G [32] that results(under UV light) in yellow or pink spots, respectively. Both dyes can beeasily removed if the polarity of the solvent is changed or the detectedlipid is passed through a short column. Similar results can be obtainedwith primuline [33], which provides detection limits comparable torhodamine (low nanomole range). Interestingly, primuline can beremoved by exposing the TLC plate to high vacuum and is suitable forsubsequent identification of separated lipids by MS [34].

It is known that polyunsaturated lipids give intense darkening underconditions employed in silver ion TLC. This is explained by the reductionof Agþ to colloidal silver [23]. However, this effect depends on the sol-vents used for the separation and is most marked in the presence ofaromatic hydrocarbons, such as toluene.

14.2.2.2 Destructive and Nonspecific Reagents

Treating the entire TLC plate with a corrosive reagent (e.g., 50%H2SO4)and subsequent charring (about one hour at 120 �C) is an established,

14. SEPARATION OF (PHOSPHO)LIPIDS BY THIN-LAYER CHROMATOGRAPHY382

classical method of lipid detection [35]. However, although not yetinvestigated in detail, saturated and unsaturated lipids require differenttimes to react. Measuring the intensities of the resulting black spots is alsoa widely used quantitative measure of individual lipids (detection limitsabout 25e50 ng per lipid class) [36].

14.2.2.3 Destructive and Specific Reagents

Many reagents (such as ninhydrin that reacts with the amino residue ofPE) that react selectively with a particular functional group of phospho-lipids to generate a colored product are known. A detailed survey of thesereagents is beyond the scope of this chapter but is available in Refs [2,37]and from the excellent Internet site www.cyberlipid.org. It should benoted also that complete oxidation of phospholipids (eluted from thesilica gel) and subsequent phosphate determination (normally accordingto Bartlett [38]) is another established quantitative method. This methodallows an unequivocal identification of phospholipids and their differ-entiation from other lipids. In a recent study selected dyes were comparedin terms of their achievable detection levels [39]. The most sensitive stainwas 0.2% amido black 10B in 1 M NaCl. About 15 ng for diacylglycerols(DAGs), TAGs, and PS, about 100 ng for FFA, and about 500 ng of phorbolesters were obtained. This is a clear indication that standards are anabsolute necessity if quantitative data are to be obtained.

14.3 APPLICATIONS

Lipids represent a very complex class of biomolecules. In addition to theheadgroup that determines the (phospho)lipid class, the fatty acid chaincomposition and its linkage type (acyl, alkyl, or alkenyl) add to thecomplexity. Thus, it is obvious that a single separation step is normally notsufficient to resolve all individual lipid species. However, this informationis readily available if normal-phase TLC (i.e., separation of the differentlipid classes) is combinedwithMSdetection of the individual lipid species.Therefore, TLCeMS coupling is nowadays a hot topic of research [40].

The aim of this section is to provide a survey of the most importantlipid classes and how they can be identified in complex mixtures. Lipidsare sorted according to increasing complexity, i.e., the discussion will startwith FFA and end with phospho- and glycolipids.

14.3.1 Fatty Acids

The concentration of FFA in biological tissues and body fluids ismodest except for organs responsible for fat metabolism, such as the liver[41]. The majority of fatty acids are esterified with alcohols, particularly

14.3 APPLICATIONS 383

glycerol. It is common practice to hydrolyze the related lipids and todetermine the released FFA. Although this is regularly the domain of gaschromatographyemass spectrometry (GC–MS) [42], FFA analysis may beperformed also by TLCdeven if the achievable accuracy is higher forGCeMS.

14.3.1.1 Determination of Differences in Chain Length andNumber of Double Bonds

Our focus will be on normal-phase TLC because it is more widely usedthan reversed-phase TLC for this application [43]. However, silver nitrate-impregnated TLC plates are mandatory to differentiate FFA that differ intheir double bond content and to distinguish cis and trans configurations[44]. Using silver nitrate-impregnated silica gel 60 TLC plates, it waspossible to isolate polyunsaturated FFA fractions as their methyl esters tofacilitate subsequent determination by GCeMS [45]. Tolueneeacetonitrile(97:3 v/v) was used to develop the plates and resulted in an excellentseparation between dienes, trienes, and tetraenes. It is particularlyremarkable that even saturated FFA can be differentiated by TLC [46].This method requires, however, initial conversion of the FFA to themonodansyl cadavaride derivatives [46]. Using methanoleacetonitrileetetrahydrofuran (18:2:1 v/v/v), the following Rf values were obtained:20:0 (0.28), 17:0 (0.45), and 15:0 (0.58). Using the x:y nomenclature, where“x” represents the number of carbon atoms, while “y” denotes the numberof double bondsdwithout specifying the positions of the double bonds.

Unfortunately, one-dimensional TLC is not suitable for the separationof complex FFA mixtures that have to be analyzed by two-dimensionalTLC. A suitable solvent for the first dimension development ishexaneediethyl ether (9:1 v/v) and hexaneediethyl ether (2:3 v/v) for thesecond dimension development.

14.3.1.2 Oxidation Products of FFA

Highly unsaturated FFA experience much higher interest than satu-rated or moderately unsaturated FFA. This is particularly true forarachidonic acid (AA, 20:4) which is released by the enzyme phospholi-pase A2 from phospholipids. AA is the educt for many physiologicallyimportant products, such as leukotrienes, thromboxanes, etc. In addition,AA is a major target for reactive oxygen species (ROS) generated by in-flammatory conditions [47]. An in-depth discussion of these aspects isbeyond of the scope of this review. However, the reader should be awarethat this is a challenging topic due to (1) the large variety of ROS that aregenerated under in vivo conditions and, thus, the complex product pat-terns and (2) the limited stability of some primary products [47]. Rao et al.[48] described a method to separate selected metabolites of AA on silicagel G plates. The mobile phase for the isolation of thromboxane B2 was

14. SEPARATION OF (PHOSPHO)LIPIDS BY THIN-LAYER CHROMATOGRAPHY384

diethyl etheremethanoleacetic acid (135:3:3 v/v/v). In addition, hy-droxyl acids could be separated by using petrol etherediethyl ethereacetic acid (60:39:1 v/v/v).

Threo- and erythro-isomers of vicinal dihydroxy esters could beseparated on silica gel impregnated with boric acid using hexaneediethylether (60:40 v/v) as the mobile phase, in which, the threo isomer migratesmore rapidly than the erythro isomer.

The hydrogenation of unsaturated fat (fat hardening) plays a signifi-cant industrial role but is accompanied by the partial isomerization of thecis configuration into the trans form. Since many enzymes recognizeexclusively compounds with a cis configuration, trans compounds areassumed to have harmful effects on human health. Therefore, the differ-entiation of cis and trans isomers by silver ion TLC has many importantapplications [49].

14.3.2 Cholesterol and Cholesteryl Esters

Cholesterol is (in addition to phospholipids, such as PC) an importantcomponent of biomembranes, while cholesteryl esters are important forthe transport of water insoluble FFA in the form of LP. Oxidized LP havebeen implicated as important in atherosclerosis research [4]. Afluorescence-based method to detect cholesterol in amounts as low as 5 ngwas described in 1996 [50]. Organic extracts of human LP were separatedon silica layers with hexaneediethyl ethereacetic acid (80/15/1 v/v/v)and afterward incubated in a filipin (a strong polyene fluorophore) sus-pension. The measured fluorescence intensity was linear between 5 and3000 ng cholesterol. This approach is also suitable to determine oxidizedforms of cholesterol. Combining TLC and GCeMS is a powerful tool foranalysis of cholesteryl ester hydroperoxides [51]. Less than 1 nmol wasdetectable on silica gel TLC plates developed with n-hexaneediethylethereacetic acid (70/30/1 v/v/v) [51].

Cholesterol is an abundant constituent of food and assumed to beaffected by processes such as cooking and freezing. Accordingly, there issignificant interest in the determination of cholesterol oxidation products.TLC of the nonsaponifiable constituents of meat extracts was performedon silica gel with hexaneediethyl ether to separate oxysterols from thenative sterols. After elution of the oxysterols, a second development withhexaneediethyl ethereethyl acetate (1:1:1 v/v/v) was performed toseparate the sterols. Subsequently, the sample was extracted from thelayer and derivatized for detailed composition analysis by GC [52].Cholesterol oxides can be identified also by TLC [53]. Finally, TLC is alsosuitable for the analysis of bile acids. These are major metabolites ofcholesterol and facilitate its elimination in the feces by the formation ofmicelles that solubilize the cholesterol in the bile [54].

