1094

INSTRUMENTATION

SPE methodologies for the separation of lipids

Solid phase extraction (SPE) is asample preparation techniquethat frequently is used for isola-

lion and fractionation of lipids. It israpidly replacing traditional samplepreparation techniques. e.g .. liquid-liquid extraction, liquid-solid columnchromatography. and thin-layer chro-matography (TLC). due to the ease ofuse. speed. cost effectiveness, anddecreased requirements for solventusage.

This ankle will describe briefly theprinciples and general applications ofSPE for lipid analysis. In addition, thereader is referred 10 two recentreviews of the use and applications ofSPE for lipid separation (1.2) and toresources provided by $PE manufac-turers (e.g., 3,4). The $PE separationof oxygenated lipid metabolites (i.e.,prostaglandins and leukotricnes), lipidperoxidarion products, and steroidprofiling, although important in acomplete lipid analysis, is beyond thescope of this article.

Principles, applications of SPESmall, commercial columnsprepncked with a variety of solid sta-tionary phases were introduced in thelate 1970s. The earliest applicationsof SPE for lipid analysis used silica,bonded aminopropyl. and CI8reverse-phase columns for the isola-tion and separation of various lipidclasses from foods and biological tis-sues (5-7). These phases continue tobe widely used for lipid analyses andare discussed in greater detail in thefollowing sections. Alumina, macrop-orous carbon and ion exchange phasesalso have found recent application forthe isolation and fractionation oflipids (8-12).

A number of solid phases are com-mercially available (Figure I), andnew SPE cartridges are continuallybeing introduced-an indication of thewidespread interest and utilization ofSPE for sample preparation. Many ofthe newer cartridges offer mixed modeseparation mechanisms (13,14) andcontain proprietary stationary phases(15.16) which are designed for specif-ic applications. Among the newer car-

This article was prepared bySusan E. Ebeler and Jolm O.Eheter: of the Department ofViticulture and Enology,University of California, Davis.Davis, CA 95616.....- J.D. EbeIer

the blend to a syringe barrel column,and eluting with selected solvents orsolvent mixtures. This technique wasused to isolate and fractionate lipidsfrom Escherichia coli and Mycobac-terium paratuberculosis without theneed for separate lysis and fractiona-tion steps (18). Mycobacteria typicallyhave thick cell walls which are hard todisrupt without severe treatments suchas antibiotics. detergents, or sonica-tion. Using MSPD. cells appeared tobe completely lysed and the cellularcomponents were well dispersed overthe C 18 panicles providing a homoge-neous blend for subsequent lipid frac-tionation (18). The MSPD is a single-step procedure that is chemically andphysically mild and offers manypotential applications for the analysisof lipids in a variety of matrices.

Silica sorbents. Many SPE lipidanalyses are performed following apreliminary extraction of the sampleinto chloroform or other similar non-polar solvents, making silica a logicalsorbern choice for further lipid separa-tion and purification steps. The polarsnanot groups adsorb polar com-pounds, and analyte retention and elu-tion are related directly to solventpolarity. Ion-exchange effects. due tothe mildly acidic nature of the silicaparticles, also influence separations onsilica-based sorbenrs. Silica adsorbsvarying amounts of water, which mayaffect separations. The columns mayrequire either drying prior to analysisor the use of solvents with controlled-moisture contents (19).

In an early application. silica SPE

[continued on pag~ 1096,

rrtdges. restricted access media orshielded hydrophobic phases offerpotential advantages for the analysisof biological samples because thesample can be injected directly ontothe cartridge without fouling the sta-tionary phase (16). Proteins and otherhigh molecular weight compounds areunretained while smaller moleculesare retained by normal HPLC sorptionmechanisms. Several manufacturersare preparing SPE-like cartridges withthese shielded hydrophobic phases.These cartridges hold great potentialfor lipid separations; however few lit-erature reports of the application ofrestricted access medi and other newSPE sorbents for lipid analyses haveemerged.

