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Review ArticleChromatographic Methods in the Separation of Long-ChainMono- and Polyunsaturated Fatty Acids

MaBgorzata DoBowy and Alina Pyka

Department of Analytical Chemistry Faculty of Pharmacy Medical University of Silesia in Katowice 4 Jagiellonska41-200 Sosnowiec Poland

Correspondence should be addressed to Alina Pyka apykasumedupl

Received 15 July 2014 Accepted 7 October 2014

Academic Editor Hasan Uslu

Copyright copy 2015 M Dołowy and A Pyka This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

This review presents various chromatographic systems TLC HPLC GC and also SFC developed for identification and accuratequantification of long-chain mono- and polyunsaturated fatty acids from different samples with emphasis on selected literaturewhich was published during last decade Almost all the aspects such as preseparation step of fatty acids (cis and trans) stationaryphase solvent system and detection mode are discussed

1 Introduction

Long-chain fatty acids (LC-FA) are organic compounds inwhich the hydrocarbon chain length may vary from 10 to 30carbons The hydrocarbon chain can be saturated or unsat-urated (contains one or more double bonds) Based on thenumber of double bonds unsaturated fatty acids are classifiedinto the following groups [1 2]

(i) monounsaturated fatty acids (monoenoic acidsMUFA) containing one double bond for exampleoleic acid

(ii) polyunsaturated fatty acids (polyenoic acids PUFA)having two or more double bonds for example 120574-linolenic acid

(iii) eicosanoids which are derived from polyenoic fattyacids for example prostaglandins

Recent literature data indicate that both monounsatu-rated and polyunsaturated fatty acids are biological importantcompounds which play a significant role for the living organ-isms [3ndash17] Human feeding studies during the last ten yearsdemonstrate that PUFA as well as MUFA are the main com-ponents of cholesterol-lowering diet [4 7 9] Moreover therehas been a much interest in the effect of MUFA and PUFAon immune and inflammatory system [13] Among various

monounsaturated fatty acids the most popular is oleic acid(C181n-9) It is found in plants (eg olive oil) animals andmicroorganisms Olive oil consumption has benefit for colonand breast cancer prevention [8] The current studies showthat oleic acid plays important role in prevention of coronarydisease (ability to reduce LDL-cholesterol) [7 9] Polyun-saturated fatty acids similar to monounsaturated fatty acidsare widely distributed in nature [18] There are three classesof unsaturated fatty acids common in human tissues [5]the 120596-3 (n-3 PUFA) 120596-6 (n-6 PUFA) and 120596-9 (n-9 PUFA)fatty acids To the group of discussed unsaturated fatty acidsbelongs also demospongic acid a mixture of very long-chain fatty acids mainly C

24ndashC30

with the atypical 59-diunsaturation system It exists in microorganisms marineinvertebrates and terrestrial plants [19] The main sources ofomega-3 are fishes some plants and green algae [20] Greenoleaginous algae are the potential source of the following 120596-3acids eicosapentaenoic (EPA) docosahexaenoic (DHA) andalso arachidonic acid (AA) from 120596-6 group [21ndash23] Omega-6 PUFA are present in high concentration in grains as wellas in many seeds and meats From this reason we can noticean increase in the human consumption of seafood duringseveral last years Oleaginous microorganisms as alternativesources of PUFA to others such as animal oil productshave been widely studied Marine fungoid protists (Thraus-tochytrids) like Schizochytrium have been found to be as

Hindawi Publishing CorporationJournal of ChemistryVolume 2015 Article ID 120830 20 pageshttpdxdoiorg1011552015120830

2 Journal of Chemistry

a novel excellent DHA and EPA 120596-3 fatty acids producers[24 25] An excellent review paper performed by Nicholsdemonstrates that many microorganisms including marinebacteria have been considered the major de novo producer ofn-3 PUFA [26] Available literature data suggest that over thepast several years extensive research has been made for theproduction of PUFA by fungi [27 28] As it was reported byArjuna among various microorganisms an optimal source ofomega-6 polyunsaturated fatty acids specifically 120574-linolenicacid (GLA) can be certain fungi [27]

It is well known that PUFA can affect many physio-logical processes including cardiovascular neurological andimmune functions as well as cancer Consumption of oilsrich in n-3 LC-PUFA during pregnancy reduces the risk forearly premature birth [12] Studies with nonhuman primatesand human newborns indicate that DHA is essential for thenormal functional development of the retina and brain par-ticularly in premature infants [13 29] A new paper preparedby Kaczmarski et al demonstrated the significant role oflinoleic acid and also 120572-linolenic acid in some symptoms ofatopic dermatitis [17]Therefore it could be noted that PUFAare important nutraceutical and pharmaceutical targets [22]

Until today there is a lack of knowledge about thefunction of LC-PUFA in mammalian tissues and cells inwhich they are found However it was stated that very long-chain polyunsaturated fatty acids are known to accumulate intwo types of major genetic peroxisomal diseases Zellwegersyndrome and X-linked adrenoleukodystrophy (X-ALD)characterized by neurodegenerative phenotypes [30 31] Thestudy of Hama and coworkers showed existence of many LC-PUFA types in specimens from Zellweger patients suggestingthe possibility of a new biomarker for peroxisomal diseases[31] Review paper performed by Agbaga and coworkers [32]summarized the current knowledge of VLC-PUFA to theirfunctional role in the retina which are highly enriched inPUFA with special emphasis on the elongases responsiblefor their synthesis by ELOVL4 protein Interest in LC-PUFAwas rekindled in 1987 after LC-PUFA were initially detectedin bovine retinas by Aveldano [33] Effect of retinal LC-PUFA on rod and cone photoreceptors was also describedby Bennett et al [34] Another paper prepared by Butovichconfirmed that among differentmammalian tissuesmeibumis an exceptionally complex mixture of various fatty acids[35]

Because most of LC-PUFA including omega-3 andomega-6 fatty acids cannot be synthesized in enough quantityby the humanorganism theymust be supplied in the dietTheproblemof supplementation of LC-PUFAand the explorationof a new (alternative to oil fish) source of LC-PUFA (egmarine microalgae) is widely described for few years in manypapers For this reason there is a need to find an effectiveand rapid method for identification and quantification ofnewly developed long-chain mono- and polyunsaturatedfatty acids in plants and seafood A set of various tools such aschromatographic methods is also needed to complete the fullstructural characteristic of PUFA in mammalian samples forexample in human plasma brain retina or meibum This isimportant for the purpose of clinical diagnosis

Hence this review presents various chromatographicsystems TLC HPLC GC and also SFC suitable for pre-separation and accurate quantification of long-chain mono-and polyunsaturated fatty acids from different samples withemphasis on selected literature which was published duringlast decade

2 Thin-Layer Chromatography (TLC) ofLong-Chain Mono- and PolyunsaturatedFatty Acids

Saturated and unsaturated long-chain fatty acids (MUFAand PUFA) are basic structural elements of lipids Thereforechromatographic determination of fatty acids compositionby TLC including LC-MUFA and LC-PUFA content ismandatory for lipids analysis in food agricultural andalso in biological samples [36ndash39] Moreover the mono-and polyunsaturated fatty acids can be chromatographicallydetermined by TLC in lipids from aquatic organisms (egmarine and freshwater fishes shell fishes and marine algae)[40]

It is well known that thin-layer chromatography is a clas-sical method of separation identification and quantificationof fatty acids [41] The literature survey from the last decadededicated to lipids analysis indicates that among differentanalytical methods thin-layer chromatography (TLC) and itsmodern version high performance thin-layer chromatogra-phy (HPTLC) are still very important tool in lipids and alsoin fatty acids analysis As it was reported by Fuchs et al [41]there aremany advantages whichmake TLC very competitivewithHPLC (high performance liquid chromatography) in thefatty acids field such as simplicity of use less expensive costsmall consumption of solvents in comparison with HPLCavailability of analysis of several samples in parallel andpossibility of easy visualization of unsaturated fatty acids afterTLC fractionation by use of suitable dyes [41] Shermarsquos reportregarding TLC analysis in food and agriculture samples con-firmed that quantitative HPTLC equipped with densitometercan produce results comparable to those obtained by theuse of gas chromatography (GC) or high performance liquidchromatography (HPLC) [42]

Modern topic in TLC analysis of mono- and polyunsat-urated fatty acids is the use of high performance thin-layerchromatography in combination with mass spectrometricdetection for example HPTLC-MALDI-TOFMS [43ndash48]Research studies indicate that MS detection is a powerfultool for the identification of TLC spots in more detail incomparison with traditional staining methods [41]

Many types of stationary phases classified as normal (NP)and reversed (RP) are used for TLC analysis of fatty acids(MUFA and PUFA)Themost popularNP layers are silica gelalumina cellulose starch polyamides and kieselguhr [42]Of all above-mentioned stationary phases the best is silicagel which can be additionallymodified by impregnationwithdifferent agents Reversed-phase TLC is usually performed onchemically bonded RP-18 RP-2 or RP-8 layers [42]

Numerous research papers by Nikolova-Damyanova andother authors showed that the main reason which explains

Journal of Chemistry 3

why TLC plays a significant role in fatty acid analysis is avail-ability of various commercial and home-made adsorbentsincluding impregnated TLC plates [36 41 49ndash51] Impreg-nation of TLC plates with a proper reagent can improvethe resolution of different classes of organic compoundsincluding fatty acids Among different modifications of sta-tionary phases used in TLC impregnated silica gel is a verysuitable adsorbent for MUFA and PUFA analysis [41]

21 Separation of MUFA and PUFA with the Use of Impreg-nated TLC Plates One of the primarily used TLC impreg-nating procedure to separate fatty acids in complex lipidsamples was silver ion TLC (Ag-TLC) Detailed informationon Ag-TLC of saturated and unsaturated fatty acids wasperformed in several papers and books [36 49ndash51] Silver ionchromatography is based on the ability of Ag+ to form weakreversible charge transfer complexes with 120587 electrons of thedouble bonds of unsaturated fatty acids [36 52]The retentionof long-chain unsaturated fatty acids depends on the strengthof complexation with Ag (I) on the number of double bondsand their configuration and also on the distance betweendouble bonds Literature data indicate that both home-madeand precoated glass plates are used in Ag-TLC [51] Gen-eral procedures of preparation of the stationary phases forsilver ion chromatographic techniques have been surveyedby Momchilova and Nikolova-Damyanova in 2003 [53]Among various available TLC adsorbents silica gel is themain supporting material [53] Impregnation of thin layer isperformed by spraying or immersing the plate in solutionof silver nitrate at concentration of 05ndash20 (in the caseof immersion) while for the spraying procedure 10ndash40solution of silver nitrate is recommended [51 53]Themobilephase used in argentationTLCusually consists of two or threecomponents for example hexane petroleum ether benzeneand toluene Moreover small amount of acetone diethylether ethanol methanol or acetic acidmay be added to thesemobile phases [40] Of various visualizing agents of spotsthose which are most popular for Ag-TLC of fatty acids are50 ethanol solution of sulfuric acid phosphomolybdic acidand a mixture of copper-acetate-phosphoric acid Anothervisualizing method is spraying the plates with fluorescentindicator by 2101584071015840-dichlorofluorescein in ethanol and nextviewing the spots under UV light [36] General migrationrules in Ag-TLC analysis of fatty acids were described byNikolova-Damyanova and coworkers [53 54] According toNikolova-Damyanova suggestions the retention of fatty acidswith more than one double bond depends on the distancebetween the bonds and the elution order is as follows sep-arated double bonds fatty acids gt interrupted double bonds gtconjugated double bonds longer chain unsaturated fattyacids (LC-PUFA) are held less strongly than shorter chainfatty acids and fatty acids with trans double bonds are heldless strongly than fatty acids with cis double bonds [53 54]

One of the first Ag-TLC analyses of fatty acids wasperformed by Wilson and Sargent in 1992 to separate PUFAhaving physiological interest PUFArsquos methyl esters wereseparated on silica gel 60 TLC plates impregnated withAgNO

3 The plates were developed with toluene-acetonitrile

(97 3 vv) Visualization of the spots was made by

the use of 3 copper acetate-8 orthophosphoric acid It wasstated that this technique is particularly useful for metabolicstudies of the chain elongated PUFA [55] In another workWilson and Sargent showed that silver nitrate-impregnatedTLC plates were helpful in separation of monounsaturatedfatty acids (as methyl esters) from polyunsaturated and alsofrom saturated fatty acids respectively in metabolic studiesof fatty acids by human skin fibroblasts [56] Next work byLin et al indicated that silica gel and hexane-chloroform-diethyl ether-acetic acid (80 10 10 1 vvvv) were goodin analysis of docosahexaenoic acid (22 6 DHA) in thespermatozoa of monkeys [57] Individual phospholipidsfrom this sample were separated by another system suchas chloroform-methanol-petroleum ether-acetic acid-boricacid (40 20 30 10 18 vvvvv) Other literature dataconfirmed that argentate silica gel chromatography enabledobtaining the high purity eicosapentaenoic acid extractedfrom microalgae and fish oils [58] The recent literaturereviews which were focused on TLC chromatography showthat among different chromatographic materials Ag-TCM-TLC (silver-thiolate silica gel) is very stable (in comparisonwith highly light sensitive Ag-TLC plates) for TLC analysisof unsaturated organic compounds including MUFA andPUFA Dillon et al [59] confirmed that Ag-TCM-TLC systemoperates similar to Ag-TLC by separating fatty acids onthe degree of unsaturation (number of double bonds) Theresults of this analysis are comparable to those obtained byAg-TLC Ag-TCM-TLC method was used to analyze somepolyhydrocarbons and also methyl esters of unsaturated fattyacids containing from 0 to 6 double bonds in form of methylesters Amixture consisting of hexane-ethyl acetate (9 1 vv)was used as mobile phase Under these conditions completeseparation of fatty acids with 0ndash5 double bonds was observedResolution of fatty acids consisting of 6 double bonds fromothers was not achieved in this case [59]

The results presented in this section show that variouscommercial silica gel plates were used in separation ofunsaturated fatty acids but some of them are not suitable forderivatization process by methylation of fatty acids on TLCplates and next their quantification by gas chromatographybecause it causes the loss of separated fatty acids [60]Methylation procedure of fatty acids after their previousfractionation on argentate silica gel was used in the analysesof unsaturated fatty acids from lipid-rich seeds In this casea mixture of hexane-diethyl ether-acetic acid and 70 30 1(vvv) was used as a mobile phase The plates were sprayedwith 2101584071015840-dichlorofluorescein ethanolic solution and nextidentified under UV lamp (at 365 nm) [60] The impactof Ag-silica gel on TLC analysis of methylated fatty acidsfor example c9t11-CLA (isomer of linoleic acid) in humanplasma as a prior step before GC quantification was showedby Shahin et al [61] Another paper prepared by Kramer etal demonstrated that the best technique to analyze the CLAand trans 18 1 isomers in synthetic and animal products isthe combination of gas chromatographywith Ag-TLC orwithAg-HPLC [62] Moreover usage of Ag-TLC in the separationof isomeric forms of EPA and DHA obtained after chemicalisomerization of them (during fish oil deodorization) may befound in a paper by Fournier et al [63]


A new application of Ag-TLC is bioanalysis A simpleand rapid TLC method for analysis of PUFA levels in humanblood was developed by Bailey-Hall et al [64]

It should be pointed out that besides the modificationof silica gel with Ag+ ions the following metal salts Cu(I)Cu(II) Co(III) and Zn(II) can be used for impregnation ofTLC plates [41 65] Another type of impregnating agent forTLC analysis of fatty acids (MUFA and PUFA) is boric acid Itwas stated that the metabolites of arachidonic acid were sat-isfactorily separated on silica gel impregnated with boric acidas complexing agent and by mobile phase hexane-diethylether (60 40 vv) [41 65] Next modification of stationaryphase which has impact on resolution effect of fatty acids andtheir derivatives such as metabolites (eg phospholipids) isEDTA and mobile phase containing chloroform-methanol-acetic acid water in volume composition of 75 45 3 1 [4166] Another work showed that efficient separation of fivedifferent phospholipids could be achieved by impregnationof TLC plates with 04 ammonium sulfate A mixture ofchloroform-methanol-acetic acid-acetone-water in volumecomposition of 40 25 7 4 2 was suitable for this procedure[67]

Besides the above-presented TLC system in normal phase(NP-TLC) unsaturated fatty acids and their metabolitescould be separated on RP-TLC plates One of the firstreports which are focused on RP-TLC analysis of PUFA wasmade by Beneytout and coworkers in 1992 [68] Beneytoutet al separated arachidonic acid and its metabolites onreversed-phase layer The plates were silica gel coated withphenylmethylvinylchlorosilane A mixture of heptane-methyl formate-diethyl ether-acetic acid (65 25 10 2vvvv) was applied as mobile phase [68]

22 2D-TLC of MUFA and PUFA Two dimensional TLC(2D-TLC) is one of the newly developed powerful tools toseparate various lipids mixture and fatty acids coming fromlipids It is known that 2D-TLC improved the quality of sepa-ration but it is much more time consuming in comparisonwith very popular 1D-TLC [41] Literature review showedthat 2D-TLC is rather a method of choice for separationof lipids from cell membrane polyphosphoinositides andalso of lipid oxidation products in mixture This analysisis usually performed on silica gel impregnated with mag-nesium acetate (75) and by solvent system chloroform-methanol-ammonia (5 25 5 vvv) in the first direction andchloroform-acetone-methanol-acetic acid water (6 8 2 21vvvv) in the second dimension [69]

23 Detection of Spots and Quantification Methods of MUFAand PUFA Detection of fatty acids by TLC method is basedon their visualization by binding to a dye As is was reportedin excellent review by Fuchs et al [41] a lot of visualizingreagents suitable for detection of fatty acids are describedin the literature Among them the most popular reagentsare iodine vapors 2101584071015840-dichlorofluorescein rhodamine6G which produce coloured spots and also primulinewhich gives sensitivities in the nanomole range [41] Incase of PUFA intense darkening is achieved after theirseparation on AgNO

3impregnated TLC plates (as an effect

of reduction of Ag+ to colloid silver) but this method ofdetection required the presence of aromatic hydrocarbon asmobile phase component [70] Other visualizing reagentsare as the following sulfuric acid potassium dichromatein 40 sulfuric acid or 3ndash6 solution of cupric acetatein phosphoric acid Moreover detection of different fattyacids is possible by PMA (phosphomolybdic acid) and bysulfuryl chloride vapors [41] Visualization of fatty acidspots is performed by spraying or dipping the plates insolution of respective visualizing agents Next the spots areobserved under UV light or identified by densitometry Formore detailed characterization of fatty acids which have beenseparated by thin-layer chromatography TLC combinedwithmass spectrometer (TLC-MS) may be used In this methodthe spots are eluted from the chromatographic plates withrespective solvents and next obtained fatty acids are analyzedby MS Applying of TLC coupled with MS enables highresolution of identified peaks Moreover there is no need toextract sample from the plates prior to this analysis [41] Anovelty in thin-layer chromatographic instrumentation is aTLC in combination with MALDI MS spectrometer (TLCMALDI) [44 71 72] This technique is rather fast andprovides spectra that can be relatively simply analyzed andtolerates high sample contamination [41] The detectionlimit of fatty acids determined by TLC MALDI mightbe less than 1 nanogram [41] It was stated that TLCMALDI could be satisfactorily applied to very complexlipid mixture (eg extracts from stem cells) [47] Forexample by means of combined thin-layer chromatographyand MALDI-TOFMS analyses of the total lipid extractof the hyperthermophilic archaeon Pyrococcus furiosuswere performed [45] Next modern trend in analysisof lipid profile is the use of TLC method coupledwith FID (flame ionization detector) [73] ChromarodIatroscan TLC-FID was successfully used in the analysis oflipid classes and their constituents of fatty acids extractedfrom seafood As it was described in the paper by Sinanoglouet al [73] Iatroscan is an instrument that combines TLCresolution with capacity of quantification by FID EfficientTLC-FID separation can be achieved by addition of polarsolvent system without changing the stationary phaseHowever this apparatus allows analyzing in a short time (2-3 hr) in comparisonwithGC orHPLC about 30 samples [73]