14.3 APPLICATIONS 385

14.3.3 Glycerides

TAGs are extremely important for nutrition and the optimum storageof excessive energy in the organism since fat tissue is nearly free of water.Additionally, TAGs (normally from vegetable oils, such as palm or oliveoil) are also important chemicals in the cosmetic and pharmaceutical in-dustries. In contrast, DAGs are important second messenger molecules.Therefore, the condensation products between glycerol and FFA are ofsignificant interest.

14.3.3.1 Acylglycerols

The enzyme lipoprotein lipase (LPase) hydrolyzes the TAG moiety ofLP and its activity is of importance to establish risk factors associatedwith atherosclerosis [55]. Physiologically relevant LPase, however, gen-erates a mixture of different isomers: first 1,2- and 2,3-DAGs that arefurther converted into 2-monoacylglycerols (MAGs). Finally, 2-MAGsundergo isomerization to 3-MAGs that are subsequently converted intoFFA and glycerol. Fortunately, all relevant products can be separated bynormal-phase TLC. The mobile phase hexaneediethyl ethereacetic acid(70:30:1 v/v/v) enables the separation of TAGs, FFA, 1,2- and 1,3-DAGs,and MAGs to be achieved. The approximate Rf values for these com-pounds are 0.7, 0.45, 0.26, 0.23, and 0.05, respectively [56]. Using diethyletherehexaneemethanol (65:35:3, v/v/v) and Na2CO3-impregnatedsilica gel layers, FFA, MAGs, DAGs, and TAGs result in Rf values of 0.0,0.18, 0.79e0.85, and 0.98, respectively [57]. It is important to note that themigration of the acyl groups from the sn-2 position to the sn-1 and sn-3positions may occur if protic solvents (such as water or alcohols) are used[58]. Acyl migration, however, can be suppressed using boric acid-impregnated silica gel plates. Keep in mind that acyl migration canalso occur in detergents [59] that are sometimes used to extract lipids orto characterize phospholipids by 31P NMR spectroscopy [60].

14.3.3.2 Separation by Degree of Unsaturation

As already discussed, silver nitrate-impregnated silica gel layers aresuitable for the separation of fatty acids that differ in the number ofdouble bonds [25]. Since oleic acid (18:1), linoleic acid (18:2), and linolenicacid (18:3) predominate in TAGs from vegetable oils [61], up to ninedouble bonds may occur within a TAG. Denoting S¼ saturated,M¼monoenoic, D¼ dienoic, and T¼ trienoic acids, the following orderof Rf values (illustrated in Figure 14.2) is typically obtained:

SSS > SSM > SMM > SSD > MMM > SMD > MMD > SDD > SST

> MDD > SMT > MMT > DDD > SDT > MDT > DDT > STT

> MTT > DTT > TTT

SSS

SSM

SMM

SSDMMMSMDMMDSDD

MDD + SMTMMTDDD

SDT + MDT

DDT

(a) (b)

FIGURE 14.2 Schematic separation of soybean TAGs on silica gel G impregnated with10% (w/w) silver nitrate. Plate (a) was developed with chloroformemethanol (99:1 v/v)while plate (b) was developed with chloroformemethanol (96:4 v/v). Abbreviations:S¼ saturated, M¼monoenoic, D¼ dienoic and T¼ trienoic acid glycerol esters. Reprintedwith modification and permission from Christie and Han, Ref. [1].

14. SEPARATION OF (PHOSPHO)LIPIDS BY THIN-LAYER CHROMATOGRAPHY386

However, this separation quality can be hardly achieved by a singleTLC development. It is common practice, therefore, to separate the lesspolar fractions with hexaneediethyl ether (80:20 v/v) or chloroformemethanol (197:3 v/v) first and subsequently the remaining fractions withmore polar solvents such as diethyl ether alone or chloroformemethanol(96:4 v/v) [1].

In many papers a description such as “TAG 54:3” can be found. Usingthis nomenclature, all three fatty acyl residues are combined into a singlehypothetical residue. This is done because the determination of the po-sitions of the individual fatty acid substituents is even today a challengingtask. Enzymatic or chemical degradation of the TAG of interest is nor-mally needed. Unfortunately, most lipases are not regiospecific [62]. Thisalso applies for pancreatic lipase that is normally used on account of itsavailability and relatively low cost.

The analysis of the TAG composition of vegetable oils to establishauthenticity is an increasing issue in the European Union owing to theadulteration of edible vegetable oils (such as virgin olive oil). TLC is auseful method for this purpose. For instance, silver ion TLC of eightsamples of sunflower oil (with different linoleoyl contents) with petrolethereacetone (25:1 v/v) and petrol ethereacetoneeethyl acetate (100:5:2or 50:3:2 v/v/v) exhibited the expected differences for the fatty acid side-chain compositions [63]. Combining TLC and GCeMS enabled Myheret al. [64] to quantify more than 100 TAG species in a butter sample.Changes in oil compositions induced by frying can be monitored also by

14.3 APPLICATIONS 387

TLC [65]: however, silver ion TLC and reversed-phase TLC should becombined to obtain detailed information about the double bond contentsand the different chain lengths, respectively.

Many common mobile phases contain solvents with strongly differentpolarities. This may result in “demixing” leading to poor reproducibility ofindividual TLC separations. The stable solventmixture (dichloromethaneeethyl acetateemethanoleacetic acid (27:22:38:13 v/v/v/v)) [66] isparticularly useful for the determination of the TAG composition ofvegetable oils.

14.3.4 SL and Glycolipids

SL are “markers” of various diseases (see Ref. [67] for a review). SL arealso common components of human skin [68]. Sphingomyelin, the mostabundant, will be discussed later since it is normally detected togetherwith common phospholipids. Chloroformemethanolewater mixtures(ratios between 70:30:4 and 50:40:10 v/v/v) are typically used for neutralSL, while gangliosides containing sugar or sialyl residues require theaddition of salts for their separation. Mixtures such as 2-propanole6 Maqueous ammoniaemethyl acetate (15:5:1 v/v/v) are typically used toseparate glycated SL (up to four carbohydrate residues). Figure 14.3provides an illustration of a typical separation. Even isomeric glycopyr-anose residues, such as glucose and galactose, can be differentiated usingthis method [69]. It is also remarkable that normal fatty acid and2-hydroxyfatty acid residues lead to a splitting of the separated zones andcan, thus, be differentiated.

Galactosylceramide

Lactosylceramide

Globotriaosylceramide

GlobotetraosylceramideGangliotetraosylceramide

Glycosylceramide

FIGURE 14.3 Schematic separation of neutral sphingolipids by HPTLC on silica gelusing 2-propanole6 M aqueous ammoniaemethyl acetate (15:5:1 v/v/v) as mobile phase.Reprinted with modification and permission from Christie and Han, Ref. [1].

14. SEPARATION OF (PHOSPHO)LIPIDS BY THIN-LAYER CHROMATOGRAPHY388

Chloroformeacetoneemethanoleacetic acidewater (46:17:15:14:8 v/v/v/v/v) and chloroformemethanoleacetic acid (65:25:10 v/v/v) areestablished solvent systems for the identification of sulfatides, which arecharacteristic components of selected tissues (brain) or cells (spermatozoa).Up to nine different sulfatides were identified in kidney cell extracts [70]using normal-phase HPTLC.

Complex lipid extracts (for instance from HL-60 cells [71]) require theuse of two-dimensional TLC. The cells are initially extracted with chlor-oformemethanol (2:1 v/v) and then separated in the first dimensionwith chloroformemethanolewater (65:25:4 v/v/v) and subsequentlywith tetrahydrofuranedimethoxymethaneemethanolewater (10:6:4:1,v/v/v/v) in the second dimension. Although excellent separation qualitycan be achieved, glycolipids are very challenging compounds. Oneparticular problem is that newly discovered glycolipids often containvery long oligosaccharide chains that can significantly change the chro-matographic properties. Fortunately, monoclonal antibodies directedagainst specific glycosphingolipids are now available. This simplifiestheir detection [72].