SPE sorbents generally utilize rela-tively large particle and pore sizes(30-60 mm, 60A), and nnalytes areeluted at tow pressures. The sorbernsare typically packed in syringe-likepolyethylene or polypropylene tubes;however. SPE disks are gaining popu-larity (16,17). With disks. the packingis generally embedded or impregnatedinto a PTFE or fiberglass network oronto a membrane (17), a design thatprevents the channeling problems thatcan occur with packed beds. In addi-tion, the disks are thin and have alarge cross-sectional area providingfaster now rates and shorter analysistimes (17). The number of phases cur-rently available in the disk format islimited, however (17).

Matrix solid-phase dispersion(MSPD) is another relatively newtechnique which involves grindingbiological samples with CIS or otherbonded silica supports. transferring

INFORM. VOl. 7. no. 10 (October 1996)

1096

INSTRUMENTATION

Mod. Pb.le Struct1lH

Silica1

• Si-OHI

Cy~'I

• SI-C3HeCNI

"'"I

- Si-CaHeOCH2CH2I I Ialal

Aminopropyl··I

• Si.C3HeNH2I

Cl1 or ODSI

- SI-C1SH31I

C,I

- Si.CaH17I

C,1

- SI-C HI ,.

I""",I .SI·O

BemenesulfODlle - ~i.CI'\-O-SOI-

PropylsulfOlWCI

- Si-C3HeS03'

~I +

""'"" - Si-C3He N (CH~3

• May abo be used II • R:w:rsc phase•• May also be used asan ioo e~ phase

Agure 1. Some eommen;:~lIy available SPE solid phases

(continued/rom page 1094)

columns were used to isolate phos-phatidylcholine (PC) and phos-phatidylelhanolamine (PE) from thenonpolar lipids of chocolate (5).Recoveries for PC ranged from 90 1099% with excellent precision (I to7.8% coefficient of variation). Earlystudies also showed that silica SPEwas an excellent sample preparationtechnique for isolating microgram andnanogram quantities of phospholipidsand neutral lipids from limitedamounts of biological samples(20-22).

In a comparison of SPE and tradi-tional silica column chromatography.

Juaneda and Rocquelin (23) found noquantitative differences in recovery orreproducibility between the two meth-ods for the separation of nonphospho-rous lipids and phospholipids from ratheart, liver, and kidney tissues. How-ever, the SPE procedure effectivelyseparated the two fractions in approxi-mately five minutes, compared to sev-eral hours required for the traditionalcolumn procedure. Similarly, Sebedioet al. (24) found that SPE and a stan-dardized IUPAC-AOAC silica columnchromatographic procedure gave simi-lar results for the fractionation of non-polar and polar lipids from commer-cial frying oils. Roemen and van derVusse (25) felt that the SPE separation

of the free fatty acids from the polarlipids in heart tissue extracts was moreeffective than traditional column chro-matographic techniques. Using silicaSPE, no hydrolysis of fatty acids fromthe phosphoglycerides or other esteri-fied fatty acid classes occurred, aproblem previously reported withether silica column chromatographicmethods. This advantage may be dueto the much shorter separation time ofthe SPE method.

More complex lipid separations arealso possible on silica SPE columns.The number of fractions obtained andtheir chemical compositions aredependent on the solvent systemsused. Hamilton and Comai (26,27)developed an SPE procedure whichwas effective in isolating cholesterolester, triglyceride (TG), free fatty acid(FFA), cholesterol, and acidic PE andphosphatldylinositcl and neutral (PC.sphingomyelin, lyso-PC) phospholipidfractions from human serum, ratserum and liver, and cultured smoothmuscle cells. Using different elutionsolvents, Ingalls et al. (28) isolatedsimilar fractions from plasma withrecoveries of 80-91 %.

lanero and Burghardt (29) reportedthat MTBE-based solvent mixturesutilized with the procedures of Hamil-ton and Comai were effective in sepa-rating PE from the choline-containingphospholipids but resulted in loss ofapproximately 60% of the biologicactivity of platelet-activating factor(PAF), an important choline phospho-lipid. These authors showed thatsequential elution of rat heartextracts-first with chloroform/aceticacid, followed bymethanol/chloroform, and finally withmethanollchloroform/water-resultedin excellent separation of neutral non-phosphorous lipids from PE and PC,while PAF maintained full biologicactivity.