24 TLC Separation of cis and trans Isomers of MUFA andPUFA Since it was reported that the saturated fatty acidsindicate correlationwith cardiovascular diseases unsaturatedfatty acids have been recommended for replacement ofsaturated fatty acids in a diet For this reason an increaseof interest in unsaturated fatty acids such as n-6 and n-3 fatty acids is observed It is known that the discussedunsaturated fatty acids form specific geometrical isomersThey can be trans or cis depending on the orientation ofdouble bond Of all polyunsaturated fatty acids the transPUFA consisting of C18 C20 and C22 chain lengths areusually part of the human diet Thus it is very importantto detect and quantify them in food products One ofthe most popular method obtaining the mono- di- andtriunsaturated fatty acids in form of geometrical isomers


(cis and trans) respectively is thermal or chemical process[63] Synthesis of trans isomers is usually made by foodmanufacturing (refining hydrogenation) For instance transisomers are formed during deodorization (crucial step ofrefining) of vegetable or fish oils As it was reported byFournier et al [63] the methodologies regarding accurateseparation and quantification of trans isomers of mono-di- and triunsaturated fatty acids by chromatographic meth-ods were developed in the last decade Ag-TLC is oneof the most powerful chromatographic techniques widelyapplied to separate cis and trans isomers of LC-PUFA becauseit is characterized by simplicity low cost and efficiencyGeometrical isomers are separated according to their numberof double bonds The efficacy of Ag-TLC for separation ofEPA and DHA isomers was confirmed by Fournier et al in2006 [63] For this purpose TLC plates precoated with silicagel and impregnated by AgNO

3were used A mixture of

toluene-methanol in the volume composition 85 15 wasapplied as a mobile for resolution of EPA mono- di- tri-tetra- penta-trans and DHA hexa-trans isomers [63] Inanother work in order to determine the profile of cis andtrans isomers of CLA in liver by the TLC method a mixtureof trichloromethane-n-hexane-glacial ethanoic acid 65 35 1(vvv) and also silica gel plates were applied Rhodaminesolution was used as a visualizing agent In further steps aftermethylation of separated fatty acids they were quantified byGC method [74]

Next paper indicated that preparative silver nitrate thin-layer chromatography was successfully applied to analyze theciscis-octadecadienoic acid (18 2) in commercial samplesof bovine butter fat The detected by Ag-TLC ciscis-59-18 2 isomer was found for the first time in butter fat [54]In another food study ninety-three commercial samples ofBulgarian butter fats manufactured evenly through the yearwere subjected to quantitative silver nitrate-TLC of fatty acidscomponents (as isopropyl esters) with particular attention totransmonoenoic fatty acid content [75] All performed resultsindicate that argentationTLC should be an effective analyticaltechnique in fractionation and identification of geometricisomers of unsaturated fatty acids such as PUFA

Table 1 shows the most efficient TLC systems used forseparation and fractionation of fatty acids from variousmatrices

3 High Performance LiquidChromatography (HPLC)

There are few excellent original papers and reviews untiltoday which are focused on usage of liquid column chro-matography for the analysis of fatty acids (saturated andunsaturated) and also their related substances in biologicalfood and drugs samples [18 93ndash96] In these papers prin-ciples of HPLC including the sample preparation mobilephases stationary phases and detectionmethodswerewidelyperformed Among the earlier review papers only three ofthem survey knowledge of long-chain fatty acids One ofthem prepared by Rao et al [94] showed the LC-PUFA anal-ysis with use of HPLC but since 1974 until 1995 Next papersupported by Rezanka and Votruba [96] demonstrated

the use of chromatography including HPLC for analysis ofvery long-chain fatty acids from 1982 to 2001 The thirdreview prepared byKolanowski and coworkers [97] describedthe important instrumental methods such as HPLC andalso GC for analysis of omega-3-long-chain polyunsaturatedfatty acids but in food only Lack of the overview about thenew achievements in HPLC analysis of mono- and polyun-saturated fatty acids with an emphasis on the analysis oflong-chain polyunsaturated fatty acids such as omega-3 andomega-6 causes that there is a necessity to perform a com-plete literature survey from 2002 to 2013 (last decade) withemphasis on application of HPLC for the analysis of all bio-logical important LC-MUFA and LC-PUFA at the analyticallevels in various matrices This paper highlights the modernachievements of the HPLC including the principles of MUFAand PUFA analysis in different matrices separation modesand the new detection systems which enable quantificationof LC-PUFA at nanogram level The current knowledge ofHPLC analysis of mono- and polyunsaturated fatty acid wasdescribed on the basis of the works which were publishedduring the past decade (2002ndash2013)

The use of HPLC for the separation and quantificationof fatty acids increased since 1950 when it was applied forfirst time by Haward and Martin for analysis of some fattyacids [98] From this time until today a very quick progressin HPLC analysis of all fatty acids including LC-MUFA andLC-PUFA is observed It is known that based on natureof stationary phase (solid or liquid) the high performanceliquid chromatography is divided into [18] liquid-liquidchromatography (LLC) adsorption chromatography (LSC)and reversed-phase chromatography (RP) which is a combi-nation of LSC and LLC

Among above-mentioned high performance liquid chro-matographic techniques RP-HPLC plays a key role in LC-PUFA analysis [94] This technique allows separating thosefatty acidswhich cannot be separated by normal phaseHPLCAs it was early reported by Rao et al [94] the retention of fattyacids in RP-HPLC depends on the polarity of the stationaryphase mobile phase and chemical structure of examinedfatty acids In general the retention time is proportional tothe chain length and the number of double bonds present inexamined fatty acids Additionally the influence of geometricisomerization of fatty acids plays a very important role inreversed-phase HPLC analysis It was reported that the cisisomers of the fatty acids are generally eluted before transisomers Similar effect is observed in case of the differencesin number of carbons in studied fatty acidsThose with smallamount of carbons (short chain) are eluted first in compar-ison with long-chain fatty acids [94] The current literatureindicates that the reversed-phase HPLC is one of the popularmethods used in the field of fatty acids analysis of MUFA andPUFA because its simplicity reproducibility and credibilityThe results obtained by HPLC are usually comparable withthose determined using GC method Moreover the timeof analysis is comparable to GC technique [94] Howeverdevelopment of new detector mode such as flame ionizationdetector (FID) or electrochemical detector (ED) enhances theapplicability of HPLC for MUFA and PUFA analysis [93]


Table1Survey

ofsomes

electedTL

Ccond

ition

susefulfor

separatio

nandidentifi

catio

nof

MUFA

andPU

FAfro

mvario

ussamples

Analyte

Stationary

phase

Mob

ileph

ase

Remarks

Reference

Extracto

flipidsc

omingfro

maquatic

organism

scon

tainingPU

FAPlainsilicag

elplate

Hexane-diethylether-acetic

acid

955

1(vvv)

Thep

latesw

eres

prayed

with

sulphu

ricacid

andnext

analyzed

densito

metric

allyat60

0nm

[40]

Hum

anplasmalipidsc

ontaining

linoleica

cid

Microscop

eplates

coated

with

silicag

elG


acid

9010

1(vvv)

Thes

potswerev

isualized

bysolutio

nof

2101584071015840-dichlorofl

uoresceinin

ethano

lorb

ysulfu

ricacid

respectiv

ely[61]

Deodo

rized

fishoilcon

taining

eicosapentaeno

icand

docosahexaenoica

cid

Argentatesilicag

elTo

luene-methano

l85

15(vv)

Ag-TL

Cmetho

d[63]

Seafoo

dcontaining

saturatedand

unsaturatedfatty

acids

Silicag

elcoated

Chromarod

Iatro

scan

Hexane-diethylether-fo

rmicacid

99105(vvv)

Chloroform

-methano

l-water

54

1(vvv)

TLC-

FIDmetho

d(TLC

coup

ledwith

flameion

izationdetector)

[73]

Milk

samplec

ontaininglin

oleica

cid

Argentatesilicag

elHexane-diethylether

9010(vv)

Ag-TL

Cin

combinatio

nwith

UVdetectionat234nm

the

spotsw

erev

isualized

bysolutio

nof

2101584071015840-dichlorofl

uorescein

inmethano

l[76]

Cell

lipid

extractcon

tainingPU

FASilicag

elplates

Chloroform

-methano

l-acetic

acid-w

ater

6050

14(vvv)

Heptane-diethylether-aceticacid

6040

2(vvv)

TLC-

densito

metryafte

rdevelo

ping

thep

latesw

ere

impregnatedwith

cupricacetatea

ndph

osph

oricacid

[77]

Fatty

acidsisolatedfro

mlip

id-rich

seeds

TLCplates

(silica

gel60)


acid

7030

1(vvv)

TLCwith

UVdetectionat365n

methanolicsolutio

nof

2101584071015840-dichlorofl

uoresceinwas

used

tovisualizethe

spots

[78]

Fatty

acidsseparated

from

oleosomes

ofbeachpea

Silicag

elTL

CHexane-diethylether-acetic

acid

8515

2(vvv)

TLCmetho

diodine

vapo

rswereu

sedto

visualizethe

spots

[79]


31 Derivatization Methods of Fatty Acids for HPLC AnalysisThe most important problem in HPLC study of LC-MUFAand also LC-PUFA is a need to detect them at lower concen-tration levels such as nanograms or picogramsDerivatizationof fatty acids can improve the importance for HPLC analysisparameters such as sensitivity precision selectivity and also alimit of detection and quantification [18 95] Different deriva-tization methods used for quantitative and qualitative deter-mination of fatty acids were widely described in review paperby Rosenfeld in 2002 [99] Generally the kind of derivatizingagent depends on the type of detector applied in PUFA anal-ysis including UV absorption fluorescence light scatteringand refractive index detectors [94]

UV-VIS detector is the most popular detection systemapplied in liquid chromatography because it is sensitive andspecific In 1983 Aveldano and coworkers [100] have appliedreversed-phase high pressure liquid chromatography withUV detection on octadecylsilyl column to separate a mixtureof underivatized unsaturated and saturated fatty acids andtheir methyl esters coming from mammalian tissues

Generally in order to facilitate the detection by UV-VIS absorption the following derivatizing reagents for fattyacids are used [93] phenacyl bromide (PB) p-bromo-phenacyl bromide (BPB) p-chlorophenacyl bromide (CPB)p-nitrophenacyl bromide which are used in analysis offatty acids in oils standards and blood in the range fromng to pmol [97] Naphthyl esters (obtained by 2-naphthylbromide p-nitrophenacyl and p-dichlorophenacyl allow forthe detection of picograms fatty acids in standard mixture2-Nitrophenylhydrazine (NPH) and 2-bromoacetophenone(or 120572p-dibromoacetophenone BAP) convert the fatty acidsinto specific derivatives which can be detected in biologi-cal samples (serum) with detection limits in fmoles PNB(p-nitrobenzyl) and p-methylthiobenzyl chloride (MTBC)derivatives of fatty acids with detection limits in pmoles areimportant in coconut oil analysis Ideal derivatizing reagentfor chiral separation of fatty acids is 35-dinitrophenyl iso-cyanate (DNPI) [96]

Next very popular detection system applied in HPLC isfluorescence detector which has higher sensitivity in compar-ison with UV-VIS detector For this reason it could quantifythe fatty acids composition at picomole and femtomole levelSeveral fluorescent reagents are used for the derivatizationof fatty acids such as 9-diazomethylanthracene (9-DMA)and 9-anthryldiazomethane (ADAM) Anthryl methyl estersof fatty acids can be easily detected in human plasma andserum in picomoles Other more sensitive derivatives thanADAM are the esters of pyrenyldiazomethane (PDAM)Coumarins and 9-aminophenanthrene (9-AP) are importantin detection of fatty acids in environmental and in biolog-ical samples in the range from pmoles to fmoles Otherderivatives like dansyl piperazines and acetamides of fattyacids can be detected in serum below 100 fmol range [9394 97] Recent study performed by Wang and coworkers in2013 indicates that using a new fluorescent labeling reagentnamed 1357-tetramethyl-8-butyrethylendiamine-difluoro-boradiaza-s-indacene (TMBB-EDAN) a sensitive and rapidHPLC technique for the determination of fatty acidsin biosamples (eg human serum) was developed With

the proposed procedure the limit of detection of fatty acidderivatives was in the 02ndash04 nM range [101]

Another type of fatty acids detection is chemilumi-nescence This method is based on the prelabeling of thendashCOOH groups with a proper chemiluminogenic reagent Inpractice luminal and its related compound isoluminal havebeen widely utilized as chemiluminescence derivatizationreagents for fatty acids because of their chemiluminescentproperties Described detection system is efficient for thesensitive detection of fatty acids in human serum and plasmain picomoles [93]

In order to detect the substances with oxidizing or reduc-ing properties including fatty acids an electrochemical detec-tor (ED) may be used This detection mode enables measureof fatty acids in complex biological samples containing dif-ferent components at nanogram levels To obtain the highersensitivity the following derivatizing reagents for ED can beapplied p-aminophenol 24-dimethoxyaniline 2-bromo-21015840-nitroacetophenone ferrocene derivatives 24-dinitrophenyl-hydrazine and 35-dinitrobenzoyl chloride Clinical applica-tion of HPLC-ED is showed in the paper by Kotani et al [80]In this work determination of plasma fatty acids includingPUFA such as arachidonic acid and also linoleic by highperformance liquid chromatography coupled with electro-chemical detector (HPLC-ED) was performed [80] Thisprocedure may be found suitable for monitoring plasma fattyacids in diabetic patients

Among different types of detectors used inHPLC analysisof fatty acids very useful is that detector which requiresnone of the preliminary derivatization procedures of studiedcompounds like for example evaporative light-scatteringdetector (ELSD) or mass spectrometer (MS) [93] As it wasdescribed in paper by Lima and Abdalla [93] evaporativelight-scattering detector is sensitive tomass of vaporized ana-lyte and its operation system is not limited by the absorptioncharacteristics of the individual components and nature ofthe eluent Thus ELSD may be used to solve the problem ofseparation of fatty acids which have weak absorbance groups[93]

32 Mass Spectrometry Detection of Fatty Acids Applicationof HPLC-MS in analysis of fatty acids is known only from thelast decade Mass spectrometer is extensively used in analysisof fatty acids especially in case of biological samples Oneof the main advantages of HPLC-MS method is possibilityto analyze nonvolatile compounds including fatty acids [93]Numerous ionization and detection modes can be appliedfor fatty acids analysis such as electrospray ionization (ESI)atmospheric pressure chemical ionization (APCI) and alsotime of flight (TOF) A new possibility in HPLC-mass spec-trometry is tandem MS (HPLC-MS-MS) or combination ofelectrospray ionization with tandemmass spectrometer suchasHPLC-ESI-MS-MS techniqueThemajor advantage of LC-ESI is the great separation of fatty acids in complex matriceslike blood plasma HPLC-MS-MS is a powerful tool indetermination of double bond position or branched points inthe chain of unsaturated fatty acids [93] Next HPLCmethoddeveloped for detection of long-chain fatty acids is HPLC-MSmethod combinedwithAPCI whichwas applied in detection


of very long-chain fatty acids in form of picolinyl esterscoming from sugar cane wax [102] HPLC-MS coupled withthree ionization systems electron impact (EI) atmosphericpressure chemical ionization and electrospray ionizationwas suitable for the accurate determination of PUFA and alsotheir oxidative metabolites such as eicosanoids in biologicalsamples in the range of pg [103] According to papersprepared by Rezanka et al which were focused on fattyacids analysis in lower organisms liquid chromatographymass spectrometry coupled with ionization mode APCI wasthe most efficient method applied for identification andquantification of very long-chain polyunsaturated fatty acidsfrommarine organisms (which have ability to produce VLC-PUFA) such as marine dinoflagellates Amphidinium carteraeand green algae Chlorella kessleri [81 104ndash106] and alsoin oil obtained from Ximenia fruits (raw material forcosmetic industry) [107] As it was performed in thesepapers HPLCMS-APCI system consisted of Hichrom column(HIRPB-250AM) and gradient solvent programwith acetoni-trile (MeCN) dichloromethane (DCM) and propionitrile(EtCN) which were suitable for separation 13 of picolinylesters of VLCPUFA from lower organisms Excellent separa-tion of methyl esters of unsaturated fatty acids coming fromfreshwater crustaceanwas achieved by the samemobile phaseand the chromatographic column packed with octadecylsilylphase (Supelcosil LC-18) [108] An extensive review of avery long polyunsaturated published by Rezanka and Siglerindicates that the modern analytical methods such as HPLC-MS make the detection and identification of VLC-PUFApossible in different classes of lipids includingmicrobial king-doms and fungi [109] Reversed-phase HPLC with gradientelution of solvent system containing acetonitrile and chlo-roform and equipped with light-scattering detector (ELSD)was used to purify and identify the methyl esters of C16ndashC28 PUFA including octacosaoctaenoic acid in milligramquantities from marine microalgae [82] A simple methodbased on reversed-phase ion-pair high performance liquidchromatography (RP-HPLC) was used successfully to sepa-rate the variousmonoepoxides of eicosatrienoic arachidoniceicosapentaenoic and docosahexaenoic acids [110] Thesecompounds were easily identified by liquid chromatographymass spectrometry (HPLC-MS) with atmospheric pressurechemical ionization (APCI) in nanogram range [110] Thiswork demonstrated that themethod based on APCI-MS cou-pled with HPLC is highly reliable for the analysis of variousmonoepoxides of PUFA in their metabolism study AnotherHPLC system such as nonaqueous reversed-phase highperformance liquid chromatography (NARP-HPLC) withatmospheric pressure chemical ionization (APCI-MS) wassuitable for detection of 5 unsaturated polymethylene inter-rupted fatty acids such as cis-59-octadecadienoic (taxoleic)cis-5912-octadecatrienoic (pinolenic) cis-511-eicosadienoic(keteleeronic) and cis-51114-eicosatrienoic acids (sciadonic)isolated from conifer seed oils (obtained from EuropeanLarch Norway Spruce and European Silver Fir) [111] Com-bination of two chromatographic methods such as TLCand HPLC was used to analyze n-3 fatty acids in fish oildietary supplementsThe EPA andDHA fraction obtained bymeans of Ag-TLC method were further analyzed by HPLC