14.3.5 Phospholipids

Phospholipids constitute an important class of biomolecules, of whichglycerophospholipids (GPLs) are of particular significance. All GPLsconsist of a glycerol backbone, esterified with two fatty acids and onemolecule of phosphoric acid. The resulting phosphatidic acid (PA) mayformally react with different alcohols to give products, such as PC and PE,or negatively charged phospholipids, such as PS, phosphatidylglycerol, PI,and polyphosphoinositides (PPI) (e.g., PIP2 (phosphatidylinositol-bisphosphate)), Figure 14.4. In addition, even more complex phospho-lipids, such as cardiolipin (CL) that may be considered as two PA unitslinked by one glycerol molecule, are possible [73]. One aspect is importantregarding nomenclature: “PC”, for instance, implies the presence of twoester residues [74]. As there are also alkyl and alkenyl lipids in biologicalsystems, the more general term “glycerophosphocholine” should be used,even if “PC” is often (incorrectly) used to indicate all species.

Since phospholipids are ubiquitous compounds, there is an increasinginterest in using either them or compounds derived from them, such aslysolipids that lack one fatty acid residue, as markers for disease [41].Normal-phase TLC is commonly used to separate phospholipid mixturesand numerous excellent solvent systems have been developed [75].Detection limits of about 20 ng per phospholipid are easily achieved [76].There are one- and two-dimensional TLC methods to separate complexphospholipid mixtures and are discussed below.

CH2 CH2 N(CH3)3O~

OH

O

OH OH

OHHO

~

O CH2 2

OH

C

H

CH OH~

NH3

O CH2 C

H

COO~ O CH2 CH2 3NH~

O

O

OH

OHPO

OO

P OO

O

O

O

POO

O

POO

O

~

CH2

CH

CH2

PO

OOO

C O

O

R

C O

O

R

Phosphatidic acid (PA)

PIP PIP2

PI

PS

PG PC

PE

PIP3

FIGURE 14.4 Selected structures of glycerophospholipids relevant to this review. Allcompounds are basically derived from glycerol by esterification at two positions with fattyacids (“R” represents varying fatty acyl residues) and in the third position with phosphoricacid. The resulting phosphatidic acid (PA) can formally react again with a variety of smallorganic molecules. Accordingly, the following compounds are formed: phosphatidylethanol-amine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylserine (PS),and phosphatidylinositol (PI). PI can be further phosphorylated on the inositol ring to givephosphatidylinositolmonophosphate (PIP), phosphatidylinositol-bisphosphate (PIP2) andphosphatidylinositol-trisphosphate (PIP3).

14.3 APPLICATIONS 389

14. SEPARATION OF (PHOSPHO)LIPIDS BY THIN-LAYER CHROMATOGRAPHY390

14.3.5.1 One-Dimensional Separations

One-dimensional TLC is regularly used if neutral or zwitterionic phos-pholipids are of particular interest. The separation quality achievable byone-dimensional TLC was illustrated more than 40 years ago for plasmaand erythrocyte extracts [77]. Silica gel impregnated with 7.5% (w/w)magnesium acetate in combination with chloroformemethanoleammonia(65:25:4 v/v/v) was used for the separation. If only small quantities ofacidic phospholipids are expected chloroformemethanolewater (25:10:1v/v/v) is widely used [78]. However, PS and PE, as well as PI and PC,mayinterfere under these conditions. A solvent system commonly usedfor complex mixtures of negatively charged phospholipids is methylacetatee2-propanolechloroformemethanol containing 0.25%aqueousKCl(25:25:25:10:9 v/v/v/v/v) [79]. This system is suitable for many complexlipid mixtures although PA and PE are not well resolved. A comparison ofeight different solvent systems for the separation of phospholipids by one-dimensional TLC has been performed [80]. Chloroformemethanolewater (65:25:4 v/v/v) was recommended as providing the best overallseparation quality for phospholipids. The effect of different solvent sys-tems on the separation quality is illustrated in Figure 14.5. It should benoted, however, that excellent results are only obtained if great care is

(a) (b) (c)

DPG

PE

PGSMLPC

GL

PE

PIPS

PCSMLPC

CMH

SQDGGSu

PAPGDPG

PE

FIGURE 14.5 Schematic HPTLC separations of complex lipid mixtures from animal tis-sues. (a) Chloroformemethanolewater (25:10:1 v/v/v); (b) methyl acetatee2-propanolechloroformemethanole0.025% KCl (25:25:25:10:9 v/v/v/v/v); (c) first development withpyridineehexane (3:1 v/v) and second development in the same direction withchloroformemethanolepyridinee2 M ammonia (35:12:65:1 v/v/v/v). Abbreviations: DPG,diphosphatidylglycerol; GL, glycolipid; GSu, glycolipid sulfate; CMH, ceramidemonohexoside; SQDG, sulfoquinovosyldiacylglycerol; LPC, lysophosphatidylcholine; PE,phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phos-phatidylserine; PC, phosphatidylcholine; PA, phosphatidic acid; SM, sphingomyelin. Reprin-ted with modification and permission from Christie and Han, [1].

14.3 APPLICATIONS 391

taken regarding the water content of the solvent mixture and the activity ofthe TLC plates. This important topic has been reviewed recently [3].Another important point is that the thorough drying of the plate (betweendifferent developments) is crucial for consistent separation quality [81].If glycolipid identification is also of interest, a two-step TLC is advisable.The phospholipids are first separated from other lipids and the glycolipidsare separated in a second development. Reasonable resolution isobtained using diisobutyl ketoneeacetic acidewater (40:25:3.7 v/v/v).Chloroformeethanolewateretriethylamine (30:35:6:35 v/v/v/v) isanother useful solvent system for this separation [29].

Although the resolution achievable by one-dimensional TLC is limited,it is remarkable that all major lipid classes from human plasma and crudeliver extracts can be resolved in a single TLC separation following pre-development over a short distance with chloroformemethanolewater(65:30:5 v/v/v) to remove protein-bound lipids. Afterward, the TLC platewas fully developed with hexaneediethyl ethereformic acid (80:20:1.5v/v/v) [82]. Although the lipid concentration of urine is comparativelylow, TLC is sufficiently sensitive to characterize the lipid composition ofhuman urine. A very sensitive detection technique is to spray the platewith copper sulfate reagent with subsequent charring [83].

14.3.5.1.1 DETERMINATION OF ENZYMATIC ACTIVITY

Phospholipases A2, C, and D generate lysophospholipids, diac-ylglycerols, and phosphatidic acids, respectively (Figure 14.6). Oneimportant (clinical) task is the determination of the related enzyme

Phosphatidicacid

LPL andfree fatty acids

OPOO

OH2C

–

HC

PLC PLDPLA2

C

Choline, ethanolamine,serine or inositol

e.g., (20:4)

O CO

H2C

e.g., (18:0)

Diacylglycerols

OO

Highly unsaturated fatty acid

Saturated fatty acid

FIGURE 14.6 Schematic diagram of in vivo generation of lipid-derived second messen-gers from a selected phospholipidmolecule, alongwith the enzymes involved: phospholipaseA2 (PLA2), phospholipase C (PLC), and phospholipase D (PLD). LPL, lysophospholipid.

14. SEPARATION OF (PHOSPHO)LIPIDS BY THIN-LAYER CHROMATOGRAPHY392

activity by determining the consumption of the substrate and/or thegeneration of related products, which can be accomplished using TLC. Anelegant phospholipase C-related study was performed by Goldfine et al.[84]: these authors observed different rates of digestion for phospholipidsusing phospholipase C. Organic solvent extracts were separated usingchloroformemethanoleacetic acid (65:25:8 v/v/v) and gave an excellentseparation between the phospholipids and related diacylglycols [84].