Bonded aminopropyl sorbents. Thesurface silanol groups of silica can bereacted with siloxane derivatives toform bonded phases. A number ofbonded phases are available (Table I)and normally are classified as nonpo-lar (reverse phase), polar (normalphase), or ion exchange. The bondedphases can be either monomeric orpolymeric depending on the number

INFORM. Vol. 7. no. 10 (OCtober 1996)

1097

with analytes having polar functionalgroups than with nonpolar analytes.The amino group also can impartweak anion exchange properties sothat separations based on ionexchange mechanisms are possible.

Because of their polarity, amino-propyl columns have found wideapplications for lipid analyses. A basicmethod for the rapid separation ofindividual neutral and polar lipidclasses using bonded aminopropylSPE cartridges was developed byKaluzny et al, (7), and modificationsof this method continue to be widelyused (30-32). Using the separationscheme described by Kaluzny, neutrallipids are eluted and subsequently sep-arated on a second column with sol-vents of increasing polarity. FFA andphospholipids bind to the columnmainly via ionic interactions. Theseionic interactions are removed bychanging the pH of the elution sol-vents. "Piggy-backing" two amino-propyl columns, so that eluant fromone column was placed onto anothercolumn and further fractionated,yielded complete separation of a crudelipid extract into eight fractions: neu-tral lipids, FFA, phospholipids,cholesterol (or other sterol) esters,TO, cholesterol (or other sterols),

diglycerides, and monoglycerides.Recovery of the lipid class standardswas 96-101% with less than 2% con-tamination between fractions.

Kaluzny et al. (7) compared thisSPE procedure to a standard prepara-tive TLC separation for the fractiona-tion of bovine adipose tissue extracts.Average recovery for all lipid classeswas approximately 100% for the SPEprocedure compared to 80% for theTLC procedure. Similarly. Prasad etel. (33) reported SPE recoveries ofgreater than 90% for arachidonatecompared to less than 70% recoveryby TLC procedures; other saturatedand monosaturated fatty acids alsoshowed losses in the TLC procedure.but these losses were not as significantas those for arachidonate. Otherauthors (34). however. have reportedinconsistent TO recoveries (80-90%)with the SPE procedure of Kaluzny etal. When used to isolate FFA fromdairy products, aminopropyl columnsseemed to avoid many of the problemspreviously reponed with other columnchromatographic procedures. includ-ing hydrolysis of glycerides and inter-ference from lactic acid (8,35). In arecent application, aminopropylcolumns were used to concentratepolyunsaturated fatty acid methyl

A typica. ~Id phase axtrKtlon e.rtridge.The c.rtrldgoI .hown (;OIIQin. 100 mg of •CHI,...,.,... phase eorbenL

of layers of the bonding reagent on thesilica surface (I). The choice ofmonomeric or polymeric phases isdependent on the specific application(1,13),

AminopropyJ bonded phases arepolar and are therefore classified asnormal-phase sorbents (Table I).Hydrogen bonding to the primaryamine results in a stronger interaction

For inforrnotion circle '114

INFORM. Voi. 7. no. 10 (October 1996)

1098

INSTRUMENTATION

method for the separation of neutrallipids. glycolipids, and phospho-lipids from wheat flour using a com-bination of silica and aminopropyJbonded-phase columns. Wheal flourlipid extracts were placed on a silicaSPE column and eluted with solventcombinations of increasing polarity

esters from the saturated and monoun-saturated esters in fish oil (36). Thismay prove a useful alternative to crys-tallization, urea adduct fractionation,and fractional distillation methods forconcentration of polyunsaturated fattyacid methyl esters .