The chromatographic columnODS (39mm times 30 cm 10 120583m)mobile phase containing tetrahydrofuran-acetonitrile-water-acetic acid 25 35 75 04 (vvvv) and photodiode arraydetector in the range of 190ndash240 nm were used in thisanalysis [112] HPLC system equipped with analytical columnSupelcosil C18 (46mm times 25 cm) and photodiode arraydetector (DAD) was used for the analysis of hydroperoxyPUFA as the products of peroxidation coming from humanplasma Amixture of acetic acid-acetonitrile-tetrahydrofuran52 30 18 (vvv) as mobile phase was used The analysiswas monitored at 200ndash300 nm Quantity control by appliedprocedure of obtained derivatives of PUFA may be useful asclinicalmarkers of oxidative stress on biological systems [113]In another paper gradient reversed-phase liquid chromatog-raphy (C18 ODS 25 cm times 046 cm 5 120583m) by use of methanolwater and universal ELSD detector allowed for separationand fractionation of saturated unsaturated and oxygenatedfree fatty acids as methyl esters extracted from seeds ofCrepis alpina and Vernonia anthelmintica respectively [114]This work showed that reversed-phase C18 was suitablefor purification and fractionation of PUFA before theirquantification by GC-MS Besides HPLC system equippedwith above-mentioned types of detector modes such as ECDMS ELSD DAD and UV a fluorescence detector is alsovery useful [115 116] which enabled precise determinationof long-chain polyunsaturated fatty acids including linolenicarachidonic eicosapentaenoic and docosahexaenoic acids inhuman serum at low concentrations like fmoles For examplethe method described in 1986 by Yamaguchi et al performedthe conversion of LC-PUFA into fluorescent derivativesby reaction with 3-bromomethyl-67-dimethoxy-1-methyl-2(1H)-quinoxalinone Separation process was performed ona reversed-phase column (YMC Pack C8) with an isocraticelution using aqueous 72 (vv) acetonitrile [115] Currentliterature shows that HPLC with different solvent systems issuitable in analysis of novel hydroxyl fatty and phosphoricacid esters of 10-hydroxy-2-decenoic acid (9-HAD) comingfrom the royal jelly of honeybees (Apis mellifera) HPLCanalysis was carried out on Diaion HP-20 Sephadex LH-20and Cosmosil 75C18-OP column systems Different mobilephases containing acetonitrile were applied A refractiveindex detector was applied to monitor the elution profile ofexamined fatty acids [117]

33 Silver Ion HPLC in Quantification and Identificationof Geometric Isomers of Fatty Acids As it was reportedby Nikolova-Damyanova and Momchilova [118] silver ionHPLC (Ag-HPLC) is widely applied by many scientists asa preliminary step for the fractionation of complex mixtureof fatty acids into their groups prior to quantitative analysisby GC method Resolution of fatty acids by means of Ag-HPLC is achieved according to the number and geometryof double bonds [118] Separation is based on the reversibleformation of a weak charge-transfer complex between a silverion and a double bond The main problem in Ag-HPLC isintroduction of Ag+ into this systemThus similar to the caseof Ag-TLC the first attempt was performed on column whichwas laboratory packed with slurry of the stationary phase(eg silica gel) impregnated with AgNO

3 Second method is


to add silver ion solution (AgNO3) into mobile phase Third

method is to use commercially available silica based cationexchange column (Nucleosil 5 SA) or the column whichis produced by Chrompack (ChromSpher 5 lipids) Thesecommercial column systems give much better results (betterreproducibility) of fatty acids analysis than those laboratoryprepared [118] Another excellent review of the chromato-graphic methods used to analyze geometrical and positionalisomers of fatty acids by Aini et al [119] showed that thefollowing factors affect resolution of fatty acids in silver ionHPLC such as the impregnation method of column mobilephase composition and also column temperature [119] Thechoice of mobile phase composition used in Ag-HPLC isusually toluene based acetonitrile based hexane based ordichloromethane based [118 120] Good results have beenachieved by the use of isopropanol and tetrahydrofurane asa modifier The lower column temperature generally resultsin shorter elution time in Ag-HPLC Moreover the improve-ment in resolution of monoenoic and polyenoic fatty acidswas obtained by converting them into phenacyl benzyln-propyl n-butyl and ethyl isopropyl esters [52] Sep-aration by Ag-HPLC coupled with UV detector of cis-and trans-octadecanoic acids described by Momchilova andNikolova-Damyanova [83] indicated that the efficiency ofthe separation increases in the following order phenethyl ltphenacyl lt p-methoxyphenacyl esters Yet retention and res-olution of these fatty acids by Ag-HPLC could be affected bysmall changes of dichloromethane in mobile phase Amongvarious chromatographic systems the best resolution of cis-and trans-positional isomers of octadecenoic acid after con-verting them into p-methoxyphenacyl esters was achieved ona silver ion column by isocratic elution with a mobile phasecontaining hexane-dichloromethane-acetonitrile in volumecomposition of 60 40 02 (vvv) [120] Similar mobilephase was used by Momchilova and Nikolova-Damyanovain 2000 to estimate the chromatographic properties of posi-tional isomers of octadecenoic fatty acids after conversingthem to 2-naphthyl 2-naphthylmethyl and 9-anthrylmethylderivatives [83] According to the results obtained inthis paper dichloromethane-acetonitrile 100 0025 (vv) asmobile phase provided better resolution of 9-anthrylmethylderivatives of 6- 9- and 11ndash18 1 High resolution by Ag-HPLC was obtained for the positional isomers of PUFA suchas eicosapentaenoic acid docosahexaenoic acid and alsodocosapentaenoic acid coming from triacylglycerols con-taining PUFA The hexane-isopropanol-acetonitrile solventsystem was suitable for the separation of these compounds[84] Isolation of some PUFA from edible oils by argentatesilica gel chromatography (Ag-TLC and Ag-HPLC) was per-formed by Guil-Guerrero et al [121] Using this method theisomers of the following fatty acids linoleic acid 120572-linolenic120574-linolenic and stearidonic acid and eicosapentaenoic anddocosahexaenoic acid have been isolated in form of methylesters from linseed sunflower and borage seed oils and fromshortfin mako liver oil Similarly like it was described inthe previous part Ag-HPLC was useful for analysis of eicos-apentaenoic and docosahexaenoic acid geometrical isomersformed during fish oil deodorization [63] Obtained resultsshowed that this technique cannot be used to determine

the isomers in fish oil which have been formed at the temper-ature higher than 180∘C (eg at 220∘C) because the interfer-ence between obtained isomers especially di-transDHA andall-cis EPA was observed Efficient analysis of trans isomersof conjugated linoleic acid in synthetic and animal prod-ucts (from pigs chicken meat) was achieved also by chro-matographic techniques including Ag-HPLC [62] Recentliterature indicates that quantification of separated by Ag-HPLC geometric isomers of fatty acids is mainly performedby GC-MS or by GC coupled with FID (flame-ionizationdetector) FID UV and refractive detector are usuallyused for direct quantification of isomers of fatty acids asmethyl esters As it was reported by Nikolova-Damyanovaand Momchilova [118] aromatic esters of fatty acids can bedetected by Ag-HPLC combined with UV-VIS detector in therange of 0ndash200120583g The paper prepared in 2013 by Sun andcoworkers demonstrates that a liquid chromatographyin-line ozonolysismass spectrometry (LCO

3-MS) is a practical

and easy to use new approach to direct determination of dou-ble bond position in lipids presented in complex mixture Inorder to test this method in complex lipid extracts a sampleof bovine fat with known amount of positional isomers andcistrans isomers of unsaturated fatty acids was analyzedThemain advantage of the in-line ozonolysis is its applicabilityin combination with various mass spectrometers withoutinstrumental modification [122]

Survey of some selected HPLC conditions useful forseparation and identification of MUFA and PUFA fromvarious samples is listed in Table 2

4 Gas Chromatography (GC)

Among different chromatographic methods such as TLCor HPLC which are described in detail in this paper gaschromatography (GC) is the most popular for fatty acidsanalysis As it was reported byCasal andOliveira GC analysisof fatty acids is the first chromatographic method known infatty acids analysis for 60 years [123] From this time to 2002year numerous original and review papers were performedwith emphasis on a gas chromatographic analysis of thecommon and uncommon fatty acids [85 123ndash125]

The aimof ourworkwas to describe present-dayGC anal-ysis of fatty acids belonging to mono- and polyunsaturatedgroup (MUFA and PUFA) whose biological significance hasincreased in recent years This paper was prepared on thebasis of the papers coming from the last decade (2002ndash2013)

41 Derivatization Methods for GC Analysis of Fatty AcidsThe main step in GC analysis of fatty acids is the processof conversion of these compounds into suitable volatilederivatives [123] The most frequently applied derivatives arealkyl derivatives (methyl- ethyl- and isopropyl-) obtainedby means of esterification of fatty acids This procedurefacilitates volatility of fatty acids and also improves theirseparation and sensitivity to GC detectors For this aim thefollowing esterification methods are applied [123] methyla-tion catalyzed byH+ esterification catalyzed byOHminus esterifi-cation with benzyl alcohol (p-bromobenzyl p-methylbenzyland p-nitrobenzyl iodide)methylationwith diazomethane in


Table2Survey

ofsomes

electedHPL

Ccond

ition

susefulfor

separatio

nandidentifi

catio

nof

MUFA

andPU

FAfro

mvario

ussamples

HPL

Csyste

mMob

ileph

ase

Colum

nDetectio

nmod

eMatrix

Remarks

Reference

Ag-HPL

CIsocratic

solventsystem

hexane-acetonitrile

99703(vv)

Chromspher5

Lipidcolumn

(250times46mmV

arianNetherla

nds)

UV-VIS

detector

Deodo

rized

fishoilcon

taining

EPAD

HAG

LAL

AA

LA

SDand

otherP

UFA

Detectio

nlevela

[63]

HPL

C-EC

DEthano

l-acetonitrile

2080(vv)

ODS100R

P-18

column

LiCh

rospher

250m

mtimes4m

m5120583m

phase

particle

Electro

chem

icaldetectorsaturated

calomelele

ctrodedetectio

npo

tentialminus

415m

V

Hum

anplasmacontaining

vario

usfatty

acidsincluding

arachido

nica

cidandlin

oleic

andoleica

cids

Detectio

nlevel

50ndash160

0pm

oles

[80]

HPL

C-MS-APC

I

Theg

radientsolvent

program

was

usedM

eCNEtCND

CM60

30

10(vvv)

MeC

NEtCND

CM30

40

30(vvv)

Hichrom

columnHIRPB

-250

AM

250m

mtimes21m

m5120583m

phase

particle

Picolin

ylestersweres

eparated

byuseo

fquadrup

lemassspectrometer

fittedwith

anatmosph

ericpressure

chem

icalionizatio

nsource

Dinofl

agellateAm

phidinium

carterae

containing

VLC

-PUFA

upno

nacosahexaenoica

cid

Detectio

nlevela

[81]

RP-H

PLC-

ELSD

Theisocraticelu

tionof

MeC

NH

2Owas

used975

25

(vv)fl

owrateof

mob

ileph

ase

15mlm

in

RPcolumn(A

lltim

aC18Alltech

250m

mtimes46m

m)5120583

mph

ase

particle

Methyleste

rsPU

FAwered

etected

byevaporativelight-scatte

ring

detector

(ELSD)

Marinea

lgae

Schizochytriu

mspcon

tainingDHAE

PAand

theird

erivatives

Detectio

nlevelmg

[82]

Ag-HPL

CDichlorom

ethane-acetonitrile

100

0025(vv)

Nucleosil100-5SA

(250

mmtimes46m

m)Hichrom

Re

adingUK

UV-VIS

detectorthe

working

wavele

ngth

was

250n

mforthe

anthrylm

ethyland

naph

thyl

deriv

atives

andalso

275n

mforthe

naph

thylmethyld

erivatives

ofexam

ined

octadeceno

icacid

References

tand

ards

ofpo

sitionalisomerso

foctadeceno

icacid

andtheir

deriv

atives

(este

rs)

Detectio

nlevel120583g

[83]

Ag-HPL

C

Gradientsolvent

syste

mconsistso

fhexane-isop

ropano

l-acetonitrile

350

100

275

(vvv)

350

100

10(vvv)

Chrompack

5Lipidcolumn

(250

mmtimes46m

mN

etherla

nds)

UV-VIS

detector

thew

orking

wavele

ngth

was

206n

mAPC

I-MS

Oilprod

uced

byam

arine

microbe

containing

PUFA

(DHAE

PA)

Detectio

nlevela

[84]

a Not

describ

edby

authorsMeC

N-acetonitrileE

tCN-propion

itrileand

DCM

-dichlorom

ethane


ethereal solution or with boron trichloride (BCl3-MeOH)

methylation by means of tetramethylammonium (TMAH)hydroxide or trimethylsulfoniumhydroxide (TMSH) deriva-tization with tert-butyldimethylsilyl (tBDMSi) or dimethy-loxazolidine (DMOX) and also cyanomethyl derivatization

Among the above-presented derivatization proceduresthe most important for GC-MS analysis of mono- andpolyunsaturated fatty acids are DMOX derivatives and picol-inyl derivatives which are widely applied for the determina-tion of double sites in unsaturated fatty acids A rapid andinexpensive method for separation of very long-chain fattyacids is cyanomethyl derivatization

42 Instrumentation of GC In gas chromatography of fattyacids a variety of columns with different properties such aspolar and nonpolar are available Of all the most frequentlyapplied in fatty acids analyses are the polar columns Thefused silica columns allow improvements in the separation ofPUFA for example fromfish oil samples Another type of sta-tionary phases used for fatty acids analysis is polar polyesterssuch as Carbowax column for example PEG (polyethyleneglycol) which is commercially available as Carbowax-20MCP-Wax 52CB DB-Wax and so forth Next example of polarstationary phase used in GC is cyanopropyl polysiloxanesuch as commercially available HP-88 CP-Sil88 BPX70 SP-2340 and SP-2560 As it was described in Casal and Oliveirareport [123] the high polarity of the cyanosilicone phasesallows the separation of geometrical isomers (cis and trans)of mono- and polyunsaturated fatty acids [123]The literaturereview indicates that packed GC columns (glass tubing) withstationary phase containing usually from6 to 5mmdiametersare still in use in fatty acid analysis Among them the mostpopular is Chromosorb series Others which belong to polarstationary phases are organosilicon polyesters (EGSS-X)butanediol succinate (BDS) and diethylene glycol succinate(DECS) [121 123]

Of different GC detectors which show high sensitivity(from nanograms to femtograms) such as NPD (nitrogenphosphorus-sensitive detector) FDP (flame photometricdetector) ELCD (electrolytic conductivity detector) and PID(photoionization detector) FID (flame ionization detector)and also mass detector (MS) are commonly used in fattyacids analysis [124] FID allows detecting the organic sub-stances including unsaturated fatty acids mono- and poly- inpicograms The carrier gases used in this method are nitro-gen helium and hydrogen GC-MS detection is a powerfultool in GC analysis of fatty acids (saturated and unsaturated)coming from different sources such as sea water wood andfish oil The fact that MS is suitable for the localization ofdouble bond in polyunsaturated fatty acids is important Anefficient detection method in study of very long-chain fattyacid esters in biological sample (blood urine) and wax istandem spectrometry MSMS [18] Recently a novel trend inGC detection is coupling two different detectors in parallelto the outlet of column for example FID-ELCD Moreoverapplication of GC analysis with FT-IR or NMR technique canfacilitate the determination of functional groups and struc-tural studies of fatty acids in the case of their geometricalisomers [18]

43 GC-Mass Spectrometry (GC-MS) of Fatty Acids BesidesGC-FID mass spectrometry coupled to GC (GC-MS) isthe most powerful tool for identification of mono- andpolyunsaturated fatty acids This technique requires deriva-tization of them into picolinyl esters pyrrolidines or 44-dimethyloxazolidines derivatives respectively [123] Casaland Oliveira confirmed that the picolinyl esters are verysuitable in MS analysis of MUFA and PUFA because theyoffer a specific mass fragmentation pattern [123] In 2005Destaillats et al have shown thatmethyl ester derivatives usedfor routine quantification by GC-FID can be also used forstructural investigation by GC-MS of long-chain polyunsat-urated fatty acids (eg metabolites of rumelenic acid) [85]An improvedmethod of GC-MS for the identification of verylong-chain highly unsaturated fatty acids with more than 22carbon atoms (C

28) such as octacosaheptaenoic acid (28 7

n-6) and octacosaoctaenoic acid (28 8 n-3) in seven marinedinoflagellate species was developed by Monsour et al [86]In another work the total lipids of three crustacean speciescoming from Lake Baikal were analyzed for long-chainpolyunsaturated fatty acids such as 24 3 (n-6) 24 3 (n-3) 24 5 (n-3) 26 3 (n-6) 26 5 (n-6) and also 28 7 (n-6) by use of Ag-TLC and GC-MS [87] In further studyautomated GC-MS was applied to identify polyunsaturatedfatty acids in algal and microbial cultures (some freshwateralgae and in filamentous cyanobacterium) A novel GC-MS method developed by Akoto et al in 2005 enableddetection the polyunsaturated fatty acids after their previousderivatization into corresponding methyl esters By meansof the described method unique PUFA in some green algaeand cyanobacterium which play a role of biomarkers inecological and food studies can be determined [88] Anotherwork demonstrated that GC-MS can be successfully usedfor quantitative analysis of total n-3 and n-6 fatty acids inhuman plasma Six PUFA were esterified and next quantifiedby the use of GC-MS and also by means of reference spec-trophotometric method GC analysis was performed on DB-23 fused silica column (30m times 032mm 025mm) Heliumwas applied as carrier gas This study showed that applyingof GC-MS in quantification of PUFA (n-3 and n-6) givesthe results with the high accuracy In addition the statisticalevaluation of both analytical methods indicated that there isno significant difference between GC-MS and spectropho-tometric method [126] In the next paper the content ofseventy-two unsaturated fatty acids (polyenoic) present inmantle muscle and viscera of themarine bivalveMeganguluszyonoensis was determined [127] The GC analysis coupledto FID was performed on capillary column (Supelco 30m times032mm 025mm) Helium was used as carrier gas at aflow rate of 15mLmin DMOX derivatives of examinedfatty acids were analyzed by means of GC-MS equippedalso with Omegawax-320 capillary column (Supelco 30m times032mm 025mm) The spectra were recorded at ionizationenergy of 70 eV Another example indicates the usefulnessof GC-MS in analysis of a new octadecatrienoic acid as acomponent of Chrysanthemum zawadskii Herb (seed oil)To confirm the structure of the new unusual fatty acidsdifferent chemical derivatizations (preparation of methylesters pyrrolidine picolinyl esters and dimethyloxazolidine


derivatives) were applied [128] Capillary GC analysis wascarried out using GC chromatograph equipped with FID anda 50m times 025mm column CP-Sil 88 by means of hydrogenas a carrier gas at a flow rate of 2mLmin GC-MS analysis offatty acid derivatives were performed on silica fused columnSupelcowax 10 (30m times 025mm 025 120583m) Helium was usedas a carrier gas [128] The reviewed papers showed that arapid GC-MS method is the most frequently used techniquefor the quantification and identification of geometric isomersof long-chain PUFA including CLA [129] A series of CLAwith double bond positions with all possible geometries(cistrans transcis ciscis and transtrans) were convertedinto methyl esters and next chromatographically analyzed inthe low femtomole range for diagnostic purpose (in clinicalstudy) [130] Performed GCmethod should bemore accurateto direct quantification of total trans-octadecenoic acid infood samples (eg milk) Results presented by Destaillatset al [131] confirmed that the isomeric profile of trans-octadecenoic acid in milk fat determined by GLC in combi-nation with Ag-TLC may be found as the reference method