Phospholipases A2 activities were determined similarly by Wang andGustafson [28]. Different phospholipids (PS, PE, PI, PC, and SM) andthree lysophospholipids (LPS, LPE, and LPC) were separated on silica gelimpregnated with 0.4% (w/w) ammonium sulfate using chloroformemethanoleacetic acideacetoneewater (40:25:7:4:4:2 v/v/v/v) as themobile phase and subsequently detected by iodine staining. Finally, it wasshown recently that a simple TLC assay allows the determination ofceramide concentration and phospholipases D activity [85]. Methods forchromatographic enzyme activity determination were recently reviewed[86] as well as the use of labeled compounds [87].

14.3.5.1.2 PHOSPHOLIPID OXIDATION

This is a very important topic since all inflammatory diseases (endingwith “itis” such as “arthritis”) are accompanied by the generation of ROS.This topic has been comprehensively reviewed recently [88]. Since manyROS react in a diffusion-controlled manner, the focus is normally on the“bulk” lipids, for example, PC hydroperoxides and products derivedfrom them. These can be isolated by TLC from oxidized lysophospholi-pids using chloroformemethanolewater (10/5/1 v/v/v) [89]. The alde-hyde group present in PC subsequent to oxidation [47] was visualized byspraying with Schiff’s reagent while the hydroperoxides were detected byspraying with potassium iodide and starch.

The detection of lipid oxidation products in turkey meat was accom-plished by TLC of the phospholipid hydroperoxides and their parentphospholipids (SM, PC, and PE) [90]. Hydroperoxide detection wasperformed by dipping the developed TLC plate into a freshly preparedsolution of N,N-dimethyl-p-phenylenediamine and detecting the productby scanning densitometry at 654 nm.

TLC is also useful for the separation of headgroup-modified phospho-lipids, such as PE. Prior to reaction with HOCl (leading to the generation ofchloramines), PEwas elutedwith chloroformemethanoleacetic acid (80:12:8 v/v/v) and FFAwith diethyl etherepetrol ethereacetic acid (70:30:1 v/v/v) [91]. Air-dried plates were sprayed with ninhydrin and heated at 100 �Cto visualize the amine group of PE, or were charred at 180 �C to visualize alllipids. HOCl reacts with the fatty acid residues with formation of chloro-hydrins while the amino group is converted into a chloramine [91].Therefore, the differentiation between the individual products is important.

”tratS“

(a) (b)

0:61EPL

EPLP

CPLP

0:61CPL

m/z [Th]

FIGURE 14.7 Positive ion MALDIeTOFmass spectra of the individual fractions of an air-oxidized mixture of PC 16:0/18:2 (1-Palmitoyl-2-linoleoyl-sn-phosphatidylcholine, PLPC) andPC 16:0/18:2 (1-Palmitoyl-2-linoleoyl-sn-phosphatidylethanolamine, PLPE). The developedTLC plate from which the spectra were recorded is shown on the left. Lane (a) representsa PLPC/PLPE/LPC (lysophosphatidylcholine) 16:0/LPE (lysophosphatidylethanolamine)16:0 mixture as control while lane (b) represents a sample of air-oxidized PLPC/PLPE.Positions of the acquiredmass spectra directly from the TLCplate are labeled by numbers. Ionsare labeled according to their m/z ratios and the most prominent peaks are structurallyassigned. The structure of the most abundant ion (m/z¼ 454.1) from the primuline dye isalso indicated (left top). Due to the presence of the sulfonic acid residue, primuline is detectedwith low sensitivity as a positive ion. The reasons of the dark background in lane (b) are so farunknown. PC, phosphatidylcholine; LPE, lysophosphatidylethanolamine; TLC, thin-layerchromatography. Reprinted from Ref. [92].

14.3 APPLICATIONS 393

Finally, it was shown that TLC combined with matrix-assisted laserdesorption and ionization (MALDI) MS detection gives good results forthe oxidation products of polyunsaturated PE or PC species illustrated inFigure 14.7 [92].

14.3.5.2 Two-Dimensional Separations

Two-dimensional TLC is an excellent tool for the separation of complexlipid mixtures. A typical example (lipids from brain mitochondria) isshown in Figure 14.8 [93]. However, two-dimensional TLC has seriousdrawbacks also: first, only a single sample can be investigated by two-dimensional TLC. Second, the simultaneous application of the sampleand a lipid standards mixture is impossible. Due to these disadvantages,multiple developments in a single dimension are often used as an

1

2

FIGURE 14.8 Typical two-dimensional HPTLC separation of total lipids extracted fromthe cortical P2 fraction from brain mitochondria. The silica plate was first developed with asolvent system consisting of chloroformemethanole28% ammonia (65:25:5 v/v/v). Afterdrying the plate was developed in the orthogonal direction with a solvent system consistingof chloroformeacetoneemethanoleglacial acetic acidewater (50:20:10:10:5 v/v/v/v/v).Phospholipids were visualized by exposure to iodine vapor. “NL” means neutral lipids suchas triacylglycerols. CL, cardiolipin; PE, phosphatidylethanolamine; PC, phosphatidylcho-line; PI, phosphatidylinositol; PS, phosphatidylserine; FFA, free fatty acids Sph, sphingo-myelin. Reprinted with modification and permission from Ref. [93].

14. SEPARATION OF (PHOSPHO)LIPIDS BY THIN-LAYER CHROMATOGRAPHY394

alternative to two-dimensional TLC. In this case, a solvent mixture of highelution power is used first, followed by further developments ofincreasing length with solvents of decreasing elution power. Thisapproach confers the advantage of first concentrating the sample com-ponents while the gradient development helps to overcome resolutionproblems. This approach is often used for glycolipid analysis [94].

Two-dimensional TLC is the method of choice to screen for PPI incomplex mixtures [95]: After extraction with chloroformemethanoleHCl,aliquots are subjected to normal-phase TLC on silica gel impregnatedwith 1% (w/w) potassium oxalate. The first development withchloroformemethanole4.3 M ammonia (90:65:20 v/v/v) is used toseparate the PPI. After drying, a second development with chloroformemethanoleconcentrated ammonia (130:50:10 v/v/v) was used to separatelysophosphatidylethanol from PC. Next, the plate was rotated anddeveloped in the orthogonal direction with chloroformemethanoleaceticacidewater (100:30:35:3 v/v/v/v) to resolve the remaining phospho-lipids. The individual lipids were visualized by charring [95].

Two-dimensional TLC is also useful for the separation of lipid oxida-tion products in complex lipid mixtures [96]. Two-dimensional TLC of PC,

14.4 MALDI FOR MS DETECTION 395

PE, PI, PS, SM, CL, LPC, and LPE was performed on silica gel impreg-nated with 7.5% (w/w) magnesium acetate using chloroformemethanoleammonia (5:25:5 v/v/v) for the first development andchloroformeacetoneemethanoleacetic acidewater (6:8:2:2:1 v/v/v/v/v)for the second development in the orthogonal direction. Afterward thio-barbituric acid reactive substances were identified in individual fractionsto determine the extent of oxidation. This approach allowed the deter-mination of whether all phospholipids are equally sensitive to oxidation.

14.3.6 Phosphoinositides

PPI are involved in signal transduction and are of significant physio-logical interest. Unfortunately, PPI are difficult to analyze because theyoccur in only very small amounts and are difficult to extract with organicsolvents on account of their high polarity. Accordingly, radioactivelabeling with 32P (on the phosphate residues) or 3H (on the inositol ring) isoften used to detect low abundant PPI with sufficient sensitivity. Theseparation of PPI from residual phospholipids is not difficult (due to theirincreased polarity) and can be performed in one- or two-dimensional TLC[97]. One-dimensional TLC, for example, was able to resolve the majorityof major and minor phospholipid species in extracts from human eryth-rocytes and platelets on silica gel with chloroformemethanoleacetic acid(55:25:5 v/v/v). Normal-phase TLC is also capable of resolving differentPI isomers phosphorylated in the 3-, 4-, or 5 position [98]. The mostcommon method uses TLC plates impregnated with boric acid and1-propyl acetatee2-propanoleethanole6% aqueous ammonia (3:9:3:9v/v/v/v) as the mobile phase as shown in Figure 14.9.

14.4 MALDI FOR MS DETECTION

MALDI is fast, simple and has the advantage of producing nearlyexclusively singly charged ions. In addition, MALDI is tolerant of rela-tively high sample contamination from salts and other materials. Formethodological details see Ref. [99] or the second edition of the excellentbook by Franz Hillenkamp and Jasna Peter-Katalini�c [100].