Prieto et al . (37) developed a

Derived Irom . .. Coconut Oil (Whole CUIand Froctionaled)Canola Oil (Oleic Acid)Linseed Oil (Linolenic Acid)Soya (Stearic Acid)Sunllower Oil (Oleic Acid)

Twin RiversTechnologies

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780 Washington Street. Quincy. Massachusetts. 02169Tel (617) 472-9200 Fax (617) 472-5460

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to separate steryl esters, TO, FFA,diglycerides, monoglycerides, mono-galactosylmono- and monogatacro-syldiglycerides (MOMO andMODO), digalactosylmono- anddigalactosyldiglycerides (DGMGand DODO), and PC and Iyso-PC.N-acyl-phosphatidy lethanolami ne(NAPE) and N-acyl-Iysophos-phatidyethanolamine (NALPE),which coeluted on the silica column,were placed on an aminopropyl SPEcolumn and completely separatedwith mixtures ofch Ioroform/met ha nolla mmoni u mhydroxide. Cross-contamination ofthe phospholipid fractions, as deter-mined by HPLC, was less than 14%.Recovery of the phospholipidsranged from 81-87%. Separation ofthe lipid classes using only anaminopropyl column was also evalu-ated; however, the monoglyceridesand monogalactosylglyceride frac-tions, as well as the PC and LPCfractions, were poorly separated onthis column.

C 18 bonded-phase sorbents.Reverse-phase separations on C 18sorbcnts. while not as common as nor-mal-phase separations, also have beenemployed for lipid fractionations.Lipids are retained selectively by thenonpolar CI8 sortent: therefore polar,non lipid components can be easilyremoved and the lipids can be frac-tionated by eluting with increasinglynonpolar solvents. Reverse-phaseseparations may be particularly suitedfor isolation of lipids dissolved or dis-persed in aqueous or other polar sol-vents, eliminating the need for a pre-liminary extraction into an organicsolvent. For example, Pempkowiak(38) isolated short- and long-chainfatty acids directly from distilledwater and sea water using CI8 SPEcolumns. Salts present in sea waterdid not influence the separation effi-ciency. Using CI8 SPE, PC, cerebro-sides. sulfatides, and gangliosideswere isolated from polar precursorsand enzymes in aqueous, in vitrobiosynthetic mixtures (39). The proce-dure was effective in removing water-soluble precursors without loss of thelipid products and was easier andmore effective than Folch extractions

INFORM.Vol. 7. no. 10(October 1990)

1099

or other similar procedures. Fattyacyl-CoA esters also have been isolat-ed successfully using CIS cartridges(40-43), however the recent develop-ment of oligonucleotide SPE car-tridges may provide a more rapid andhighly specific technique for isolationof these compounds (44).

Argentation $PE. Argentationchromatography may be used to sepa-rate lipid materials on the basis of thenumber, type, and position of theunsaturated centers. Briefly, this typeof chromatography takes advantage ofthe fact that compounds withethylenic or acetylenic bonds interactweakly with silver ions. The nature ofthe interaction is not completelyunderstood but appears to involveoverlap of the olefin p-orbitals and thed- and s-orbital of the silver ion. Ster-ic factors affect the ease of overlap,and the double bond remains essen-tially intact during cornplexing (45).

Argentation chromatographyinvolves impregnating a sorbent, usu-a\ly silica, with silver ions, generallyin the form of aqueous silver nitrate.The method has been applied to col-umn chromatography, TLC. andHPLC separations of unsaturatedlipids. Silver ion or argentation chro-matography has been extensivelyreviewed (45,46).

Christie (47) showed that smallcolumns, packed with a bonded ben-zenesulfonate ion-exchange medium,could be loaded with silver ions;importantly the amount of silver need-ed was far less than required for tradi-tional TLC or column techniques. Thesilver-ion SPE cartridges achievedexcellent separation of fatly acidmethyl esters (FAMEs), cholesterolesters, and TO based on the degree ofunsaturation in the fatty acid moiety.In addition, the fractions were notcontaminated with silver ions (47-52).Although silver ion SPE cartridgescurrently must be impregnated by theanalyst, their potential usefulnesseventually may lead to their commer-cial availability.