The recently published paper by Liu and coworkersindicates that among various analytical techniques GC-MSmethod using newer generation of detection system such assingle quadrupole mass detector is high sensitive new toolfor studies of n-3n-6 PUFA in small samples of humanand mouse retina These studies suggest that the methoddeveloped by Liu et al could be satisfactorily applied in thetreatment of the pathological process of macular degenera-tions and dystrophies [132]

44 GC-FID Analysis (Gas Chromatography Coupled withFlame Ionization Detector) of Fatty Acids The currentresearch shows that flame ionization mode of detection is themost common analytical method used in routine quantitativeanalysis of fatty acids in various matrices because it is robustand of low cost in comparison with mass spectrometry [123]The studies conducted by Schreiner confirmed the accuracyand robustness of GC-FID technique for quantification ofLC-PUFA [133] In order to check the robustness of gaschromatographicmethod in combination with FID for quan-tification of PUFA various chromatographic systems wereused by Schreiner Thus this work is a guideline whichexplains how to achieve the accuracy 17 and robustness ofGC-FID method During the last decade there is an increasein the application of GCFID in monitoring of omega-3 andomega-6 fatty acids in various matrices samples includinghuman and nutritional According to Bocking and coworkersPUFA including the two presented above fatty acids andlinoleic acid could be satisfactorily quantified using GC-FIDin serum for clinical diagnosis (eg in case of cardiovasculardiseases) and in quality control of food for example cowrsquosmilk [89] Another example of application of GC-FID inanalysis of biological samples shows the paper performed byBlaha et al [134] The described GC-FID study of selectedMUFA and PUFA (eg CLA EPA and DHA) and theirmetabolites were analyzed in human blood under the follow-ing conditions fused silica capillary column SP2330 (30m times025mm) helium as a carrier gas and temperature of columnin the range from 120∘C to 230∘C The developed method

plays a significant role in coronary atherosclerosis studyQuantitative aspect of GC-FID bioanalysis of PUFA inplasma lipoproteins was also widely described by Tvrzickaand coworkers [135] Novel possibilities in GC-FID analysisare to use this technique for determining of medium andlong-chain polyunsaturated fatty acids in clinical (parenteral)formulations [136] The data presented in this paper indicatethat GC-FID can be easily used for accurate determinationof PUFA composition in lipid emulsion Most of the papersregarding the use of gas chromatography for analysis ofpolyunsaturated fatty acids such as linoleic acid and theirgeometrical isomers present in dairy fat (eg milk fat) rec-ommend the column type Carbowax such as Supelcowax 10[137]Themain advantage of this column is a better resolutionof the linolenic and eicosadienoic acid isomers [137] Kramerand Cruz-Hernandez and their coworkers indicated thatbesides polyethylene glycol capillary column 100-m CP-Sil88 column (100 cyanopropyl polysiloxane) could be verysuitable in PUFA analysis including CLA isomers in milkfat [76 137] Both helium and hydrogen were used as thecarrier gases respectively Gas chromatograph was equippedwith FIDThe profile of polyunsaturated fatty acids especiallytrans isomers in food samples is usually analyzed by gaschromatographyThe official method recommends the use offatty acids in form of methyl esters [138] In 2000 Timminsand coworkers developed a method for quantification ofomega-3 from marine oils as ethyl esters This analysis wasconducted with gas chromatograph coupled with FID onan Omegawax 320 fused silica capillary column (30m times032mm 025 120583m) The carrier gas was helium at a pressure63 kPa [139] Profile of polyunsaturated trans fatty acids infood products (eg peanut oil) including preparation of fattyacid methyl esters was successfully determined with a highlypolar column CP-Sil 88 Moreover the methylation proce-dure allows accurate isolation of geometrical isomeric oflinolenic acid [140] Tyburczy et al studied the trans polyun-saturated fatty acids in selected fast food such as hamburgerpizza chicken cheese and others [90] This fact showsusage of GC-FID method in monitoring of the trans fattyacid level in some restaurant foods The current researchindicates the usefulness of GC-FID in quantification of somemono- and polyunsaturated fatty acids (eg oleic linoleicand linolenic) in oils of wheat germ and seeds produced asby-products [91] The content of long-chain polyunsaturatedfatty acids like eicosapentaenoic acid and docosahexaenoicacid in different assortments of smokedAtlanticmackerel andBaltic sprats was determined by the use of gas chromatographequipped with FID [92] The results demonstrate that theapplied GC-FID system is a powerful analytical tool incontrol of the PUFA content in sprats smoked fish whichare the source of n-3 and n-6 fatty acids A recent paper hasshown GC-FID determination of fatty acid methyl esters inbiodiesel bland using an ionic liquid stationary phase [141]In this work the two 19-di(3-vinyl-imidazolium)nonanebis(trifluoromethyl)sulfonylimidate stationary phase capil-lary columns SLB-IL-100 (30m times 025mm 025120583m) and onSLB-IL-100 (12m times 010mm 008120583m) have been evaluatedto determine the profile of fatty acids including eicosanoiclinoleic oleic and linolenic in biodiesel blend


45 Gas Chromatography in Analysis of Geometrical and Posi-tional Isomers of Fatty Acids As it was described in previoussection regarding TLC and HPLC methods the analysis ofgeometrical isomers and position of double bonds may givea great impact on nutritional study especially in food qualitycontrol To obtain good results in analysis of geometrical iso-mers of mono- and polyunsaturated fatty acids they shouldbe converted into suitable derivatives To determine thenumber of double bonds the GC equipped with MS detectoris enough [119] Among various derivatives those whichensure good results in identification of double bond positionby GC-MS technique are 44-dimethyloxazolidine (DMOX)derivatives and picolinyl esters [119] In the case ofmonoenoicacids conversion into dimethyl disulphide is satisfactory Inaddition identification of fatty acids double bond positionis possible by GC coupled with MSMS detector whichrequires the derivatization of the fatty acids into proper com-pounds for example PTSA derivatives (p-toluenesulfonicacid derivatives) [142] To themost popular stationary phaseswhich enable satisfactory resolution and positional charac-teristic of double bonds in unsaturated fatty acids belongscyanopropyl capillary column such as CP-Sil 88 in case ofMSMS detection or Supelco 2560 capillary column whichis suitable for GC-FID analysis [141]

46 GC times GC (Two-Dimensional Gas Chromatography)Two-dimensional gas chromatography is a new chromato-graphic technique which recently plays a very important rolein resolution of fatty acids in complex samples such as lipidscoming from marine biota (eg algae) According to Casaland Oliveira the separation process by two-dimensionalgas chromatography (2D-GC) is carried out on the twocolumns with different polarity [123] For example in 2011 Guand coworkers performed a comprehensive two-dimensionalanalysis of fatty acids including PUFA in marine biota [143]The results described in this paper show that in GC times GCmethod using an apolar polydimethylsiloxane times polar ionicliquid column combination an excellent resolution of unsat-urated fatty acids complementary to GC-MS identificationwas achieved Another work performed by Manzano andcoauthors [144] confirmed that GC times GC system using anapparatus equipped with capillary flow technology and aFID has been satisfactorily used to separate a large numberof fatty acids including the eight isomers of the linolenicacid and also the linoleic isomers All examined fatty acidswere obtained from broccoli leaves by use of supercriticalfluid extraction (SFE) The results of this experiment showedthat SFE technique and GC times GC analysis with FID orMS detector are suitable to study fatty acids [144] Thecurrent literature in emphasis onmono- and polyunsaturatedfatty acids analysis in different matrix indicates that besidesthe widely used chromatographic techniques such as TLCHPLC andGC equippedwith various detectors the relativelynew method is gas chromatography coupled to isotoperatio mass spectrometer (known as IRMS) [145] As it wasreported by Meier-Augenstein [145] this technique may beused in fatty acids analysis in complex biological samplesand it is alternative to the typical detection systems appliedin chromatographic analysis Another new solution in gas

chromatographic analysis of unsaturated fatty acids is to useGC in combination with infrared spectroscopy based ontransmission (GC-IR) or on attenuated reflection (GC-ATR)[146] Both techniques are refined in quantification of transfatty acids in food products (margarines vegetable oils)

Survey of some selected GC conditions useful for sep-aration and identification of some MUFA and PUFA fromdifferent matrices is listed in Table 3

5 Supercritical Fluid Chromatography (SFC)

A brief review of the chromatographic studies of LC-MUFAand LC-PUFA indicated that among traditional GC tech-nique and HPLC method an alternative or complementarymethod to both of the mentioned techniques is supercriticalfluid chromatography (SFC) Supercritical fluid chromatog-raphy is known as hybrid of gas and liquid chromatographyIn contrast to GC and HPLC SFC allows the use of higherflow rates low temperature no derivatization and also uni-versal detectors such as FID ELSD orMS [147]This methodleads to greater efficiency in short time analysis times andreduced consumption of organic solvents Moreover as wasaccurately described by Senorans and Ibanez [148] it permitsthe separation and determination of thermally labile (egpolyenic fatty acids) low volatile and polar fatty acids (eghydroxy fatty acids) which cannot be analyzed by HPLCor GC without derivatization For this reason in the lastfew years the use of SFC in fatty acid analysis has attractedincreasing interest in the food sector [147 148]

Supercritical fluid chromatography was usually per-formed with pure carbon dioxide (CO

2) as the mobile phase

Nowadays SFC is often carried out in subcritical conditionsand inmany cases CO

2is modified with an organic solvent in

order to increase the solubility of polar compounds [147ndash149]SFC separation of fatty acids has been performed by

means of different chromatographic columns such as opentubular packed and packed capillary The open tubularcolumns are preferred for separations when high efficienciesare required and also for complex samples [147ndash149]

Different detection modes are applied in SFC analysissuch as UV-VIS detectors ELSD (evaporative light scatteringdetector) andMS detector (mass spectrometer) but themostwidely used in SFC is MS detector It was stated that SFCanalysis combined with MS detector provided very goodlevels of sensitivity Similar to liquid chromatography (HPLC-MS) atmospheric pressure chemical ionization (APCI) andelectrospray ionization (ESI) are the most popular ionizationmethods recommended for SFC-MS analysis Moreoverthere is no limitation of mass analyzer that could be coupledto SFC system [147ndash149]

All important parameters such as mobile phase compo-sition columns and the detection mode systems needed toSFC analysis of fatty acids were accurately described in reviewpapers by Senorans and Ibanez in 2002 and also byBernal andcoworkers in 2013 [148 149] Excellent review study by Bernalet al of the analysis of fatty acids in foods demonstrates thatnew trend in SFC analysis is SFC times SFC system The useof two-dimensional SFC (2D-SFC) or SFC coupled to RP-HPLC improves the separation of very complex mixtures


Table3Survey

ofsomes

electedGCcond

ition

susefulfor

separatio

nandidentifi

catio

nof

MUFA

andPU

FAfro

mvario

ussamples

Analyte

GCcolumn

Derivatization

Identifi

catio

nqu

antifi

catio

nRe

ference

Liverlipidsfrom

ratcon

taining

rumelenicacid

(cis-9tra

ns-11andcis-15

183)isomer

oflin

olenicacid

BPX-

70capillary

columnSG

EMelb

ourne(50

mtimes

032

mm025120583

m)heliu

mwas

used

ascarrierg

asun

derfl

ow24m

Lmin

44-dimethyloxazolid

ine

(DMOX)

andmethyl

esterd

erivatives

Selectiveq

uadrup

olem

assd

etectoru

nder

ionizatio

nvoltage

of70

eVat230∘C

[85]

Thea

lgae

extractcon

taining28

7(n-6)

andalso

288

(n-3)

Polarc

olum

nBP

X-70(50

mtimes032

mm025120583

m)

nonp

olarH

P-1capillarycolumn(50mtimes032

mm

017120583m)h

elium

was

used

ascarrierg

asDMOXderiv

atization

Massspectrometer

(Fiso

nsM

D-800)

underion

izationvoltage

of35

eVat200∘C

[86]

Extractsfro

mthreec

rustaceanspecies

comingfro

mLake

Baikalcontaining

n-3

andn-6PU

FA

Supelco

wax

10column(60mtimes025

mm025120583

m)the

temperature

program

was

from

100∘Cto

280∘C

The

carrierg

aswas

heliu

matflo

wrateof

70cm

s

Methyleste

rsand

oxazolines

ofPU

FAMassspectrometer

with

chem

ical

ionizatio

n(FinniganMAT

1020B)

[87]

Extractsfro

mgreenalgaea

ndcyanob

acteriu

mAfusedHP-5M

S(A

gilent)capillarycolumn(30mtimes

025

mm01120583m)heliu

mwas

used

asmob

ileph

ase

Methyleste

rsof

PUFA

Iontrap

massspectrometer

(ITD

-MS)

was

used

[88]

Clinicalform

ulations

containing

PUFA

Capillary

columnZe

bron

ZB-W

AXplus

(30mtimes025

mm025120583

m)

Methyleste

rsof

PUFA

GC-

FIDsyste

m[89]

Food

samples

containing

saturatedand

unsaturatedfatty

acids(MUFA

and

PUFA

)cis

andtra

ns

SP-2560CP

Scolumn

(30mtimes025

mm020120583

m)hydrogen

was

used

asa

carrierg

as

Methyleste

rsof

MUFA

andPU

FAGC-

FIDsyste

m[90]

Oilextractsfro

mgrapes

eedandwheat

germ

richin

linolenicandlin

oleica

cids

DB-23

(032

mmtimes30

m)temperature

programming

150ndash

230∘C

carrierg

aswas

heliu

mMethyleste

rsof

linolenic

andlin

oleica

cid

GC-

FIDsyste

m[91]

Smoked

mackerelm

eatcon

tainingEP

andDHA

Colum

nRtx2330

(105

mtimes025

mm02120583m)

temperature

programming30ndash210∘C

Methyleste

rsof

EPand

DHA

GC-

FIDsyste

m[92]


A preparative supercritical fluid chromatography was appliedfor PUFA separation to obtain enriched fractions of EPA fromfish oil mixture Silica gel as the stationary phase and carbondioxide as the supercritical fluid were used Under theseconditions the best purity in EP-EE (eicosapentaenoic ethylester) reaches 95 with a 246 yield [149] Recent meta-bolomic investigations show that the SFC-MS enables thesimultaneous rapid analysis of lipids with varied structureApplying cyanopropyl bonded silica gel-packed column theresolution of lipid mixture from the leaf of Catharanthusroseus resulted in satisfactory separation of lipidmixture withtime less than 15 minutes The data obtained in this studyconfirm that SFC system is a powerful tool for studies on lipidmetabolomic because it is useful as a fingerprinting methodnot only for the screening of various lipids but also for thedetailed profiling of their individual components [150 151]

6 Conclusions

This literature review which is focused on the applicationof the chromatographic methods such as TLC HPLC GCand also SFC in analysis of mono- and polyunsaturated fattyacids (MUFA PUFA) in different matrices shows that allof the presented chromatographic methods are suitable inpreseparation and quantification of these compounds Thechoice of the chromatographic systems depends on the typeof the sample to be analyzed and on the aim of chromato-graphic analysis (problem which should be solved by meansof this technique) For instance in order to obtain completefractionation of ldquotranscisrdquo fatty acids from different samplesthe traditional Ag-TLC or argentation HPLC (Ag-HPLC) isused The column chromatography such as GC and HPLCmay be used in further steps of chromatographic analysisof unsaturated fatty acids (MUFA and PUFA) especially todetermine the quantity of separated fatty acids In the caseof geometrical isomers (cis and trans) and determining ofthe number and position of double bonds present in studiedunsaturated fatty acids Ag-HPLC and various GC systemsare popular Current literature indicates that the innova-tions which are implemented into chromatographic systemsinclude modified stationary phases new derivatizing agentsfor fatty acids and also developing of novel detection sys-tems allowing sensitive and reproducible fatty acid analysisfrom complex samples Moreover the new chromatographicsystems combined with other instrumental methods such asMS MSMS IR or two-dimensional GC enable analysis ofa large number of MUFA and PUFA in lower quantitieswhich is required for example in food analysis The resultspresented in our paper confirm that the chromatographicmethods are still a powerful tool in analysis of mono-and polyunsaturated fatty acids

Conflict of Interests

The authors declare no conflict of interests regarding thepublication of this paper

Acknowledgments

This research was financed by the Medical University ofSilesia as part of statutory research in 2014 Project no KNW-1-006N40

References

[1] D Voet and J G Voet Biochemistry John Wiley amp Sons NewYork NY USA 4th edition 2010

[2] R Murray K M Botham P J Kennelly et al Harperrsquos Illus-trated Biochemistry McGraw-Hill Education Toronto Canada29th edition 2012

[3] P Rise F Marangoni and C Galli ldquoRegulation of PUFAmetabolism pharmacological and toxicological aspectsrdquo Pros-taglandins Leukotrienes and Essential Fatty Acids vol 67 no 2-3 pp 85ndash89 2002

[4] A Sellmayer and P C Weber ldquoPolyunsaturated fatty acids andcardiovascular risk Interference at the level of gene expressionrdquoThe Journal of Nutrition Health and Aging vol 6 no 4 pp 230ndash236 2002

[5] L Arab ldquoBiomarkers of fat and fatty acid intakerdquo Journal ofNutrition vol 133 supplement 3 pp 925Sndash932S 2003

[6] P Benatti G Peluso R Nicolai and M Calvani ldquoPolyunsatu-rated fatty acids biochemical nutritional and epigenetic prop-ertiesrdquo Journal of the American College of Nutrition vol 23 no4 pp 281ndash302 2004

[7] W Karmally ldquoBalancing unsaturated fatty acids whatrsquos theevidence for cholesterol loweringrdquo Journal of the AmericanDietetic Association vol 105 no 7 pp 1068ndash1070 2005

[8] O Ozyilkan D Colak Z Akcali and B Basturk ldquoOlive fruit ofpeace against cancerrdquoAsianPacific Journal of Cancer Preventionvol 6 no 1 pp 77ndash82 2005

[9] A Lapointe C Couillard and S Lemieux ldquoEffects of dietaryfactors on oxidation of low-density lipoprotein particlesrdquo TheJournal of Nutritional Biochemistry vol 17 no 10 pp 645ndash6582006

[10] J S Perona R Cabello-Moruno and V Ruiz-Gutierrez ldquoTherole of virgin olive oil components in the modulation ofendothelial functionrdquo The Journal of Nutritional Biochemistryvol 17 no 7 pp 429ndash445 2006

[11] P D Biondo D N Brindley M B Sawyer and C J Field ldquoThepotential for treatmentwith dietary long-chain polyunsaturatedn-3 fatty acids during chemotherapyrdquoThe Journal of NutritionalBiochemistry vol 19 no 12 pp 787ndash796 2008

[12] B Koletzko E Lien C Agostoni and et al ldquoThe roles of long-chain polyunsaturated fatty acids in pregnancy lactation andinfancy review of current knowledge and consensus recom-mendationsrdquo Journal of Perinatal Medicine vol 36 no 1 pp5ndash14 2008