14.4.1 Glyco- and Sphingolipids

Considerable progress has been made recently in the TLCeMALDIMSof Glyco- and sphingolipids [101]. In place of a detailed survey we willmention some landmarks since this topic was recently reviewed [102]. It isremarkable that only very little fragmentation occurs when the analytesare directly desorbed from a conventional TLC platedeven if complex

1 2 3

PA

PI-4-P

PI-4,5-P2PI-4,5-P2PI-3,4-P2

PI-3,4,5-P3

PI-3P + PI-4-P

SF

PE?

PC + PS

PI

O

FIGURE 14.9 Phosphor screen autoradiography of lipid compounds separated onboric acid-impregnated HPTLC silica gel 60 layers developed with 1-propyl acetatee2-propanoleabsolute ethanole6% aqueous ammonia (3:9:3:9 v/v/v/v). 32P-labeled erythro-cyte (lane 1), 32P-labeled erythrocyte phospholipids incubated additionally with PI 3-kinase gand Mge[g-32P] ATP (adenosine triphosphate) (lane 2), and 32P-labeled A431 cell phospho-lipids (lane 3). Abbreviations: O, origin; PI 3,4,5-P3, phosphatidylinositol 3,4,5-trisphosphate;PI 3,4-P2, phosphatidylinositol 3,4-bisphosphate; PI 4,5-P2, phosphatidylinositol 4,5-bisphosphate; PI 3-P, phosphatidylinositol 3-phosphate; PI 4-P, phosphatidylinositol4-phosphate; PI, phosphatidylinositol; PA, phosphatidic acid; PC, phosphatidylcholine; PS,phosphatidylserine; PE, phosphatidylethanolamine; SF, solvent front. “?” indicates the pres-ence of an unidentified product. Reprinted from Ref. [98] with permission.

14. SEPARATION OF (PHOSPHO)LIPIDS BY THIN-LAYER CHROMATOGRAPHY396

molecules such as gangliosides are analyzed [103]. However, oneimportant aspect is the application of the matrix (normally a small organicmolecule that has the task of absorbing the laser energy) [104]. Thehomogeneity of the matrix/analyte cocrystals determines the reproduc-ibility of the measurements and the matrix layer must be applied ashomogeneous as possible without compromising the chromatographicresolution. This last point is particularly important because applying anorganic solution of the matrix may lead to the blurring of compoundswith similar Rf values. Fortunately, special spray devices that meet theserequirements are now available from several companies.

The majority of studies of glycolipids have used UV lasers with asmaller number employing infrared lasers primarily with noncommercial

14.5 MALDI FOR MS DETECTION 397

spectrometers self-built instruments. The use of infrared lasers has ad-vantages and disadvantages: on the one hand, glycerol (the most commoninfrared matrix) is a liquid and, thus, there are no problems regardinginhomogeneous matrix/analyte cocrystallization. On the other hand,infraredMALDI is characterized bymore complex adduct ions in contrastto results for UV lasers, which might complicate data analysis. Using aninfrared MALDI source, Dreisewerd and coworkers were able to showthat even minor gangliosides could be unequivocally identified on a TLCplate, as there was virtually no fragmentation of molecular ions that couldbe determined with high accuracy [105]. Similar results were obtainedusing a UV laser. In a recent study, 2,5-dihydroxybenzoic acid (DHB) inacetonitrileewater (1:1 v/v) was used as matrix for the analysis of gly-cosphingolipids. Detection limits of about 50 pmol were obtained [106]. Itis a significant advantage that antibodies, i.e., oligosaccharide-specificproteins, are often available to assist in the identification of specific gly-cosphingolipids. Using this approach in combination with TLCeMALDIMS, detection limits less than 1 ng were obtained [107]. The application ofantibodies allows the direct use of crude lipid extracts without priorseparation of glycosphingolipids in some cases.

14.4.2 Glycerophospholipids

As for glycolipids, two different approaches are in use for the identi-fication of glycerophospholipids by TLCeMALDI MS. Using an infraredlaser and glycerol as a matrix has the advantage that quantitative resultsfor selected model phospholipids (with defined acyl groups) can be ob-tained even if abundant glycerol adducts (and to a minor extent evenNaCl adducts) of the lipids were also detectable [108]. In anotherapproach the authors used basically the same setup with a nitrogen laserand 2,5-DHB as matrix [34]. An extract from hen egg yolk was usedbecause it is readily available and contains an abundance of phospho-lipids. A selected single track of a TLC-separated hen egg yolk extract andsome selected positive ion MALDI mass spectra (directly recorded fromthe TLC plate) are shown in Figure 14.10 [109]. Two aspects of this anal-ysis should be emphasized: first, even low abundant lipids (e.g., PI) thatmake up less than 1% (w/w) of the phospholipids from egg yolk can beeasily detected [34]. Thus, the detection limit is about 400 pmol [34].Second, different mass spectra are obtained that depend on the positionwhere the laser beam hits the spot of a lipid fraction. This is particularlyevident for the PE fraction where shorter and longer chain fatty acidgroups can be distinguished. This clearly indicates that changes in thefatty acid chain composition slightly affect the migration properties ofthe phospholipids. This difference would never have been resolved by thevisual inspection of the TLC plate.

740.5PE 16:0/18:1

+Na 768.5PE 18:0/18:1

+Na

790.5 [790.536]PE 18:0/20:4 + Na

812.5PE 18:0/20:4– H + 2 Na

740 760 780 800 820

PE

m/z

909.5PI 18:0/20:4

+Na

885.5PI 18:0/18:2

+Na

857.5PI 16:0/18:2

+Na

879.5 [879.500]PI 16:0/18:2–H +2 Na

860 880 900 920 940

PI

m/z

660 680 700 720 740

725.6 [725.557]SM 16:0+Na703.6

SM 16:0+H

677.5

SM

m/z

480 500 520 540 560

496.3LPC 16:0+H

524.3LPC 18:0+H

518.3 [518.322]LPC 16:0+Na

546.3LPC 18:0+NaLPC

m/z

782.6 [782.568]PC 16:0/18:1

+Na

810.6786.6

760.6PC 16:0/18:1

+H

758.6

740 760 780 800 820

PC

m/z

504.3 [504.307]LPE 18:0+Na

526.3LPE 18:0–H + 2 Na

476.3LPE 16:0+Na

460 480 500 520 540

LPE

m/z

FIGURE 14.10 Expanded region of a TLC-separated egg yolk extract and the corre-sponding positive ion MALDIeTOF mass spectra recorded directly from the indicated posi-tions on the plate. Only the relevant mass regions of each phospholipid class are shown andassignments are provided directly in the individual traces. Data given in parentheses corre-spond to theoreticalmasses andwere introduced to enable comparisons with the experimentaldata in selected cases. Note that the PE fraction provides different mass spectra, depending onthe position where the laser beam hits the PE spot. The only marked fragmentation is the lossof the headgroup of SM (leading to m/z¼ 677.5). PE, phosphatidylethanolamine; PI, phos-phatidylinositol; LPE, lysophosphatidylethanolamine; PC, phosphatidylcholine; LPC, lyso-phosphatidylcholine; SM, sphingomyelin. Reprinted with permission from Ref. [109].

14. SEPARATION OF (PHOSPHO)LIPIDS BY THIN-LAYER CHROMATOGRAPHY398

14.5 SUMMARY AND OUTLOOK 399

14.5 SUMMARY AND OUTLOOK

There is an unequivocally considerable and even increasing interest in(phospho)lipid analysis. It may even be expected that lipids will experi-ence additional interest in the future because an increasing number ofdiseases (such as atherosclerosis or rheumatic diseases) are recognized tobe accompanied by alterations of the lipid compositions of the affectedtissues and/or body fluids [110]. Another advantage is surely the ubiquityof lipids: since the same lipids occur in human and animals, the samebiomarkers can be used, and there is no need to produce differentantibodies.