[13] C Ruggiero F Lattanzio F Lauretani B Gasperini C Andres-Lacueva and A Cherubini ldquoΩ-3 polyunsaturated fatty acidsand immune-mediated diseases Inflammatory bowel diseaseand rheumatoid arthritisrdquo Current Pharmaceutical Design vol15 no 36 pp 4135ndash4148 2009

[14] J T Brenna N Salem Jr A J Sinclair and S C Cunnaneldquo120572-Linolenic acid supplementation and conversion to n-3 long-chain polyunsaturated fatty acids in humansrdquo ProstaglandinsLeukotrienes and Essential Fatty Acids vol 80 no 2-3 pp 85ndash912009


[15] P A Corsetto G Montorfano S Zava I E Jovenitti A Cre-mona and A M Rizzo ldquoEffects of n-3 PUFAs on breast cancercells through their incorporation in plasma membranerdquo Lipidsin Health and Disease vol 10 article 73 2011

[16] IM Berquin I J Edwards S J Kridel andYQChen ldquoPolyun-saturated fatty acid metabolism in prostate cancerrdquo Cancerand Metastasis Reviews vol 30 no 3-4 pp 295ndash309 2011

[17] M Kaczmarski B Cudowska M Sawicka-Zukowska and ABobrus-Chociej ldquoSupplementation with long chain polyunsat-urated fatty acids in treatment of atopic dermatitis in childrenrdquoAdvances in Dermatology and Allergology vol 30 no 2 pp 103ndash107 2013

[18] I Brondz ldquoDevelopment of fatty acid analysis by high-per-formance liquid chromatography gas chromatography andrelated techniquesrdquo Analytica Chimica Acta vol 465 no 1-2pp 1ndash37 2002

[19] J-M Kornprobst and G Barnathan ldquoDemospongic acids revis-itedrdquoMarine Drugs vol 8 no 10 pp 2569ndash2577 2010

[20] C Bigogno I Khozin-Goldberg S Boussiba A Vonshak andZ Cohen ldquoLipid and fatty acid composition of the green oleagi-nous algaParietochloris incisa the richest plant source of arachi-donic acidrdquo Phytochemistry vol 60 no 5 pp 497ndash503 2002

[21] MAHossain ldquoFish as source of n-3 polyunsaturated fatty acids(PUFAs) which one is better-farmed or wildrdquoAdvance Journalof Food Science and Technology vol 3 no 6 pp 455ndash466 2011

[22] O P Ward and A Singh ldquoOmega-36 fatty acids alternativesources of productionrdquo Process Biochemistry vol 40 no 12 pp3627ndash3652 2005

[23] L A Copeman andCC Parrish ldquoLipids classes fatty acids andsterols in seafood fromGilbert Bay southern Labradorrdquo Journalof Agricultural and Food Chemistry vol 52 no 15 pp 4872ndash4881 2004

[24] A Gupta C J Barrow and M Puri ldquoOmega-3 biotechnologythraustochytrids as a novel source of omega-3 oilsrdquo Biotechnol-ogy Advances vol 30 no 6 pp 1733ndash1745 2012

[25] V Manikan M Sahaid Kalil M H Mohd Isa and A AbdulHamid ldquoImproved prediction for medium optimization usingfactorial screening for docosahexaenoic acid production bySchizochytrium SP SW1rdquo American Journal of Applied Sciencesvol 11 no 3 pp 462ndash474 2014

[26] D S Nichols ldquoProkaryotes and the input of polyunsaturatedfatty acids to the marine food webrdquo FEMSMicrobiology Lettersvol 219 no 1 pp 1ndash7 2003

[27] A Arjuna ldquoProduction of polyunsaturated fatty acids by fungia reviewrdquo International Journal of Pharma and Bio Sciences vol5 pp 931ndash954 2014

[28] S Das P S Lyla and S Ajmal Khan ldquoFatty acid profiles ofmarine benthic microorganisms isolated from the continentalslope of Bay of Bengal a possible implications in the benthicfood webrdquo Ocean Science Journal vol 42 no 4 pp 247ndash2542007

[29] J E Karr J E Alexander and R G Winningham ldquoOmega-3polyunsaturated fatty acids and cognition throughout the life-span a reviewrdquo Nutritional Neuroscience vol 14 no 5 pp 216ndash225 2011

[30] Y Abe M Honsho H Nakanishi R Taguchi and Y FujikildquoVery-long-chain polyunsaturated fatty acids accumulate inphosphatidylcholine of fibroblasts from patients with Zellwegersyndrome and acyl-CoA oxidase1 deficiencyrdquo Biochimica et Bio-physica Acta Molecular and Cell Biology of Lipids vol 1841 no4 pp 610ndash619 2014

[31] K Hama T Nagai C Nishizawa et al ldquoMolecular species ofphospholipidswith very long chain fatty acids in skin fibroblastsof Zellweger syndromerdquo Lipids vol 48 no 12 pp 1253ndash12672013

[32] M P Agbaga N A Mandal and R E Anderson ldquoRetinalvery long-chain PUFAs new insights from studies on ELOVL4proteinrdquo Journal of Lipid Research vol 51 no 7 pp 1624ndash16422010

[33] M I Aveldano ldquoA novel group of very long chain polyenoicfatty acids in dipolyunsaturated phosphatidylcholines from ver-tebrate retinardquo Journal of Biological Chemistry vol 262 no 3pp 1172ndash1179 1987

[34] L D Bennett R S BrushM Chan et al ldquoEffect of reduced reti-nal VLC-PUFA on rod and cone photoreceptorsrdquo InvestigativeOphthalmology amp Visual Science vol 55 no 5 pp 3150ndash31572014

[35] I A Butovich ldquoLipidomics of human meibomian gland secre-tions chemistry biophysics and physiological role of meibo-mian lipidsrdquo Progress in Lipid Research vol 50 no 3 pp 278ndash301 2011

[36] B Nikolova-Damyanova ldquoFatty acids silver ion TLCrdquo in Ency-clopedia of Chromatography J Cazes Ed pp 846ndash848 Taylorand Francis New York NY USA 3rd edition 2010

[37] J C Touchstone ldquoThin-layer chromatographic procedures forlipid separationrdquo Journal of Chromatography B BiomedicalApplications vol 671 no 1-2 pp 169ndash195 1995

[38] B Nikolova-Damyanova R Velikova and L Kuleva ldquoQuanti-tative TLC for determination of the triacylglycerol compositionof sesame seedsrdquo Journal of Liquid Chromatography amp RelatedTechnologies vol 25 no 10-11 pp 1623ndash1632 2002

[39] B Nikolova-Damyanova ldquoQuantitative thin-layer chromatog-raphy of triacylglycerols Principles and applicationrdquo Journal ofLiquid Chromatography and Related Technologies vol 22 no 10pp 1513ndash1537 1999

[40] T Nakamura M Fukuda and R Tanaka ldquoEstimation ofpolyunsaturated fatty acid content in lipids of aquatic organismsusing thin-layer chromatography on a plain silica gel platerdquoLipids vol 31 no 4 pp 427ndash432 1996

[41] B Fuchs R Suszlig K Teuber M Eibisch and J Schiller ldquoLipidanalysis by thin-layer chromatographymdasha review of the currentstaterdquo Journal of Chromatography A vol 1218 no 19 pp 2754ndash2774 2011

[42] J Sherma ldquoThin-layer chromatography in food and agriculturalanalysisrdquo Journal of Chromatography A vol 880 no 1-2 pp129ndash147 2000

[43] B Fuchs K Teuber A Nimptsch et al ldquoThin-layer chro-matography with direct mass spectrometric analyte detectionmdasha review of the progress achieved in the last five yearsrdquo inHigh-Performance Thin-Layer Chromatography (HPTLC) M MSrivastava Ed pp 335ndash363 Springer NewYork NYUSA 2011

[44] B Fuchs ANimptsch R Suss et al ldquoThe analysis of brain lipidsby a directly coupled MALDITLC approachrdquo Journal of AOACInternational vol 91 pp 1227ndash1236 2008

[45] S Lobasso P Lopalco R Angelini et al ldquoCoupled TLC andMALDI-TOFMS analyses of the lipid extract of the hyper-thermophilic archaeon pyrococcus furiosusrdquoArchaea vol 2012Article ID 957852 10 pages 2012

[46] B Fuchs ldquoAnalysis of phospolipids and glycolipids by thin-layerchromatography-matrix-assisted laser desorption and ioniza-tion mass spectrometryrdquo Journal of Chromatography A vol1259 pp 62ndash73 2012


[47] B Fuchs J Schiller R Suszlig et al ldquoAnalysis of stem cell lipids byoffline HPTLC-MALDI-TOFMSrdquo Analytical and BioanalyticalChemistry vol 392 no 5 pp 849ndash860 2008

[48] B Fuchs J Schiller R Suszlig M Schurenberg and D Suckau ldquoAdirect and simple method of coupling matrix-assisted laserdesorption and ionization time-of-flight mass spectrometry(MALDI-TOFMS) to thin-layer chromatography (TLC) for theanalysis of phospholipids from egg yolkrdquo Analytical and Bioan-alytical Chemistry vol 389 no 3 pp 827ndash834 2007

[49] G Dobson W W Christie and B Nikolova-DamyanovaldquoSilver ion chromatography of lipids and fatty acidsrdquo Journal ofChromatography B Biomedical Applications vol 671 no 1-2 pp197ndash222 1995

[50] B Nikolova-Damyanova ldquoSilver ion chromatography andlipidsrdquo in Advances in Lipid MethodologymdashOne WW ChristieEd pp 181ndash237 The Oily Press Ayr UK 1992

[51] B Nikolova-Damyanova and S Momchilova ldquoSilver ion thin-layer chromatography of fatty acids A surveyrdquo Journal of LiquidChromatography and Related Technologies vol 24 no 10 pp1447ndash1466 2001

[52] B Nikolova-Damyanova W W Christie and B HerslofldquoMechanistic aspects of fatty acid retention in silver ion chro-matographyrdquo Journal of Chromatography A vol 749 no 1-2 pp47ndash54 1996

[53] S Momchilova and B Nikolova-Damyanova ldquoStationaryphases for silver ion chromatography of lipids preparation andpropertiesrdquo Journal of Separation Science vol 26 no 3-4 pp261ndash270 2003

[54] D Nikolova D Antonova I Marekov and B Nikolova-Damyanova ldquoBis-methylene-interrupted octadecadienoic fattyacids in Bulgarian bovine butter fatsrdquo European Journal of LipidScience and Technology vol 108 no 3 pp 212ndash217 2006

[55] R Wilson and J R Sargent ldquoHigh-resolution separation ofpolyunsaturated fatty acids by argentation thin-layer chro-matographyrdquo Journal of Chromatography A vol 623 no 2 pp403ndash407 1992

[56] R Wilson and J R Sargent ldquoChain separation of monounsat-urated fatty acid methyl esters by argentation thin-layer chro-matographyrdquo Journal of Chromatography A vol 905 no 1-2pp 251ndash257 2001

[57] D S Lin W E Connor D P Wolf M Neuringer and D LHachey ldquoUnique lipids of primate spermatozoa desmosteroland docosahexaenoic acidrdquo The Journal of Lipid Research vol34 no 3 pp 491ndash499 1993

[58] E H Belarbi E Molina and Y Chisti ldquoA process for highyield and scaleable recovery of high purity eicosapentaenoicacid esters frommicroalgae and fish oilrdquo Enzyme andMicrobialTechnology vol 26 no 7 pp 516ndash529 2000

[59] J T Dillon J C Aponte Y-J Tsai and Y Huang ldquoThin layerchromatography in the separation of unsaturated organic com-pounds using silver-thiolate chromatographic materialrdquo Jour-nal of Chromatography A vol 1251 pp 240ndash243 2012

[60] J M Sowa and P V Subbaiah ldquoVariable recoveries of fatty acidsfollowing the separation of lipids on commercial silica gel TLCplates selective loss of unsaturated fatty acids on certain brandsof platesrdquo Journal of Chromatography B vol 813 no 1-2 pp 159ndash166 2004

[61] A M Shahin M K McGuire M A McGuire K L Ritzen-thaler and T D Shultz ldquoDetermination of c9 t11 -CLA inmajorhuman plasma lipid classes using a combination of methylatingmethodologiesrdquo Lipids vol 38 no 7 pp 793ndash800 2003

[62] J K G Kramer C Cruz-Hernandez Z Deng J Zhou GJahreis and M E R Dugan ldquoAnalysis of conjugated linoleicacid and trans 181 isomers in synthetic and animal productsrdquoThe American Journal of Clinical Nutrition vol 79 no 6 pp1137Sndash1145S 2004

[63] V Fournier P Juaneda F Destaillats et al ldquoAnalysis of eicos-apentaenoic and docosahexaenoic acid geometrical isomersformed during fish oil deodorizationrdquo Journal of Chromatogra-phy A vol 1129 no 1 pp 21ndash28 2006

[64] E Bailey-Hall E B Nelson and A S Ryan ldquoValidation of arapid measure of blood PUFA levels in humansrdquo Lipids vol 43no 2 pp 181ndash186 2008

[65] J Flieger H Szumiło and K Giełzak-Kocwin ldquoOptimizingmodification conditions of silica gel with metal saltsrdquo Journalof Liquid Chromatography and Related Technologies vol 22 no19 pp 2879ndash2894 1999

[66] D Allan and S Cockcroft ldquoA modified procedure for thin layerchromatography of phospholipidsrdquo Journal of Lipid Researchvol 23 no 9 pp 1373ndash1374 1982

[67] W-Q Wang and A Gustafson ldquoOne-dimensional thin-layerchromatographic separation of phospholipids and lysophos-pholipids from tissue lipid extractsrdquo Journal of Chromatography- Biomedical Applications vol 581 no 1 pp 139ndash142 1992

[68] J L Beneytout D Greuet and M T Rigaud ldquoSeparationof arachidonic acid metabolites by thin-layer chromatographyusing new silicone-bonded platesrdquo Journal of High ResolutionChromatography vol 7 no 9 pp 538ndash539 1984

[69] J Pikul and F A Kummerow ldquoThiobarbituric acid reactivesubstance formation as affected by distribution of polyenoicfatty acids in individual phospholipidsrdquo Journal of Agriculturaland Food Chemistry vol 39 no 3 pp 451ndash457 1991

[70] M JMartinez-Lorenzo IMarzo J Naval andA Pineiro ldquoSelf-staining of polyunsaturated fatty acids in argentation thin-layerchromatographyrdquo Analytical Biochemistry vol 220 no 1 pp210ndash212 1994

[71] B Fuchs and J Schiller ldquoMALDI-TOF MS analysis of lipidsfrom cells tissues and body fluidsrdquo in Lipids in Health andDisease P J Quinn and X Wang Eds vol 49 of SubcellularBiochemistry pp 541ndash565 Springer Berlin Germany 2008

[72] B Fuchs K Arnold and J Schiller ldquoMass spectrometry ofbiological moleculesrdquo in Encyclopedia of Analytical ChemistryR A Meyers Ed pp 1ndash39 JohnWiley amp Sons Chichester UK2008

[73] V J Sinanoglou I F Strati S M Bratakos C Proestos PZoumpoulakis and S Miniadis-Meimaroglou ldquoOn the com-bined application of IatroscanTLC-FID andGC-FID to identifytotal neutral and polar lipids and their fatty acids extractedfrom foodsrdquo ISRN Chromatography vol 2013 Article ID859024 8 pages 2013

[74] A Zabala M P Portillo M T Macarulla V M Rodrıguezand A Fernandez-Quintela ldquoEffects of cis-9trans-11 and trans-10cis-12 CLA isomers on liver and adipose tissue fatty acidprofile in hamstersrdquo Lipids vol 41 no 11 pp 993ndash1001 2006

[75] I Marekov S Panayotova and R Tarandjiiska ldquoFatty acidcomposition and seasonal variation of trans fatty acid content inBulgarian butter fats by silver ion TLC and GCrdquo Journal of Liq-uid Chromatography and Related Technologies vol 32 no 9 pp1183ndash1195 2009

[76] C Cruz-Hernandez Z Deng J Zhou et al ldquoMethods foranalysis of conjugated linoleic acids and trans-181 isomers in


dairy fats by using a combination of gas chromatography silver-ion thin-layer chromatographygas chromatography and silver-ion liquid chromatographyrdquo Journal of AOAC International vol87 no 2 pp 545ndash562 2004

[77] S D Kramer J A Hurley N J Abbott and D J BegleyldquoLipids in blood-brain barriermodels in vitro I thin-layer chro-matography and high-performance liquid chromatography forthe analysis of lipid classes and long-chain polyunsaturated fattyacidsrdquo In Vitro Cellular amp Developmental BiologymdashAnimal vol38 no 10 pp 557ndash565 2002

[78] A Laffargue A de Kochko and S Dussert ldquoDevelopment ofsolid-phase extraction and methylation procedures to analysefree fatty acids in lipid-rich seedsrdquo Plant Physiology and Bio-chemistry vol 45 no 3-4 pp 250ndash257 2007

[79] G Chinnasamy P J Davis and A K Bal ldquoSeasonal changesin oleosomic lipids and fatty acids of perennial root nodules ofbeach peardquo Journal of Plant Physiology vol 160 no 4 pp 355ndash365 2003

[80] A Kotani T Fuse and F Kusu ldquoDetermination of plasma freefatty acids by high-performance liquid chromatography withelectrochemical detectionrdquo Analytical Biochemistry vol 284no 1 pp 65ndash69 2000

[81] T Rezanka L Nedbalova and K Sigler ldquoOdd-numbered very-long-chain polyunsaturated fatty acids from the dinoflagel-late Amphidinium carterae identified by atmospheric pressurechemical ionization liquid chromatography-mass spectrome-tryrdquo Phytochemistry vol 69 no 16 pp 2849ndash2855 2008

[82] M PMansour ldquoReversed-phase high-performance liquid chro-matography purification of methyl esters of C16-C28 polyun-saturated fatty acids inmicroalgae including octacosaoctaenoicacid [288(n-3)]rdquo Journal of Chromatography A vol 1097 no 1-2 pp 54ndash58 2005

[83] S Momchilova and B Nikolova-Damyanova ldquoSilver ion highperformance liquid chromatography of polynuclear aromaticderivatives of positionally isomeric octadecenoic fatty acidsrdquoJournal of Liquid Chromatography and Related Technologies vol23 no 9 pp 1319ndash1330 2000

[84] J J Han Y Iwasaki and T Yamane ldquoUse of isopropanolas a modifier in a hexane-acetonitrile based mobile phasefor the silver ion HPLC Separation of positional isomers oftriacylglycerols containing long chain polyunsaturated fattyacidsrdquo Journal of High Resolution Chromatography vol 22 pp357ndash361 1999

[85] F Destaillats J-L Sebedio O Berdeaux P Juaneda and PAngers ldquoGas chromatography-mass spectrometry determina-tion of metabolites of conjugated cis-9trans-11cis-15 183 fattyacidrdquo Journal of Chromatography B Analytical Technologies inthe Biomedical and Life Sciences vol 820 no 1 pp 15ndash22 2005

[86] M P Mansour J K Volkman D G Holdsworth A E Jacksonand S I Blackburn ldquoVery-long-chain (C28) highly unsaturatedfatty acids in marine dinoflagellatesrdquo Phytochemistry vol 50no 4 pp 541ndash548 1999