Hopefully, we were able to provide sufficient evidence that (HP)TLC isa powerful tool for lipid analysis and can be applied to all relevant lipidclasses of physiological and diagnostic interest. Although many otheranalytical methods [111] can be used for the same purpose, (HP)TLC isaccepted as a time-saving and economical method that may be used witha minimum of trouble shootings. Finally, (HP)TLC may be applied to“suspicious” samples (for instance, from food or cosmetics) that mayeasily plug or even damage an HPLC column.

The scope of the hyphenation of HPTLC to other analyticaltechniquesdparticularly MSdappears to hold considerable promise forthe analysts who previously had reservations concerning the use of planarchromatography. Although a lot of different methods are alreadycommercially available, further significant progress can be expected inthis field. So far, there are basically methods based on the extraction ofanalytes of interest prior to MS and different desorption methods thatallow the characterization of analytes directly on the TLC plate. Of course,the selection of the most appropriate method depends on the analyticalproblem and access to particular instrument platforms. To date, methodsbased on extraction seem to providemore reliable quantitative data, whiledesorption methods provide higher resolving power. For instance, lipidswith different acyl groups can be identified within a single spot on a TLCplate. This obviously opens a new dimension that makes HPTLC highlycompetitive with more common LCeMS methods.

Acknowledgments

The authors wish to thank all colleagues and friends who helped them in writing this review.Particularly the kind and helpful advice of Dr Suckau andDr Schurenberg (Bruker Daltonics,Bremen) as well as Dr Griesinger and Dr Mattheis (Merck KGaA, Darmstadt) is gratefullyacknowledged.

This work was supported by the German Research Council (DFG Schi 476/12-1 and FU771/1-2 as well as TR 67 project A2 & A8) and the Federal Ministry of Education andResearch of the Federal Republic of Germany (“The Virtual Liver”, 0315735).

14. SEPARATION OF (PHOSPHO)LIPIDS BY THIN-LAYER CHROMATOGRAPHY400

References

[1] Christie WW, Han X. Lipid analysis. 4th ed. Bridgwater: Woodhead PublishingLimited; 2010, ISBN 0955251249.

[2] Fuchs B, Suss R, Teuber K, Eibisch M, Schiller J. Lipid analysis by thin-layerchromatography e a review of the current state. J Chromatogr A 2011;1218:2754e74.

[3] Handloser D, Widmer V, Reich E. Separation of phospholipids by HPTLC e aninvestigation of important parameters. J Liq Chromatogr Relat Technol 2008;31:1857e70.

[4] Touchstone JC. Thin-layer chromatographic procedures for lipid separation.J Chromatogr B 1995;671:169e95.

[5] Dynska-Kukulska K, Ciesielski W. Methods of extraction and thin-layer chromatog-raphy determination of phospholipids in biological samples. Rev Anal Chem 2012;31:43e56.

[6] Ettre LS. Chapters in the evolution of chromatography. 1st ed. London: ImperialCollege Press; 2008.

[7] Fuchs B, Suß R, Nimptsch A, Schiller J. Matrix-assisted laser desorption and ionizationtime-of-flight mass spectrometry (MALDI-TOFMS) directly combinedwith thin-layerchromatography (TLC) e a review of the current state. Chromatographia 2009;69:95e105.

[8] Zellmer S, Lasch J. Individual variation of human plantar stratum corneum lipids,determined by automated multiple development of high-performance thin-layerchromatography plates. J Chromatogr B 1997;691:321e9.

[9] Hahn-Deinstrop E. Applied thin-layer chromatography. 2nd ed.Weinheim:Wiley-VCH;2007.

[10] Leray C. Introduction to lipidomics. 1st ed. Boca Raton: CRC Press; 2013.[11] Yeung T, Ozdamar B, Paroutis P, Grinstein S. Lipid metabolism and dynamics during

phagocytosis. Curr Opin Cell Biol 2006;18:429e37.[12] Carrapiso AI, Garcıa C. Development in lipid analysis: some new extraction tech-

niques and in situ transesterification. Lipids 2000;35:1167e77.[13] Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J

Biochem Physiol 1959;37:911e7.[14] Iverson SJ, Lang SL, Cooper MH. Comparison of the Bligh and Dyer and Folch

methods for total lipid determination in a broad range of marine tissue. Lipids2001;36:1283e7.

[15] Folch J, LeesM, Sloane Stanley GH. A simple method for the isolation and purificationof total lipids from animal tissues. J Biol Chem 1957;226:497e509.

[16] Colborne AJ, Laidman DL. Extraction and analysis of wheat phospholipids. Phyto-chemistry 1975;14:2639e45.

[17] Hara A, Radin NS. Lipid extraction of tissues with a low-toxicity solvent. AnalBiochem 1978;90:420e6.

[18] Gunnlaugsdottir H, Ackman RG. Three extractionmethods for determination of lipidsin fish meal: evaluation of a hexane/isopropanol method as an alternative tochloroform-based methods. J Sci Food Agric 1993;61:235e40.

[19] Rose HG, Oklander M. Improved procedure for the extraction of lipids from humanerythrocytes. J Lipid Res 1965;6:428e31.

[20] Honeyman TW, Strohsnitter W, Scheid CR, Schimmel RJ. Phosphatidic acid and phos-phatidylinositol labelling in adipose tissue. Relationship to the metabolic effects ofinsulin and insulin-like agents. Biochem J 1983;212:489e98.

[21] Pietruszko R, Gray GM. The products of mild alkaline and mild acid hydrolysis ofplasmalogens. Biochim Biophys Acta 1962;56:232e9.

REFERENCES 401

[22] Ruiz-Gutierrez V, Perez-Camino MC. Update on solid-phase extraction for the anal-ysis of lipid classes and related compounds. J Chromatogr A 2000;885:321e41.

[23] Nikolova-Damyanova B. Retention of lipids in silver ion high-performance liquidchromatography: facts and assumptions. J Chromatogr A 2009;1216:1815e24.

[24] Ellis SR, Hughes JR, Mitchell TW, in het PanhuisM, Blanksby SJ. Using ambient ozonefor assignment of double bond position in unsaturated lipids. Analyst 2012;137:1100e10.

[25] Dobson G, ChristieWW, Nikolova-Damyanova B. Silver ion chromatography of lipidsand fatty acids. J Chromatogr B 1995;671:197e222.

[26] Ando Y, SatakeM, Takahashi Y. Reinvestigation of positional distribution of fatty acidsin docosahexaenoic acid-rich fish oil triacyl-sn-glycerols. Lipids 2000;35:579e82.

[27] Abidi SL. Separation procedures for phosphatidylserines. J Chromatogr B 1998;717:279e93.

[28] Wang WQ, Gustafson A. One-dimensional thin-layer chromatographic separation ofphospholipids and lysophospholipids from tissue lipid extracts. J Chromatogr 1992;581:139e42.

[29] Leray C, Pelletier X, Hemmendinger S, Cazenave JP. Thin-layer chromatography ofhuman platelet phospholipids with fatty acid analysis. J Chromatogr 1987;420:411e6.

[30] Wang Y, Krull IS, Liu C, Orr JD. Derivatization of phospholipids. J Chromatogr B 2003;793:3e14.

[31] Palumbo G, Zullo F. The use of iodine staining for the quantitative analysis of lipidsseparated by thin layer chromatography. Lipids 1987;22:201e5.

[32] Kishimoto K, Urade R, Ogawa T, Moriyama T. Nondestructive quantification ofneutral lipids by thin-layer chromatography and laser-fluorescent scanning: suitablemethods for “lipidome” analysis. Biochem Biophys Res Commun 2001;281:657e62.

[33] White T, Bursten S, Federighi D, Lewis RA, Nudelman E. High-resolution separationand quantification of neutral lipid and phospholipid species in mammalian cells andsera by multi-one-dimensional thin-layer chromatography. Anal Biochem 1998;258:109e17.

[34] Fuchs B, Schiller J, Suss R, Schurenberg M, Suckau D. A direct and simple method ofcoupling matrix-assisted laser desorption and ionization time-of-flight mass spec-trometry (MALDI-TOF MS) to thin-layer chromatography (TLC) for the analysis ofphospholipids from egg yolk. Anal Bioanal Chem 2007;389:827e34.

[35] Kritchevsky D, Davidson LM, Kim HK. Quantitation of serum lipids by a simpleTLC-charring method. Clin Chim Acta 1973;46:63e8.

[36] Klein TR, Kirsch D, Kaufmann R, Riesner D. Prion rods contain small amounts of twohost sphingolipids as revealed by thin-layer chromatography and mass spectrometry.Biol Chem 1998;379:655e66.

[37] Sherma J, Bennett S. Comparison of reagents for lipid and phospholipid detection anddensitometric quantitation on silica-gel and C-18 reversed phase thin-layers. J LiqChromatogr 1983;6:1193e211.

[38] Bartlett GR. Phosphorus assay in column chromatography. J Biol Chem 1959;234:466e8.

[39] PlekhanovAY. Rapid staining of lipids on thin-layer chromatogramswith amido black10B and other water-soluble stains. Anal Biochem 1999;271:186e7.

[40] Cheng SC, Huang MZ, Shiea J. Thin layer chromatography/mass spectrometry.J Chromatogr A 2011;1218:2700e11.

[41] Fuchs B, Schiller J. Lysophospholipids: their generation, physiological role and detec-tion. Are they important disease markers? Mini-Rev Med Chem 2009;9:368e78.

[42] Bielawska K, Dziakowska I, Roszkowska-Jakimiec W. Chromatographic determina-tion of fatty acids in biological material. Toxicol Mech Methods 2010;20:526e37.

14. SEPARATION OF (PHOSPHO)LIPIDS BY THIN-LAYER CHROMATOGRAPHY402

[43] BhatHK,Ansari GA. Improved separation of lipid esters by thin-layer chromatography.J Chromatogr 1989;483:369e78.

[44] Nikolova-Damyanova B, Momchilova S. Silver ion thin-layer chromatography of fattyacids. A survey. J Liq Chromatogr Relat Technol 2001;24:1447e66.

[45] Wilson R, Sargent JR. High-resolution separation of polyunsaturated fatty acids byargentation thin-layer chromatography. J Chromatogr 1992;623:403e7.

[46] Frank HG, Graf R. Determination of free unsaturated C20 fatty acids in rat placentaeusing HPTLC. J Planar Chromatogr e Mod TLC 1991;4:134e7.

[47] Fuchs B, Bresler K, Schiller J. Oxidative changes of lipids monitored by MALDI MS.Chem Phys Lipids 2011;164:782e95.

[48] Rao GH, Reddy KR, White JG. Simple method for the separation of monohydroxyfatty acid metabolites of arachidonate metabolism. J Chromatogr 1982;232:176e9.

[49] Destaillats F, Golay PA, Joffre F, de Wispelaere M, Hug B, Giuffrida F, et al. Compar-ison of available analytical methods to measure trans-octadecenoic acid isomericprofile and content by gas-liquid chromatography in milk fat. J Chromatogr A 2007;1145:222e8.

[50] Smejkal GB, Hoppe G, Hoff HF. Filipin as a fluorescent probe of lipoprotein-derivedsterols on thin-layer chromatograms. Anal Biochem 1996;239:115e7.

[51] Kawai Y, Miyoshi M, Moon JH, Terao J. Detection of cholesteryl ester hydroperoxideisomers using gas chromatography-mass spectrometry combined with thin-layerchromatography blotting. Anal Biochem 2007;360:130e7.

[52] Pie JE, Spahis K, Seillan C. Cholesterol oxidation in meat products during cooking andfrozen storage. J Agric Food Chem 1991;39:250e4.

[53] Nourooz-Zadeh J. Determination of the autoxidation products from free or totalcholesterol: a new multistep enrichment methodology including the enzymic releaseof esterified cholesterol. J Agric Food Chem 1990;38:1667e73.

[54] Panveliwalla D, Lewis B, Wootton ID, Tabaqchali S. Determination of individual bileacids in biological fluids by thin-layer chromatography and fluorimetry. J Clin Pathol1970;23:309e14.

[55] Olivecrona G, Olivecrona T. Triglyceride lipases and atherosclerosis. Curr Opin Lipidol2010;21:409e15.

[56] Mangold HK, Malins DC. Fractionation of fats, oils and waxes on thin layers of silicicacid. J Am Oil Chem Soc 1960;37:383e5.

[57] Schuch R, Mukherjee KD. Rapid radio thin-layer chromatography for assay of lipase-catalyzed esterification and interesterification reactions. J Chromatogr 1988;450:448e51.

[58] Akesson B. Composition of rat liver triacylglycerols and diacylglycerols. Eur JBiochem 1969;9:463e77.

[59] Pluckthun A, Dennis EA. Acyl and phosphoryl migration in lysophospholipids:importance in phospholipid synthesis and phospholipase specificity. Biochemistry1982;21:1743e50.

[60] Schiller J, Muller M, Fuchs B, Arnold K, Huster D. 31P NMR spectroscopy of phospho-lipids: from micelles to membranes. Curr Anal Chem 2007;3:283e301.

[61] Schiller J, Suß R, Petkovi�c M, Arnold K. Triacylglycerol analysis of vegetable oils bymatrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) massspectrometry and 31P NMR spectroscopy. J Food Lipids 2002;9:185e200.

[62] Byrdwell WC. Modern methods for lipid analysis by liquid chromatography/massspectrometry and related techniques. 1st ed. Champaign: AOCS Press; 2005.

[63] Marekov I, Tarandjiiska R, Momchilova S, Nikolova-Damyanova B. Quantitativesilver ion thin layer chromatography of triacylglycerols from sunflower oilsdiffering in the level of linoleic acid. J Liq Chromatogr Relat Technol 2008;31:1959e68.

REFERENCES 403

[64] Myher JJ, Kuksis A, Marai L, Sandra P. Identification of the more complex triacylgly-cerols in bovine milk fat by gas chromatography-mass spectrometry using polar capil-lary columns. J Chromatogr 1988;452:93e118.

[65] Momchilova S, Nikolova-Damyanova B. Quantitative TLC and gas chromatographydetermination of the lipid composition of raw and microwaved roasted walnuts,hazelnuts, and almonds. J Liq Chromatogr Relat Technol 2007;30:2267e85.

[66] McSavage J, Wall PE. Optimization of a mobile phase in reversed-phase HPTLC forthe separation of unsaturated lipids in vegetable oils degraded during frying. JPC:J Planar Chromatogr 1998;11:214e21.

[67] van Echten-Deckert G. Sphingolipid extraction and analysis by thin-layerchromatography. Methods Enzymol 2000;312:64e79.

[68] Raith K, Farwanah H, Wartewig S, Neubert RHH. Progress in the analysis of stratumcorneum ceramides. Eur J Lipid Sci Technol 2004;106:561e71.

[69] Ogawa K, Fujiwara Y, Sugamata K, Abe T. Thin-layer chromatography of neutral gly-cosphingolipids: an improved simple method for the resolution of GlcCer, GalCer,LacCer and Ga2Cer. J Chromatogr 1988;426:188e93.

[70] Niimura Y, Ishizuka I. Isolation and identification of nine sulfated glycosphingolipidscontaining two unique sulfated gangliosides from the African green monkey kidneycells, Verots S3, and their possible metabolic pathways. Glycobiology 2006;16:729e35.

[71] Platt FM, Neises GR, Karlsson GB, Dwek RA, Butters TD. N-butyldeoxygalactonojir-imycin inhibits glycolipid biosynthesis but does not affect N-linked oligosaccharideprocessing. J Biol Chem 1994;269:27108e14.

[72] Meisen I, MormannM, Muthing J. Thin-layer chromatography, overlay technique andmass spectrometry: a versatile triad advancing glycosphingolipidomics. BiochimBiophys Acta 2011;1811:875e96.

[73] Eibisch M, Zellmer S, Gebhardt R, Suss R, Fuchs B, Schiller J. Phosphatidylcholine di-mers can be easily misinterpreted as cardiolipins in complex lipid mixtures: a matrix-assisted laser desorption/ionization time-of-flight mass spectrometric study of lipidsfrom hepatocytes. Rapid Commun Mass Spectrom 2011;25:2619e26.

[74] Fahy E, Subramaniam S, Brown HA, Glass CK, Merrill Jr AH, Murphy RC, et al. Acomprehensive classification system for lipids. J Lipid Res 2005;46:839e61.