[87] T Rezanka and V M Dembitsky ldquoVery long chain polyun-saturated fatty acids in crustacea of the order BathynellaceardquoBiochemical Systematics and Ecology vol 27 no 6 pp 551ndash5581999

[88] L Akoto R Pel H Irth U A T Brinkman and R J JVreuls ldquoAutomated GC-MS analysis of raw biological samplesapplication to fatty acid profiling of aquatic micro-organismsrdquoJournal of Analytical and Applied Pyrolysis vol 73 no 1 pp 69ndash75 2005

[89] C BockingWANockherM SchreinerH Renz and P I Pfef-ferle ldquoDevelopment and validation of a combined method forthe biomonitoring of omega-3-6 fatty acids and conjugatedlinoleic acids in different matrices from human and nutritionalsourcesrdquo Clinical Chemistry and Laboratory Medicine vol 48no 12 pp 1757ndash1763 2010

[90] C Tyburczy P Delmonte A R Fardin-Kia M M Mossoba JK G Kramer and J I Rader ldquoProfile of trans fatty acids (FAs)including trans polyunsaturated fas in representative fast foodsamplesrdquo Journal of Agricultural and Food Chemistry vol 60no 18 pp 4567ndash4577 2012

[91] M M M Hassanein and A G Abedel-Razek ldquoChromato-graphic quantitation of some bioactive minor components inoils of wheat germ and grape seeds produced as by-productsrdquoJournal of Oleo Science vol 58 no 5 pp 227ndash233 2009

[92] A Stołyhwo I Kołodziejska and Z E Sikorski ldquoLong chainpolyunsaturated fatty acids in smoked Atlantic mackerel andBaltic spratsrdquo Food Chemistry vol 94 no 4 pp 589ndash595 2006

[93] E S Lima and D S P Abdalla ldquoHigh-performance liquidchromatography of fatty acids in biological samplesrdquo AnalyticaChimica Acta vol 465 no 1-2 pp 81ndash91 2002

[94] M S Rao K Hidajat and C B Ching ldquoReversed-phase HPLCthe separation method for the characterization and purificationof long chain polyunsaturated fatty acidsmdasha reviewrdquo Journal ofChromatographic Science vol 33 no 1 pp 9ndash21 1995

[95] A Mehta A M Oeser and M G Carlson ldquoRapid quantitationof free fatty acids in human plasma by high-performance liquidchromatographyrdquo Journal of Chromatography B BiomedicalApplications vol 719 no 1-2 pp 9ndash23 1998

[96] T Rezanka and J Votruba ldquoChromatography of very long-chainfatty acids from animal and plant kingdomsrdquoAnalytica ChimicaActa vol 465 no 1-2 pp 273ndash297 2002

[97] W Kolanowski D Jaworska and J Weissbrodt ldquoImportance ofinstrumental and sensory analysis in the assessment of oxidativedeterioration of omega-3 long-chain polyunsaturated fatty acid-rich foodsrdquo Journal of the Science of Food and Agriculture vol87 no 2 pp 181ndash191 2007

[98] G A Haward and A J P Martin ldquoThe separation of the C12-C18 fatty acids by reversed-phase partition chromatographyrdquoJournal of Biochemistry vol 46 pp 532ndash538 1950

[99] J M Rosenfeld ldquoApplication of analytical derivatizations tothe quantitative and qualitative determination of fatty acidsrdquoAnalytica Chimica Acta vol 465 no 1-2 pp 93ndash100 2002

[100] M I Aveldano M VanRollins and L A Horrocks ldquoSeparationand quantitation of free fatty acids and fatty acid methyl estersby reverse phase high pressure liquid chromatographyrdquo Journalof Lipid Research vol 24 no 1 pp 83ndash93 1983

[101] F-H Wang X-J Xiong X-F Guo H Wang and H-S ZhangldquoDetermination of fatty acids in bio-samples based on the pre-column fluorescence derivatization with 1357-tetramethyl-8-butyrethylenediamine-difluoroboradiaza-s-indacene by highperformance liquid chromatographyrdquo Journal of Chromatogra-phy A vol 1291 pp 84ndash91 2013

[102] T Rezanka and K Sigler ldquoIdentification of very long chain fattyacids from sugar cane wax by atmospheric pressure chemicalionization liquid chromatography-mass spectroscopyrdquo Phyto-chemistry vol 67 no 9 pp 916ndash923 2006

[103] J Sajiki and J Yonekubo ldquoDetermination of free polyunsat-urated fatty acids and their oxidative metabolites by high-performance liquid chromatography (HPLC) and mass spec-trometry (MS)rdquo Analytica Chimica Acta vol 465 no 1-2 pp417ndash426 2002


[104] T Rezanka L Nedbalova and K Sigler ldquoIdentification ofvery-long-chain polyunsaturated fatty acids fromAmphidiniumcarterae by atmospheric pressure chemical ionization liquidchromatography-mass spectroscopyrdquo Phytochemistry vol 69no 12 pp 2391ndash2399 2008

[105] T Rezanka O Schreiberova T Krulikovska J Masak and KSigler ldquoRP-HPLCMS-APCI analysis of odd-chain TAGs fromRhodococcus erythropolis including some regioisomersrdquo Chem-istry and Physics of Lipids vol 163 no 4-5 pp 373ndash380 2010

[106] T Rezanka ldquoIdentification of very long chain fatty acidsby atmospheric pressure chemical ionization liquid chroma-tography-mass spectroscopy from green alga Chlorella kesslerirdquoJournal of Separation Science vol 25 no 18 pp 1332ndash1336 2002

[107] T Rezanka and K Sigler ldquoIdentification of very long chainunsaturated fatty acids from Ximenia oil by atmospheric pres-sure chemical ionization liquid chromatography-mass spec-troscopyrdquo Phytochemistry vol 68 no 6 pp 925ndash934 2007

[108] T Rezanka ldquoAnalysis of very long chain polyunsaturatedfatty acids using high-performance liquid chromatographymdashatmospheric pressure chemical ionization mass spectrometryrdquoBiochemical Systematics and Ecology vol 28 no 9 pp 847ndash8562000

[109] T Rezanka andK Sigler ldquoOdd-numbered very-long-chain fattyacids from the microbial animal and plant kingdomsrdquo Progressin Lipid Research vol 48 no 3-4 pp 206ndash238 2009

[110] M Fer S Goulitquer Y Dreano F Berthou L Corcos and YAmet ldquoDetermination of polyunsatured fatty acid monoepox-ides by high performance liquid chromatography-mass spec-trometryrdquo Journal of Chromatography A vol 1115 no 1-2 pp1ndash7 2006

[111] M Lısa M Holcapek T Rezanka and N Kabatova ldquoHigh-performance liquid chromatography-atmospheric pres-sure chemical ionization mass spectrometry and gas chroma-tography-flame ionization detection characterization of Δ5-polyenoic fatty acids in triacylglycerols from conifer seed oilsrdquoJournal of Chromatography A vol 1146 no 1 pp 67ndash77 2007

[112] J I Teng and N M M Gowda ldquoAnalysis of n-3 fatty acids infish oils by high-performance liquid chromatographyrdquo Chro-matographia vol 35 no 9-12 pp 627ndash630 1993

[113] R W Browne and D Armstrong ldquoHPLC analysis of lipid-derived polyunsaturated fatty acid peroxidation products inoxidatively modified human plasmardquo Clinical Chemistry vol46 no 6 pp 829ndash836 2000

[114] J-T Lin T A McKeon and A E Stafford ldquoGradient reversed-phase high-performance liquid chromatography of saturatedunsaturated and oxygenated free fatty acids and their methylestersrdquo Journal of Chromatography A vol 699 no 1-2 pp 85ndash91 1995

[115] M Yamaguchi R Matsunaga K Fukuda M Nakamura andY Ohkura ldquoHighly sensitive determination of free polyun-saturated long-chain fatty acids in human serum by high-performance liquid chromatography with fluorescence detec-tionrdquo Analytical Biochemistry vol 155 no 2 pp 256ndash261 1986

[116] K Nishimura T Suzuki S Momchilova K Miyashita EKatsura and Y Itabashi ldquoAnalysis of conjugated linoleic acidsas 9-anthrylmethyl esters by reversed-phase high-performanceliquid chromatography with fluorescence detectionrdquo Journal ofChromatographic Science vol 43 no 9 pp 494ndash499 2005

[117] N Noda K Umebayashi T Nakatani K Miyahara and KIshiyama ldquoIsolation and characterization of some hydroxy fattyand phosphoric acid esters of 10-hydroxy-2-decenoic acid from

the royal jelly of honeybees (Apis mellifera)rdquo Lipids vol 40 no8 pp 833ndash838 2005

[118] B Nikolova-Damyanova and S Momchilova ldquoSilver ion HPLCfor the analysis of positionally isomeric fatty acidsrdquo Journal ofLiquid Chromatography amp Related Technologies vol 25 no 13ndash15 pp 1947ndash1965 2002

[119] N M Aini M M Yusoff and H A Azhari ldquoChromatographicmethods to analyze geometrical and positional isomers of fattyacids A reviewrdquo in Proceedings of the National Conference onPostgraduate Research (NCON-PGR 09) M M Noor M MRahman and K Kadirgama Eds UMP Conference Hall pp165ndash175 Pahang Malaysia 2009

[120] S Momchilova B Nikolova-Damyanova and W W ChristieldquoSilver ion high-performance liquid chromatography of iso-meric cis- and trans-octadecenoic acids Effect of the ester moi-ety and mobile phase compositionrdquo Journal of ChromatographyA vol 793 no 2 pp 275ndash282 1998

[121] J L Guil-Guerrero P Campra-Madrid and R Navarro-JuarezldquoIsolation of some PUFA from edible oils by argentated silicagel chromatographyrdquoGrasas y Aceites vol 54 no 2 pp 116ndash1212003

[122] C Sun Y Y Zhao and J M Curtis ldquoThe direct determi-nation of double bond positions in lipid mixtures by liquidchromatographyin-line ozonolysismass spectrometryrdquo Ana-lytica Chimica Acta vol 762 pp 68ndash75 2013

[123] S Casal and B Oliveira ldquoFatty acids GC analysisrdquo in Encyclo-pedia of Chromatography J Cazes Ed pp 833ndash845 Taylor ampFrancis New York NY USA 3rd edition 2010

[124] T Seppanen-Laakso I Laakso and R Hiltunen ldquoAnalysis offatty acids by gas chromatography and its relevance to researchon health and nutritionrdquo Analytica Chimica Acta vol 465 no1-2 pp 39ndash62 2002

[125] R G Ackman ldquoThe gas chromatograph in practical analysesof common and uncommon fatty acids for the 21st centuryrdquoAnalytica Chimica Acta vol 465 no 1-2 pp 175ndash192 2002

[126] M W Muriuki G G Dumancas N Purdie and L ReillyldquoQuantification of total 120596-6 and 120596-3 fatty acids and 120596-6120596-3ratio in human serum using GC-MSrdquo LC-GC North Americavol 29 no 1 pp 60ndash65 2011

[127] H Kawashima and M Ohnishi ldquoIdentification of minor fattyacids and various nonmethylene-interrupted diene isomers inmantle muscle and viscera of the marine bivalve Meganguluszyonoensisrdquo Lipids vol 39 no 3 pp 265ndash271 2004

[128] N Tsevegsuren K Fujimoto W W Christie and Y EndoldquoOccurrence of a novel cisciscis-octadeca-3912-trienoic (ZZZ-octadeca-3912-trienoic) acid in Chrysanthemum (Tanace-tum) zawadskii Herb (Compositae) seed oilrdquo Lipids vol 38 no5 pp 573ndash578 2003

[129] A L Michaud M P Yurawecz P Delmonte B A Corl D EBauman and J T Brenna ldquoIdentification and characterizationof conjugated fatty acid methyl esters of mixed double bondgeometry by acetonitrile chemical ionization tandem massspectrometryrdquo Analytical Chemistry vol 75 no 18 pp 4925ndash4930 2003

[130] J Ecker M Scherer G Schmitz and G Liebisch ldquoA rapidGC-MS method for quantification of positional and geometricisomers of fatty acid methyl estersrdquo Journal of ChromatographyB Analytical Technologies in the Biomedical and Life Sciencesvol 897 pp 98ndash104 2012

[131] F Destaillats P-A Golay F Joffre et al ldquoComparison ofavailable analytical methods to measure trans-octadecenoic


acid isomeric profile and content by gas-liquid chromatographyin milk fatrdquo Journal of Chromatography A vol 1145 no 1-2 pp222ndash228 2007

[132] A Liu R Terry Y Lin K Nelson and P S BernsteinldquoComprehensive and sensitive quantification of long-chain andvery long-chain polyunsaturated fatty acids in small samples ofhuman and mouse retinardquo Journal of Chromatography A vol1307 pp 191ndash200 2013

[133] M Schreiner ldquoQuantification of long chain polyunsaturatedfatty acids by gas chromatography evaluation of factors affect-ing accuracyrdquo Journal of Chromatography A vol 1095 no 1-2pp 126ndash130 2005

[134] V BlahaD SolichovaD CernohorskyM Bratova PVyroubaland Z Zadak ldquoBioanalysis of PUFA metabolism and lipidperoxidation in coronary atherosclerosisrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 22 no 3 pp 563ndash5722000

[135] E Tvrzicka M Vecka B Stankova and A Zak ldquoAnalysis offatty acids in plasma lipoproteins by gas chromatography-flameionization detection quantitative aspectsrdquo Analytica ChimicaActa vol 465 no 1-2 pp 337ndash350 2002

[136] Z Xu K Harvey T Pavlina G Dutot G Zaloga and RSiddiqui ldquoAn improved method for determining medium- andlong-chain FAMEs using gas chromatographyrdquo Lipids vol 45no 2 pp 199ndash208 2010

[137] J K G Kramer C B Blackadar and J Zhou ldquoEvaluation oftwo GC columns (60-m SUPELCOWAX 10 and 100-m CP Sil88) for analysis of milkfat with emphasis on CLA 181 182 and183 isomers and short- and long-chain FArdquo Lipids vol 37 no8 pp 823ndash835 2002

[138] P Delmonte and J I Rader ldquoEvaluation of gas chromatographicmethods for the determination of trans fatrdquo Analytical andBioanalytical Chemistry vol 389 no 1 pp 77ndash85 2007

[139] A Timmins E J MacPherson and R G Ackman ldquoDirect useof methyl tricosanoate as an internal standard and overcominga potential error in the quantitation of the omega-3 long-chainpolyunsaturated fatty acids of marine oils as ethyl estersrdquo FoodChemistry vol 70 no 3 pp 425ndash426 2000

[140] P A Golay F Giuffrida F Dionisi and F Destaillats ldquoStream-lined methods for the resolution and quantification of fattyacids including trans fatty acid isomers in food products by gaschromatographyrdquo Journal of AOAC International vol 92 no 5pp 1301ndash1309 2009

[141] C Ragonese P Q Tranchida D Sciarrone and L Mon-dello ldquoConventional and fast gas chromatography analysis ofbiodiesel blends using an ionic liquid stationary phaserdquo Journalof Chromatography A vol 1216 no 51 pp 8992ndash8997 2009

[142] P Delmonte Q Hu A-R F Kia and J I Rader ldquoPreparationchromatographic separation and relative retention times ofcistrans heptadecaenoic (171) fatty acidsrdquo Journal of Chro-matography A vol 1214 no 1-2 pp 30ndash36 2008

[143] Q Gu F David F Lynen et al ldquoEvaluation of ionic liquidstationary phases for one dimensional gas chromatography-mass spectrometry and comprehensive two dimensional gaschromatographic analyses of fatty acids in marine biotardquo Jour-nal of Chromatography A vol 1218 no 20 pp 3056ndash3063 2011

[144] P Manzano E Arnaiz J C Diego et al ldquoComprehensive two-dimensional gas chromatography with capillary flow modula-tion to separate FAME isomersrdquo Journal of Chromatography Avol 1218 no 30 pp 4952ndash4959 2011

[145] W Meier-Augenstein ldquoStable isotope analysis of fatty acids bygas chromatography-isotope ratio mass spectrometryrdquo Analyt-ica Chimica Acta vol 465 no 1-2 pp 63ndash79 2002

[146] HAzzian J KGKramerA RKamalian et al ldquoQuantificationof trans fatty acids in food products by GC ATR-FTIR and FT-NIR methodsrdquo Lipid Technology vol 16 pp 229ndash231 2004

[147] L T Taylor ldquoSupercritical fluid chromatography for the 21stcenturyrdquo The Journal of Supercritical Fluids vol 47 no 3 pp566ndash573 2009

[148] F J Senorans and E Ibanez ldquoAnalysis of fatty acids in foodsby supercritical fluid chromatographyrdquoAnalytica Chimica Actavol 465 no 1-2 pp 131ndash144 2002

[149] J L Bernal M T Martin and L Toribio ldquoSupercritical fluidchromatography in food analysisrdquo Journal of ChromatographyA vol 1313 pp 24ndash36 2013

[150] G Pettinello A Bertucco P Pallado and A Stassi ldquoProductionof EPA enriched mixtures by supercritical fluid chromatogra-phy from the laboratory scale to the pilot plantrdquo Journal ofSupercritical Fluids vol 19 no 1 pp 51ndash60 2000

[151] T BambaN Shimonishi AMatsubara et al ldquoHigh throughputand exhaustive analysis of diverse lipids by using supercriticalfluid chromatography-mass spectrometry for metabolomicsrdquoJournal of Bioscience and Bioengineering vol 105 no 5 pp 460ndash469 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


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Applied ChemistryJournal of



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Analytical ChemistryInternational Journal of


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ElectrochemistryInternational Journal of



CatalystsJournal of


a novel excellent DHA and EPA 120596-3 fatty acids producers[24 25] An excellent review paper performed by Nicholsdemonstrates that many microorganisms including marinebacteria have been considered the major de novo producer ofn-3 PUFA [26] Available literature data suggest that over thepast several years extensive research has been made for theproduction of PUFA by fungi [27 28] As it was reported byArjuna among various microorganisms an optimal source ofomega-6 polyunsaturated fatty acids specifically 120574-linolenicacid (GLA) can be certain fungi [27]

It is well known that PUFA can affect many physio-logical processes including cardiovascular neurological andimmune functions as well as cancer Consumption of oilsrich in n-3 LC-PUFA during pregnancy reduces the risk forearly premature birth [12] Studies with nonhuman primatesand human newborns indicate that DHA is essential for thenormal functional development of the retina and brain par-ticularly in premature infants [13 29] A new paper preparedby Kaczmarski et al demonstrated the significant role oflinoleic acid and also 120572-linolenic acid in some symptoms ofatopic dermatitis [17]Therefore it could be noted that PUFAare important nutraceutical and pharmaceutical targets [22]

Until today there is a lack of knowledge about thefunction of LC-PUFA in mammalian tissues and cells inwhich they are found However it was stated that very long-chain polyunsaturated fatty acids are known to accumulate intwo types of major genetic peroxisomal diseases Zellwegersyndrome and X-linked adrenoleukodystrophy (X-ALD)characterized by neurodegenerative phenotypes [30 31] Thestudy of Hama and coworkers showed existence of many LC-PUFA types in specimens from Zellweger patients suggestingthe possibility of a new biomarker for peroxisomal diseases[31] Review paper performed by Agbaga and coworkers [32]summarized the current knowledge of VLC-PUFA to theirfunctional role in the retina which are highly enriched inPUFA with special emphasis on the elongases responsiblefor their synthesis by ELOVL4 protein Interest in LC-PUFAwas rekindled in 1987 after LC-PUFA were initially detectedin bovine retinas by Aveldano [33] Effect of retinal LC-PUFA on rod and cone photoreceptors was also describedby Bennett et al [34] Another paper prepared by Butovichconfirmed that among differentmammalian tissuesmeibumis an exceptionally complex mixture of various fatty acids[35]

Because most of LC-PUFA including omega-3 andomega-6 fatty acids cannot be synthesized in enough quantityby the humanorganism theymust be supplied in the dietTheproblemof supplementation of LC-PUFAand the explorationof a new (alternative to oil fish) source of LC-PUFA (egmarine microalgae) is widely described for few years in manypapers For this reason there is a need to find an effectiveand rapid method for identification and quantification ofnewly developed long-chain mono- and polyunsaturatedfatty acids in plants and seafood A set of various tools such aschromatographic methods is also needed to complete the fullstructural characteristic of PUFA in mammalian samples forexample in human plasma brain retina or meibum This isimportant for the purpose of clinical diagnosis

Hence this review presents various chromatographicsystems TLC HPLC GC and also SFC suitable for pre-separation and accurate quantification of long-chain mono-and polyunsaturated fatty acids from different samples withemphasis on selected literature which was published duringlast decade

2 Thin-Layer Chromatography (TLC) ofLong-Chain Mono- and PolyunsaturatedFatty Acids

Saturated and unsaturated long-chain fatty acids (MUFAand PUFA) are basic structural elements of lipids Thereforechromatographic determination of fatty acids compositionby TLC including LC-MUFA and LC-PUFA content ismandatory for lipids analysis in food agricultural andalso in biological samples [36ndash39] Moreover the mono-and polyunsaturated fatty acids can be chromatographicallydetermined by TLC in lipids from aquatic organisms (egmarine and freshwater fishes shell fishes and marine algae)[40]

It is well known that thin-layer chromatography is a clas-sical method of separation identification and quantificationof fatty acids [41] The literature survey from the last decadededicated to lipids analysis indicates that among differentanalytical methods thin-layer chromatography (TLC) and itsmodern version high performance thin-layer chromatogra-phy (HPTLC) are still very important tool in lipids and alsoin fatty acids analysis As it was reported by Fuchs et al [41]there aremany advantages whichmake TLC very competitivewithHPLC (high performance liquid chromatography) in thefatty acids field such as simplicity of use less expensive costsmall consumption of solvents in comparison with HPLCavailability of analysis of several samples in parallel andpossibility of easy visualization of unsaturated fatty acids afterTLC fractionation by use of suitable dyes [41] Shermarsquos reportregarding TLC analysis in food and agriculture samples con-firmed that quantitative HPTLC equipped with densitometercan produce results comparable to those obtained by theuse of gas chromatography (GC) or high performance liquidchromatography (HPLC) [42]

Modern topic in TLC analysis of mono- and polyunsat-urated fatty acids is the use of high performance thin-layerchromatography in combination with mass spectrometricdetection for example HPTLC-MALDI-TOFMS [43ndash48]Research studies indicate that MS detection is a powerfultool for the identification of TLC spots in more detail incomparison with traditional staining methods [41]

Many types of stationary phases classified as normal (NP)and reversed (RP) are used for TLC analysis of fatty acids(MUFA and PUFA)Themost popularNP layers are silica gelalumina cellulose starch polyamides and kieselguhr [42]Of all above-mentioned stationary phases the best is silicagel which can be additionallymodified by impregnationwithdifferent agents Reversed-phase TLC is usually performed onchemically bonded RP-18 RP-2 or RP-8 layers [42]

Numerous research papers by Nikolova-Damyanova andother authors showed that the main reason which explains






























Table1Survey

ofsomes

electedTL

Ccond

ition

susefulfor

separatio

nandidentifi

catio

nof

MUFA

andPU

FAfro

mvario

ussamples

Analyte

Stationary

phase

Mob

ileph

ase

Remarks

Reference

Extracto

flipidsc

omingfro

maquatic

organism

scon

tainingPU

FAPlainsilicag

elplate


acid

955

1(vvv)

Thep

latesw

eres

prayed

with

sulphu

ricacid

andnext

analyzed

densito

metric

allyat60

0nm

[40]

Hum

anplasmalipidsc

ontaining

linoleica

cid

Microscop

eplates

coated

with

silicag

elG


acid

9010

1(vvv)

Thes

potswerev

isualized

bysolutio

nof

2101584071015840-dichlorofl

uoresceinin

ethano

lorb

ysulfu

ricacid

respectiv

ely[61]

Deodo

rized

fishoilcon

taining

eicosapentaeno

icand

docosahexaenoica

cid

Argentatesilicag

elTo

luene-methano

l85

15(vv)

Ag-TL

Cmetho

d[63]

Seafoo

dcontaining

saturatedand

unsaturatedfatty

acids

Silicag

elcoated

Chromarod

Iatro

scan


rmicacid

99105(vvv)

Chloroform

-methano

l-water

54

1(vvv)

TLC-

FIDmetho

d(TLC

coup

ledwith

flameion

izationdetector)

[73]

Milk

samplec

ontaininglin

oleica

cid

Argentatesilicag


9010(vv)

Ag-TL

Cin

combinatio

nwith

UVdetectionat234nm

the

spotsw

erev

isualized

bysolutio

nof

2101584071015840-dichlorofl

uorescein

inmethano

l[76]

Cell

lipid

extractcon

tainingPU

FASilicag

elplates

Chloroform

-methano

l-acetic

acid-w

ater

6050

14(vvv)


6040

2(vvv)

TLC-

densito

metryafte

rdevelo

ping

thep

latesw

ere

impregnatedwith

cupricacetatea

ndph

osph

oricacid

[77]

Fatty

acidsisolatedfro

mlip

id-rich

seeds

TLCplates

(silica

gel60)


acid

7030

1(vvv)

TLCwith

UVdetectionat365n

methanolicsolutio

nof

2101584071015840-dichlorofl

uoresceinwas

used

tovisualizethe

spots

[78]

Fatty

acidsseparated

from

oleosomes

ofbeachpea

Silicag

elTL


acid

8515

2(vvv)

TLCmetho

diodine

vapo

rswereu

sedto

visualizethe

spots

[79]















3 Second method is













Table2Survey

ofsomes

electedHPL

Ccond

ition

susefulfor

separatio

nandidentifi

catio

nof

MUFA

andPU

FAfro

mvario

ussamples

HPL

Csyste

mMob

ileph

ase

Colum

nDetectio

nmod

eMatrix

Remarks

Reference

Ag-HPL

CIsocratic

solventsystem

hexane-acetonitrile

99703(vv)

Chromspher5

Lipidcolumn

(250times46mmV

arianNetherla

nds)

UV-VIS

detector

Deodo

rized

fishoilcon

taining

EPAD

HAG

LAL

AA

LA

SDand

otherP

UFA

Detectio

nlevela

[63]

HPL

C-EC

DEthano

l-acetonitrile

2080(vv)

ODS100R

P-18

column

LiCh

rospher

250m

mtimes4m

m5120583m

phase

particle

Electro

chem


calomelele

ctrodedetectio

npo

tentialminus

415m

V

Hum

anplasmacontaining

vario

usfatty

acidsincluding

arachido

nica

cidandlin

oleic

andoleica

cids

Detectio

nlevel

50ndash160

0pm

oles

[80]

HPL

C-MS-APC

I

Theg

radientsolvent

program

was

usedM

eCNEtCND

CM60

30

10(vvv)

MeC

NEtCND

CM30

40

30(vvv)

Hichrom

columnHIRPB

-250

AM

250m

mtimes21m

m5120583m

phase

particle

Picolin

ylestersweres

eparated

byuseo

fquadrup

lemassspectrometer

fittedwith

anatmosph

ericpressure

chem

icalionizatio

nsource

Dinofl

agellateAm

phidinium

carterae

containing

VLC

-PUFA

upno

nacosahexaenoica

cid

Detectio

nlevela

[81]

RP-H

PLC-

ELSD

Theisocraticelu

tionof

MeC

NH

2Owas

used975

25

(vv)fl

owrateof

mob

ileph

ase

15mlm

in

RPcolumn(A

lltim

aC18Alltech

250m

mtimes46m

m)5120583

mph

ase

particle

Methyleste

rsPU

FAwered

etected


ring

detector

(ELSD)

Marinea

lgae

Schizochytriu

mspcon

tainingDHAE

PAand

theird

erivatives

Detectio

nlevelmg

[82]

Ag-HPL

CDichlorom

ethane-acetonitrile

100

0025(vv)

Nucleosil100-5SA

(250

mmtimes46m

m)Hichrom

Re

adingUK

UV-VIS

detectorthe

working

wavele

ngth

was

250n

mforthe

anthrylm

ethyland

naph

thyl

deriv

atives

andalso

275n

mforthe

naph

thylmethyld

erivatives

ofexam

ined

octadeceno

icacid

References

tand

ards

ofpo

sitionalisomerso

foctadeceno

icacid

andtheir

deriv

atives

(este

rs)

Detectio

nlevel120583g

[83]

Ag-HPL

C

Gradientsolvent

syste

mconsistso

fhexane-isop

ropano

l-acetonitrile

350

100

275

(vvv)

350

100

10(vvv)

Chrompack

5Lipidcolumn

(250

mmtimes46m

mN

etherla

nds)

UV-VIS

detector

thew

orking

wavele

ngth

was

206n

mAPC

I-MS

Oilprod

uced

byam

arine

microbe

containing

PUFA

(DHAE

PA)

Detectio

nlevela

[84]

a Not

describ

edby

authorsMeC

N-acetonitrileE

tCN-propion

itrileand

DCM

-dichlorom

ethane































Table3Survey

ofsomes

electedGCcond

ition

susefulfor

separatio

nandidentifi

catio

nof

MUFA

andPU

FAfro

mvario

ussamples

Analyte

GCcolumn

Derivatization

Identifi

catio

nqu

antifi

catio

nRe

ference

Liverlipidsfrom

ratcon

taining

rumelenicacid

(cis-9tra

ns-11andcis-15

183)isomer

oflin

olenicacid

BPX-

70capillary

columnSG

EMelb

ourne(50

mtimes

032

mm025120583

m)heliu

mwas

used

ascarrierg

asun

derfl

ow24m

Lmin

44-dimethyloxazolid

ine

(DMOX)

andmethyl

esterd

erivatives

Selectiveq

uadrup

olem

assd

etectoru

nder

ionizatio

nvoltage

of70

eVat230∘C

[85]

Thea

lgae

extractcon

taining28

7(n-6)

andalso

288

(n-3)

Polarc

olum

nBP

X-70(50

mtimes032

mm025120583

m)

nonp

olarH


mm

017120583m)h

elium

was

used

ascarrierg

asDMOXderiv

atization

Massspectrometer

(Fiso

nsM

D-800)

underion

izationvoltage

of35

eVat200∘C

[86]

Extractsfro

mthreec

rustaceanspecies

comingfro

mLake

Baikalcontaining

n-3

andn-6PU

FA

Supelco

wax


mm025120583

m)the

temperature

program

was

from

100∘Cto

280∘C

The

carrierg

aswas

heliu

matflo

wrateof

70cm

s

Methyleste

rsand

oxazolines

ofPU

FAMassspectrometer

with

chem

ical

ionizatio

n(FinniganMAT

1020B)

[87]

Extractsfro

mgreenalgaea

ndcyanob

acteriu

mAfusedHP-5M

S(A


025

mm01120583m)heliu

mwas

used

asmob

ileph

ase

Methyleste

rsof

PUFA

Iontrap

massspectrometer

(ITD

-MS)

was

used

[88]

Clinicalform

ulations

containing

PUFA

Capillary

columnZe

bron

ZB-W

AXplus

(30mtimes025

mm025120583

m)

Methyleste

rsof

PUFA

GC-

FIDsyste

m[89]

Food

samples

containing

saturatedand

unsaturatedfatty

acids(MUFA

and

PUFA

)cis

andtra

ns

SP-2560CP

Scolumn

(30mtimes025

mm020120583

m)hydrogen

was

used

asa

carrierg

as

Methyleste

rsof

MUFA

andPU

FAGC-

FIDsyste

m[90]

Oilextractsfro

mgrapes

eedandwheat

germ

richin

linolenicandlin

oleica

cids

DB-23

(032

mmtimes30

m)temperature

programming

150ndash

230∘C

carrierg

aswas

heliu

mMethyleste

rsof

linolenic

andlin

oleica

cid

GC-

FIDsyste

m[91]

Smoked

mackerelm

eatcon

tainingEP

andDHA

Colum

nRtx2330

(105

mtimes025

mm02120583m)

temperature


Methyleste

rsof

EPand

DHA

GC-

FIDsyste

m[92]



6 Conclusions




Acknowledgments


References









































































































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






























Table1Survey

ofsomes

electedTL

Ccond

ition

susefulfor

separatio

nandidentifi

catio

nof

MUFA

andPU

FAfro

mvario

ussamples

Analyte

Stationary

phase

Mob

ileph

ase

Remarks

Reference

Extracto

flipidsc

omingfro

maquatic

organism

scon

tainingPU

FAPlainsilicag

elplate


acid

955

1(vvv)

Thep

latesw

eres

prayed

with

sulphu

ricacid

andnext

analyzed

densito

metric

allyat60

0nm

[40]

Hum

anplasmalipidsc

ontaining

linoleica

cid

Microscop

eplates

coated

with

silicag

elG


acid

9010

1(vvv)

Thes

potswerev

isualized

bysolutio

nof

2101584071015840-dichlorofl

uoresceinin

ethano

lorb

ysulfu

ricacid

respectiv

ely[61]

Deodo

rized

fishoilcon

taining

eicosapentaeno

icand

docosahexaenoica

cid

Argentatesilicag

elTo

luene-methano

l85

15(vv)

Ag-TL

Cmetho

d[63]

Seafoo

dcontaining

saturatedand

unsaturatedfatty

acids

Silicag

elcoated

Chromarod

Iatro

scan


rmicacid

99105(vvv)

Chloroform

-methano

l-water

54

1(vvv)

TLC-

FIDmetho

d(TLC

coup

ledwith

flameion

izationdetector)

[73]

Milk

samplec

ontaininglin

oleica

cid

Argentatesilicag


9010(vv)

Ag-TL

Cin

combinatio

nwith

UVdetectionat234nm

the

spotsw

erev

isualized

bysolutio

nof

2101584071015840-dichlorofl

uorescein

inmethano

l[76]

Cell

lipid

extractcon

tainingPU

FASilicag

elplates

Chloroform

-methano

l-acetic

acid-w

ater

6050

14(vvv)


6040

2(vvv)

TLC-

densito

metryafte

rdevelo

ping

thep

latesw

ere

impregnatedwith

cupricacetatea

ndph

osph

oricacid

[77]

Fatty

acidsisolatedfro

mlip

id-rich

seeds

TLCplates

(silica

gel60)


acid

7030

1(vvv)

TLCwith

UVdetectionat365n

methanolicsolutio

nof

2101584071015840-dichlorofl

uoresceinwas

used

tovisualizethe

spots

[78]

Fatty

acidsseparated

from

oleosomes

ofbeachpea

Silicag

elTL


acid

8515

2(vvv)

TLCmetho

diodine

vapo

rswereu

sedto

visualizethe

spots

[79]















3 Second method is













Table2Survey

ofsomes

electedHPL

Ccond

ition

susefulfor

separatio

nandidentifi

catio

nof

MUFA

andPU

FAfro

mvario

ussamples

HPL

Csyste

mMob

ileph

ase

Colum

nDetectio

nmod

eMatrix

Remarks

Reference

Ag-HPL

CIsocratic

solventsystem

hexane-acetonitrile

99703(vv)

Chromspher5

Lipidcolumn

(250times46mmV

arianNetherla

nds)

UV-VIS

detector

Deodo

rized

fishoilcon

taining

EPAD

HAG

LAL

AA

LA

SDand

otherP

UFA

Detectio

nlevela

[63]

HPL

C-EC

DEthano

l-acetonitrile

2080(vv)

ODS100R

P-18

column

LiCh

rospher

250m

mtimes4m

m5120583m

phase

particle

Electro

chem


calomelele

ctrodedetectio

npo

tentialminus

415m

V

Hum

anplasmacontaining

vario

usfatty

acidsincluding

arachido

nica

cidandlin

oleic

andoleica

cids

Detectio

nlevel

50ndash160

0pm

oles

[80]

HPL

C-MS-APC

I

Theg

radientsolvent

program

was

usedM

eCNEtCND

CM60

30

10(vvv)

MeC

NEtCND

CM30

40

30(vvv)

Hichrom

columnHIRPB

-250

AM

250m

mtimes21m

m5120583m

phase

particle

Picolin

ylestersweres

eparated

byuseo

fquadrup

lemassspectrometer

fittedwith

anatmosph

ericpressure

chem

icalionizatio

nsource

Dinofl

agellateAm

phidinium

carterae

containing

VLC

-PUFA

upno

nacosahexaenoica

cid

Detectio

nlevela

[81]

RP-H

PLC-

ELSD

Theisocraticelu

tionof

MeC

NH

2Owas

used975

25

(vv)fl

owrateof

mob

ileph

ase

15mlm

in

RPcolumn(A

lltim

aC18Alltech

250m

mtimes46m

m)5120583

mph

ase

particle

Methyleste

rsPU

FAwered

etected


ring

detector

(ELSD)

Marinea

lgae

Schizochytriu

mspcon

tainingDHAE

PAand

theird

erivatives

Detectio

nlevelmg

[82]

Ag-HPL

CDichlorom

ethane-acetonitrile

100

0025(vv)

Nucleosil100-5SA

(250

mmtimes46m

m)Hichrom

Re

adingUK

UV-VIS

detectorthe

working

wavele

ngth

was

250n

mforthe

anthrylm

ethyland

naph

thyl

deriv

atives

andalso

275n

mforthe

naph

thylmethyld

erivatives

ofexam

ined

octadeceno

icacid

References

tand

ards

ofpo

sitionalisomerso

foctadeceno

icacid

andtheir

deriv

atives

(este

rs)

Detectio

nlevel120583g

[83]

Ag-HPL

C

Gradientsolvent

syste

mconsistso

fhexane-isop

ropano

l-acetonitrile

350

100

275

(vvv)

350

100

10(vvv)

Chrompack

5Lipidcolumn

(250

mmtimes46m

mN

etherla

nds)

UV-VIS

detector

thew

orking

wavele

ngth

was

206n

mAPC

I-MS

Oilprod

uced

byam

arine

microbe

containing

PUFA

(DHAE

PA)