[75] Sherma J, Fried B. Handbook of thin layer chromatography. 1st ed. New York: MarcelDekker; 1991.

[76] Thanh NTK, Stevenson G, Obatomi D, Bach P. Determination of lipids in animaltissues by high-performance thin-layer chromatography with densitometry. JPC:J Planar Chromatogr 2000;5:375e81.

[77] Turner JD, Rouser G. Precise quantitative determination of human blood lipids bythin-layer and triethylaminoethylcellulose column chromatography. II. Plasmalipids. Anal Biochem 1970;38:437e45.

[78] Wang G, Wang T. The role of plasmalogen in the oxidative stability of neutral lipidsand phospholipids. J Agric Food Chem 2010;58:2554e61.

[79] Vitiello F, Zanetta JP. Thin-layer chromatography of phospholipids. J Chromatogr1978;166:637e40.

[80] Aloisi J, Fried B, Sherma J. Comparison of mobile phases for separation of phospho-lipids by one-dimensional TLC on preadsorbent high-performance silica-gel plates.J Liq Chromatogr 1991;14:3269e75.

[81] Ruiz JI, Ochoa B. Quantification in the subnanomolar range of phospholipids andneutral lipids by monodimensional thin-layer chromatography and image analysis.J Lipid Res 1997;38:1482e9.

[82] Kupke IR, Zeugner S. Quantitative high-performance thin-layer chromatographyof lipids in plasma and liver homogenates after direct application of 0.5-microlitersamples to the silica-gel layer. J Chromatogr 1978;146:261e71.

14. SEPARATION OF (PHOSPHO)LIPIDS BY THIN-LAYER CHROMATOGRAPHY404

[83] Massa DR, Vasta JD, Fried B, Sherma J. High-performance thin-layer chromatographicanalysis of the polar lipid content of human urine and urine from BALB/c miceexperimentally infected with Echinostoma caproni. JPC: J Planar Chromatogr 2008;21:337e41.

[84] Goldfine H, Johnston NC, Knob C. Nonspecific phospholipase C of Listeria monocy-togenes: activity on phospholipids in Triton X-100-mixed micelles and in biologicalmembranes. J Bacteriol 1993;175:4298e306.

[85] Liu G, Kleine L, Hebert RL. A direct method for the simultaneousmeasurement of cer-amide and phospholipase D activity. Prostaglandins Leukot Essent Fatty Acids 2000;63:187e94.

[86] Hendrickson HS. Phospholipase A2 and phosphatidylinositol-specific phospholipaseC assays by HPLC and TLC with fluorescent substrate. Methods Mol Biol 1999;109:1e6.

[87] Deranieh RM, Joshi AS, Greenberg ML. Thin-layer chromatography of phospholipids.Methods Mol Biol 2013;1033:1e27.

[88] Kuksis A, Suomela JP, Tarvainen M, Kallio H. Lipidomic analysis of glycerolipid andcholesteryl ester autooxidation products. Mol Biotechnol 2009;42:224e68.

[89] Itabe H, Yamamoto H, Suzuki M, Kawai Y, Nakagawa Y, Suzuki A, et al. Oxidizedphosphatidylcholines that modify proteins. Analysis by monoclonal antibody againstoxidized low density lipoprotein. J Biol Chem 1996;271:33208e17.

[90] Bruun-Jensen L, Colarow L, Skibsted LH. Detection and quantification of phospho-lipid hydroperoxides in turkey meat extracts by planar chromatography. JPC: J PlanarChromatogr 1995;8:475e9.

[91] Carr AC, van den Berg JJ, Winterbourn CC. Differential reactivities of hypochlorousand hypobromous acids with purified Escherichia coli phospholipid: formation of halo-amines and halohydrins. Biochim Biophys Acta 1998;1392:254e64.

[92] Bischoff A, Eibisch M, Fuchs B, Suß R, Schurenberg M, Suckau D, et al. A simpleTLC/MALDI method to monitor oxidation products of phosphatidylcholines andethanolamines. Acta Chromatogr 2011;23:365e75.

[93] Bayir H, Tyurin VA, Tyurina YY, Viner R, Ritov V, Amoscato AA, et al. Selective earlycardiolipin peroxidation after traumatic brain injury: an oxidative lipidomics analysis.Ann Neurol 2007;62:154e69.

[94] Muthing J. Improved thin-layer chromatographic separation of gangliosides by auto-mated multiple development. J Chromatogr B 1994;657:75e81.

[95] Mallinger AG, Yao JK, Brown AS, Dippold CS. Analysis of complex mixtures of phos-pholipid classes from cell membranes using two-dimensional thin-layer chromatog-raphy and scanning laser densitometry. J Chromatogr 1993;614:67e75.

[96] Pikul J, . Kummerow Fred A. Thiobarbituric acid reactive substance formation asaffected by distribution of polyenoic fatty acids in individual phospholipids. J AgricFood Chem 1991;39:451e7.

[97] Singh AK, Jiang Y. Quantitative chromatographic analysis of inositol phospholipidsand related compounds. J Chromatogr B 1995;671:255e80.

[98] Hegewald H. One-dimensional thin-layer chromatography of all known D-3 and D-4isomers of phosphoinositides. Anal Biochem 1996;242:152e5.

[99] Fuchs B, Suss R, Schiller J. An update of MALDI-TOF mass spectrometry in lipidresearch. Prog Lipid Res 2010;49:450e75.

[100] Hillenkamp F, Peter-Katalini�c J. MALDI MS e a practical guide to instrumentation,methods and application. 2nd ed. Weinheim: Wiley-VCH; 2013.

[101] Muthing J, Distler U. Advances on the compositional analysis of glycosphingolipidscombining thin-layer chromatography with mass spectrometry. Mass Spectrom Rev2010;29:425e79.

REFERENCES 405

[102] Fuchs B. Analysis of phospholipids and glycolipids by thin-layer chromatography-matrix-assisted laser desorption and ionization mass spectrometry. J Chromatogr A2012;1259:62e73.

[103] Ivleva VB, Sapp LM, O’Connor PB, Costello CE. Ganglioside analysis by thin-layerchromatography matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry. J Am Soc Mass Spectrom 2005;16:1552e60.

[104] Fuchs B, Schiller J. Recent developments of useful MALDI matrices for the mass spec-trometric characterization of apolar compounds. Curr Org Chem 2009;13:1664e81.

[105] Dreisewerd K, Muthing J, Rohlfing A, Meisen I, Vukeli�c Z, Peter-Katalini�c J, et al.Analysis of gangliosides directly from thin-layer chromatography plates by infraredmatrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrom-etry with a glycerol matrix. Anal Chem 2005;77:4098e107.

[106] Nakamura K, Suzuki Y, Goto-Inoue N, Yoshida-Noro C, Suzuki A. Structural charac-terization of neutral glycosphingolipids by thin-layer chromatography coupled tomatrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight MS/MS. Anal Chem 2006;78:5736e43.

[107] Souady J, Soltwisch J, Dreisewerd K, Haier J, Peter-Katalini�c J, Muthing J. Structuralprofiling of individual glycosphingolipids in a single thin-layer chromatogram bymultiple sequential immunodetection matched with direct IR-MALDI-o-TOF massspectrometry. Anal Chem 2009;81:9481e92.

[108] Rohlfing A, Muthing J, Pohlentz G, Distler U, Peter-Katalini�c J, Berkenkamp S, et al.IR-MALDI-MS analysis of HPTLC-separated phospholipid mixtures directly fromthe TLC plate. Anal Chem 2007;79:5793e808.

[109] Fuchs B, Schiller J, Suß R, Nimptsch A, Schurenberg M, Suckau D. Capabilities anddisadvantages of combined matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and high-performance thin-layer chroma-tography (HPTLC): analysis of egg yolk lipids. JPC: J Planar Chromatogr 2009;22:35e42.

[110] McIntyre TM. Bioactive oxidatively truncated phospholipids in inflammation andapoptosis: formation, targets, and inactivation. Biochim Biophys Acta 2012;1818:2456e64.

[111] Hou W, Zhou H, Elisma F, Bennett SA, Figeys D. Technological developments inlipidomics. Brief Funct Genomic Proteomic 2008;7:395e409.