Detectio

nlevela

[84]

a Not

describ

edby

authorsMeC

N-acetonitrileE

tCN-propion

itrileand

DCM

-dichlorom

ethane































Table3Survey

ofsomes

electedGCcond

ition

susefulfor

separatio

nandidentifi

catio

nof

MUFA

andPU

FAfro

mvario

ussamples

Analyte

GCcolumn

Derivatization

Identifi

catio

nqu

antifi

catio

nRe

ference

Liverlipidsfrom

ratcon

taining

rumelenicacid

(cis-9tra

ns-11andcis-15

183)isomer

oflin

olenicacid

BPX-

70capillary

columnSG

EMelb

ourne(50

mtimes

032

mm025120583

m)heliu

mwas

used

ascarrierg

asun

derfl

ow24m

Lmin

44-dimethyloxazolid

ine

(DMOX)

andmethyl

esterd

erivatives

Selectiveq

uadrup

olem

assd

etectoru

nder

ionizatio

nvoltage

of70

eVat230∘C

[85]

Thea

lgae

extractcon

taining28

7(n-6)

andalso

288

(n-3)

Polarc

olum

nBP

X-70(50

mtimes032

mm025120583

m)

nonp

olarH


mm

017120583m)h

elium

was

used

ascarrierg

asDMOXderiv

atization

Massspectrometer

(Fiso

nsM

D-800)

underion

izationvoltage

of35

eVat200∘C

[86]

Extractsfro

mthreec

rustaceanspecies

comingfro

mLake

Baikalcontaining

n-3

andn-6PU

FA

Supelco

wax


mm025120583

m)the

temperature

program

was

from

100∘Cto

280∘C

The

carrierg

aswas

heliu

matflo

wrateof

70cm

s

Methyleste

rsand

oxazolines

ofPU

FAMassspectrometer

with

chem

ical

ionizatio

n(FinniganMAT

1020B)

[87]

Extractsfro

mgreenalgaea

ndcyanob

acteriu

mAfusedHP-5M

S(A


025

mm01120583m)heliu

mwas

used

asmob

ileph

ase

Methyleste

rsof

PUFA

Iontrap

massspectrometer

(ITD

-MS)

was

used

[88]

Clinicalform

ulations

containing

PUFA

Capillary

columnZe

bron

ZB-W

AXplus

(30mtimes025

mm025120583

m)

Methyleste

rsof

PUFA

GC-

FIDsyste

m[89]

Food

samples

containing

saturatedand

unsaturatedfatty

acids(MUFA

and

PUFA

)cis

andtra

ns

SP-2560CP

Scolumn

(30mtimes025

mm020120583

m)hydrogen

was

used

asa

carrierg

as

Methyleste

rsof

MUFA

andPU

FAGC-

FIDsyste

m[90]

Oilextractsfro

mgrapes

eedandwheat

germ

richin

linolenicandlin

oleica

cids

DB-23

(032

mmtimes30

m)temperature

programming

150ndash

230∘C

carrierg

aswas

heliu

mMethyleste

rsof

linolenic

andlin

oleica

cid

GC-

FIDsyste

m[91]

Smoked

mackerelm

eatcon

tainingEP

andDHA

Colum

nRtx2330

(105

mtimes025

mm02120583m)

temperature


Methyleste

rsof

EPand

DHA

GC-

FIDsyste

m[92]



6 Conclusions




Acknowledgments


References









































































































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of















3 Second method is













Table2Survey

ofsomes

electedHPL

Ccond

ition

susefulfor

separatio

nandidentifi

catio

nof

MUFA

andPU

FAfro

mvario

ussamples

HPL

Csyste

mMob

ileph

ase

Colum

nDetectio

nmod

eMatrix

Remarks

Reference

Ag-HPL

CIsocratic

solventsystem

hexane-acetonitrile

99703(vv)

Chromspher5

Lipidcolumn

(250times46mmV

arianNetherla

nds)

UV-VIS

detector

Deodo

rized

fishoilcon

taining

EPAD

HAG

LAL

AA

LA

SDand

otherP

UFA

Detectio

nlevela

[63]

HPL

C-EC

DEthano

l-acetonitrile

2080(vv)

ODS100R

P-18

column

LiCh

rospher

250m

mtimes4m

m5120583m

phase

particle

Electro

chem


calomelele

ctrodedetectio

npo

tentialminus

415m

V

Hum

anplasmacontaining

vario

usfatty

acidsincluding

arachido

nica

cidandlin

oleic

andoleica

cids

Detectio

nlevel

50ndash160

0pm

oles

[80]

HPL

C-MS-APC

I

Theg

radientsolvent

program

was

usedM

eCNEtCND

CM60

30

10(vvv)

MeC

NEtCND

CM30

40

30(vvv)

Hichrom

columnHIRPB

-250

AM

250m

mtimes21m

m5120583m

phase

particle

Picolin

ylestersweres

eparated

byuseo

fquadrup

lemassspectrometer

fittedwith

anatmosph

ericpressure

chem

icalionizatio

nsource

Dinofl

agellateAm

phidinium

carterae

containing

VLC

-PUFA

upno

nacosahexaenoica

cid

Detectio

nlevela

[81]

RP-H

PLC-

ELSD

Theisocraticelu

tionof

MeC

NH

2Owas

used975

25

(vv)fl

owrateof

mob

ileph

ase

15mlm

in

RPcolumn(A

lltim

aC18Alltech

250m

mtimes46m

m)5120583

mph

ase

particle

Methyleste

rsPU

FAwered

etected


ring

detector

(ELSD)

Marinea

lgae

Schizochytriu

mspcon

tainingDHAE

PAand

theird

erivatives

Detectio

nlevelmg

[82]

Ag-HPL

CDichlorom

ethane-acetonitrile

100

0025(vv)

Nucleosil100-5SA

(250

mmtimes46m

m)Hichrom

Re

adingUK

UV-VIS

detectorthe

working

wavele

ngth

was

250n

mforthe

anthrylm

ethyland

naph

thyl

deriv

atives

andalso

275n

mforthe

naph

thylmethyld

erivatives

ofexam

ined

octadeceno

icacid

References

tand

ards

ofpo

sitionalisomerso

foctadeceno

icacid

andtheir

deriv

atives

(este

rs)

Detectio

nlevel120583g

[83]

Ag-HPL

C

Gradientsolvent

syste

mconsistso

fhexane-isop

ropano

l-acetonitrile

350

100

275

(vvv)

350

100

10(vvv)

Chrompack

5Lipidcolumn

(250

mmtimes46m

mN

etherla

nds)

UV-VIS

detector

thew

orking

wavele

ngth

was

206n

mAPC

I-MS

Oilprod

uced

byam

arine

microbe

containing

PUFA

(DHAE

PA)

Detectio

nlevela

[84]

a Not

describ

edby

authorsMeC

N-acetonitrileE

tCN-propion

itrileand

DCM

-dichlorom

ethane































Table3Survey

ofsomes

electedGCcond

ition

susefulfor

separatio

nandidentifi

catio

nof

MUFA

andPU

FAfro

mvario

ussamples

Analyte

GCcolumn

Derivatization

Identifi

catio

nqu

antifi

catio

nRe

ference

Liverlipidsfrom

ratcon

taining

rumelenicacid

(cis-9tra

ns-11andcis-15

183)isomer

oflin

olenicacid

BPX-

70capillary

columnSG

EMelb

ourne(50

mtimes

032

mm025120583

m)heliu

mwas

used

ascarrierg

asun

derfl

ow24m

Lmin

44-dimethyloxazolid

ine

(DMOX)

andmethyl

esterd

erivatives

Selectiveq

uadrup

olem

assd

etectoru

nder

ionizatio

nvoltage

of70

eVat230∘C

[85]

Thea

lgae

extractcon

taining28

7(n-6)

andalso

288

(n-3)

Polarc

olum

nBP

X-70(50

mtimes032

mm025120583

m)

nonp

olarH


mm

017120583m)h

elium

was

used

ascarrierg

asDMOXderiv

atization

Massspectrometer

(Fiso

nsM

D-800)

underion

izationvoltage

of35

eVat200∘C

[86]

Extractsfro

mthreec

rustaceanspecies

comingfro

mLake

Baikalcontaining

n-3

andn-6PU

FA

Supelco

wax


mm025120583

m)the

temperature

program

was

from

100∘Cto

280∘C

The

carrierg

aswas

heliu

matflo

wrateof

70cm

s

Methyleste

rsand

oxazolines

ofPU

FAMassspectrometer

with

chem

ical

ionizatio

n(FinniganMAT

1020B)

[87]

Extractsfro

mgreenalgaea

ndcyanob

acteriu

mAfusedHP-5M

S(A


025

mm01120583m)heliu

mwas

used

asmob

ileph

ase

Methyleste

rsof

PUFA

Iontrap

massspectrometer

(ITD

-MS)

was

used

[88]

Clinicalform

ulations

containing

PUFA

Capillary

columnZe

bron

ZB-W

AXplus

(30mtimes025

mm025120583

m)

Methyleste

rsof

PUFA

GC-

FIDsyste

m[89]

Food

samples

containing

saturatedand

unsaturatedfatty

acids(MUFA

and

PUFA

)cis

andtra

ns

SP-2560CP

Scolumn

(30mtimes025

mm020120583

m)hydrogen

was

used

asa

carrierg

as

Methyleste

rsof

MUFA

andPU

FAGC-

FIDsyste

m[90]

Oilextractsfro

mgrapes

eedandwheat

germ

richin

linolenicandlin

oleica

cids

DB-23

(032

mmtimes30

m)temperature

programming

150ndash

230∘C

carrierg

aswas

heliu

mMethyleste

rsof

linolenic

andlin

oleica

cid

GC-

FIDsyste

m[91]

Smoked

mackerelm

eatcon

tainingEP

andDHA

Colum

nRtx2330

(105

mtimes025

mm02120583m)

temperature


Methyleste

rsof

EPand

DHA

GC-

FIDsyste

m[92]



6 Conclusions




Acknowledgments


References









































































































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of













Table2Survey

ofsomes

electedHPL

Ccond

ition

susefulfor

separatio

nandidentifi

catio

nof

MUFA

andPU

FAfro

mvario

ussamples

HPL

Csyste

mMob

ileph

ase

Colum

nDetectio

nmod

eMatrix

Remarks

Reference

Ag-HPL

CIsocratic

solventsystem

hexane-acetonitrile

99703(vv)

Chromspher5

Lipidcolumn

(250times46mmV

arianNetherla

nds)

UV-VIS

detector

Deodo

rized

fishoilcon

taining

EPAD

HAG

LAL

AA

LA

SDand

otherP

UFA

Detectio

nlevela

[63]

HPL

C-EC

DEthano

l-acetonitrile

2080(vv)

ODS100R

P-18

column

LiCh

rospher

250m

mtimes4m

m5120583m

phase

particle

Electro

chem


calomelele

ctrodedetectio

npo

tentialminus

415m

V

Hum

anplasmacontaining

vario

usfatty

acidsincluding

arachido

nica

cidandlin

oleic

andoleica

cids

Detectio

nlevel

50ndash160

0pm

oles

[80]

HPL

C-MS-APC

I

Theg

radientsolvent

program

was

usedM

eCNEtCND

CM60

30

10(vvv)

MeC

NEtCND

CM30

40

30(vvv)

Hichrom

columnHIRPB

-250

AM

250m

mtimes21m

m5120583m

phase

particle

Picolin

ylestersweres

eparated

byuseo

fquadrup

lemassspectrometer

fittedwith

anatmosph

ericpressure

chem

icalionizatio

nsource

Dinofl

agellateAm

phidinium

carterae

containing

VLC

-PUFA

upno

nacosahexaenoica

cid

Detectio

nlevela

[81]

RP-H

PLC-

ELSD

Theisocraticelu

tionof

MeC

NH

2Owas

used975

25

(vv)fl

owrateof

mob

ileph

ase

15mlm

in

RPcolumn(A

lltim

aC18Alltech

250m

mtimes46m

m)5120583

mph

ase

particle

Methyleste

rsPU

FAwered

etected


ring

detector

(ELSD)

Marinea

lgae

Schizochytriu

mspcon

tainingDHAE

PAand

theird

erivatives

Detectio

nlevelmg

[82]

Ag-HPL

CDichlorom

ethane-acetonitrile

100

0025(vv)

Nucleosil100-5SA

(250

mmtimes46m

m)Hichrom

Re

adingUK

UV-VIS

detectorthe

working

wavele

ngth

was

250n

mforthe

anthrylm

ethyland

naph

thyl

deriv

atives

andalso

275n

mforthe

naph

thylmethyld

erivatives

ofexam

ined

octadeceno

icacid

References

tand

ards

ofpo

sitionalisomerso

foctadeceno

icacid

andtheir

deriv

atives

(este

rs)

Detectio

nlevel120583g

[83]

Ag-HPL

C

Gradientsolvent

syste

mconsistso

fhexane-isop

ropano

l-acetonitrile

350

100

275

(vvv)

350

100

10(vvv)

Chrompack

5Lipidcolumn

(250

mmtimes46m

mN

etherla

nds)

UV-VIS

detector

thew

orking

wavele

ngth

was

206n

mAPC

I-MS

Oilprod

uced

byam

arine

microbe

containing

PUFA

(DHAE

PA)

Detectio

nlevela

[84]

a Not

describ

edby

authorsMeC

N-acetonitrileE

tCN-propion

itrileand

DCM

-dichlorom

ethane































Table3Survey

ofsomes

electedGCcond

ition

susefulfor

separatio

nandidentifi

catio

nof

MUFA

andPU

FAfro

mvario

ussamples

Analyte

GCcolumn

Derivatization

Identifi

catio

nqu

antifi

catio

nRe

ference

Liverlipidsfrom

ratcon

taining

rumelenicacid

(cis-9tra

ns-11andcis-15

183)isomer

oflin

olenicacid

BPX-

70capillary

columnSG

EMelb

ourne(50

mtimes

032

mm025120583

m)heliu

mwas

used

ascarrierg

asun

derfl

ow24m

Lmin

44-dimethyloxazolid

ine

(DMOX)

andmethyl

esterd

erivatives

Selectiveq

uadrup

olem

assd

etectoru

nder

ionizatio

nvoltage

of70

eVat230∘C

[85]

Thea

lgae

extractcon

taining28

7(n-6)

andalso

288

(n-3)

Polarc

olum

nBP

X-70(50

mtimes032

mm025120583

m)

nonp

olarH


mm

017120583m)h

elium

was

used

ascarrierg

asDMOXderiv

atization

Massspectrometer

(Fiso

nsM

D-800)

underion

izationvoltage

of35

eVat200∘C

[86]

Extractsfro

mthreec

rustaceanspecies

comingfro

mLake

Baikalcontaining

n-3

andn-6PU

FA

Supelco

wax


mm025120583

m)the

temperature

program

was

from

100∘Cto

280∘C

The

carrierg

aswas

heliu

matflo

wrateof

70cm

s

Methyleste

rsand

oxazolines

ofPU

FAMassspectrometer

with

chem

ical

ionizatio

n(FinniganMAT

1020B)

[87]

Extractsfro

mgreenalgaea

ndcyanob

acteriu

mAfusedHP-5M

S(A


025

mm01120583m)heliu

mwas

used

asmob

ileph

ase

Methyleste

rsof

PUFA

Iontrap

massspectrometer

(ITD

-MS)

was

used

[88]

Clinicalform

ulations

containing

PUFA

Capillary

columnZe

bron

ZB-W

AXplus

(30mtimes025

mm025120583

m)

Methyleste

rsof

PUFA

GC-

FIDsyste

m[89]

Food

samples

containing

saturatedand

unsaturatedfatty

acids(MUFA

and

PUFA

)cis

andtra

ns

SP-2560CP

Scolumn

(30mtimes025

mm020120583

m)hydrogen

was

used

asa

carrierg

as

Methyleste

rsof

MUFA

andPU

FAGC-

FIDsyste

m[90]

Oilextractsfro

mgrapes

eedandwheat

germ

richin

linolenicandlin

oleica

cids

DB-23

(032

mmtimes30

m)temperature

programming

150ndash

230∘C

carrierg

aswas

heliu

mMethyleste

rsof

linolenic

andlin

oleica

cid

GC-

FIDsyste

m[91]

Smoked

mackerelm

eatcon

tainingEP

andDHA

Colum

nRtx2330

(105

mtimes025

mm02120583m)

temperature


Methyleste

rsof

EPand

DHA

GC-

FIDsyste

m[92]



6 Conclusions




Acknowledgments


References









































































































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of































Table3Survey

ofsomes

electedGCcond

ition

susefulfor

separatio

nandidentifi

catio

nof

MUFA

andPU

FAfro

mvario

ussamples

Analyte

GCcolumn

Derivatization

Identifi

catio

nqu

antifi

catio

nRe

ference

Liverlipidsfrom

ratcon

taining

rumelenicacid

(cis-9tra

ns-11andcis-15

183)isomer

oflin

olenicacid

BPX-

70capillary

columnSG

EMelb

ourne(50

mtimes

032

mm025120583

m)heliu

mwas

used

ascarrierg

asun

derfl

ow24m

Lmin

44-dimethyloxazolid

ine

(DMOX)

andmethyl

esterd

erivatives

Selectiveq

uadrup

olem

assd

etectoru

nder

ionizatio

nvoltage

of70

eVat230∘C

[85]

Thea

lgae

extractcon

taining28

7(n-6)

andalso

288

(n-3)

Polarc

olum

nBP

X-70(50

mtimes032

mm025120583

m)

nonp

olarH


mm

017120583m)h

elium

was

used

ascarrierg

asDMOXderiv

atization

Massspectrometer

(Fiso

nsM

D-800)

underion

izationvoltage

of35

eVat200∘C

[86]

Extractsfro

mthreec

rustaceanspecies

comingfro

mLake

Baikalcontaining

n-3

andn-6PU

FA

Supelco

wax


mm025120583

m)the

temperature

program

was

from

100∘Cto

280∘C

The

carrierg

aswas

heliu

matflo

wrateof

70cm

s

Methyleste

rsand

oxazolines

ofPU

FAMassspectrometer

with

chem

ical

ionizatio

n(FinniganMAT

1020B)

[87]

Extractsfro

mgreenalgaea

ndcyanob

acteriu

mAfusedHP-5M

S(A


025

mm01120583m)heliu

mwas

used

asmob

ileph

ase

Methyleste

rsof

PUFA

Iontrap

massspectrometer

(ITD

-MS)

was

used

[88]

Clinicalform

ulations

containing

PUFA

Capillary

columnZe

bron

ZB-W

AXplus

(30mtimes025

mm025120583

m)

Methyleste

rsof

PUFA

GC-

FIDsyste

m[89]

Food

samples

containing

saturatedand

unsaturatedfatty

acids(MUFA

and

PUFA

)cis

andtra

ns

SP-2560CP

Scolumn

(30mtimes025

mm020120583

m)hydrogen

was

used

asa

carrierg

as

Methyleste

rsof

MUFA

andPU

FAGC-

FIDsyste

m[90]

Oilextractsfro

mgrapes

eedandwheat

germ

richin

linolenicandlin

oleica

cids

DB-23

(032

mmtimes30

m)temperature

programming

150ndash

230∘C

carrierg

aswas

heliu

mMethyleste

rsof

linolenic

andlin

oleica

cid

GC-

FIDsyste

m[91]

Smoked

mackerelm

eatcon

tainingEP

andDHA

Colum

nRtx2330

(105

mtimes025

mm02120583m)

temperature


Methyleste

rsof

EPand

DHA

GC-

FIDsyste

m[92]



6 Conclusions




Acknowledgments


References









































































































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



6 Conclusions




Acknowledgments


References









































































































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



























































































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

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