Solid phase extraction vacuum manifold. Stlverel cartridge. can be analyzed at one time andfractions can be collected In teat tubes placed In the manifold chambe.r A range 01SPEcartridge sizes are COInmen:I.lly .v.lI.ble.a ahown In the lower·lell portlon of the photograph.

through. This problem can be mini-mized with careful attention to sor-bent conditioning, flow rates throughthe columns, and the choice of sol-vents for dissolving and eluting theanalytes. Most manufacturers provideguidelines for optimizing theseparameters.

A recent evaluation of lot-to-lotreproducibilities in mixed-mode solid-phase extraction columns found no sig-nificant differences between the testedlots for the analysis of drugs fromblood (53); however. lot-to-lot variabil-ity in commercial SPE cartridges hasbeen observed (54). Depending on theapplication, single lots of sorbcru froma single supplier may he necessary fora particular analysis.

Contaminant's extracted from theplastic cartridges and frits or from thesorbent are a common problem(25,33,55,56). In some cases thesecontaminants can interfere with subse-quent analyses or with the biologicalactivity of the analytes (57). Theseinterferences can be minimized byusing glass columns, stainless-steelfrits (25,33), and by rinsing the car-tridges with a variety of solvents priorto adding the sample (38.47).

Potential problems/disadvantages orSPEWhen utilizing SPE techniques, caremust be taken to avoid sample over-load and subsequent analyte break-

SummarySPE provides a flexible and selectivetechnique for the fractionation, purifi-cation. and concentration of lipids.When compared to traditional meth-ods such as column chromatography,TLC, and liquid-liquid extractions,SPE cartridges can offer improvedrecoveries and separation efficiencieswith significant cost and time savings.Usc of SPE disks may decrease sam-ple preparation times even further.SPE methods for the isolation andfractionation of lipids continue to uti-lize mainly silica, aminopropyl. andC 18 sorbents; however, the develop-ment of new phases offers the lipidchemist exciting opporruniues for thecontinuing evolution of improvedsample preparation protocols.

ReferencesI. Wachob. G.D., Solid phase

extraction of lipids, in Analysesof Fats, Oils and Lipoproteins,edited by E.G. Perkins, AmericanOil Chemist's Society, Cham-paign, Illinois, 1991, pp.122-137.

2. Ebeler, S.E., and T. Shibamoto.Overview and Recent Develop-

INFORM. Vol. 7. no. 10 (October 1996)

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INSTRUMENTATION

meets in Solid-phase Extractionfor Separation of Lipid Classes,in Lipid Chromatographic Analy-sis, edited by T. Shlbamoto, Mar-cel Dekker Inc., New York, NewYork. 1994. pp. 1-49.

3. Products Catalog and ReferenceGuide. 1995-1996. J&W Scientif-ic. Folsom. California.

4. Solid Phase Extraction Applica-tions Guide and Bibliography. 6thEdn .. Millipore Corporation, Bed-ford. Massachusetts. 1995.

5. Hurst, W.J., and R.A. Martin Jr.,The HPLC Separation and Quan-tiunion of Lecithin in Chocolate,J. Am. Oil Chern Soc.57,307-310 (1980).

6. Williams, M.A" and R.H.McCluer. The Use of Sep-PekC 18 Cartridges During the Isola-tion of Oangliosides, J. Neu-rochem. 35( I):266-269 (1980).

7. Kaluzny. M.A .. L.A. Duncan,M.V. Merrill and D.E. Epps,Rapid Separation of Lipid Classesin High Yield and Purity UsingBonded Phase Columns. J. LipidRes. 26:135-144 (1985).

8. de Jong, C .. and H.T. Badings,Determination of Free FattyAcids in Milk and Cheese. Proce-dures for Extraction, Clean Up.and Capillary Gas Chromato-graphic Analysis. JHRC & cc.13,94-98 (1990).

9. Hutta, M., E. Simunicova, D.Kaniansky, J.Tkacova and J.Brtko, lsotachophorctic Derermi-nation of Short-Chain Fatty Acidsin Drinking Water after Solid-Phase Extraction with a Carbona-ceous Sorbent, J. Chromatogr.470:223-233 (1989).

10. Salari, H., Solid-Phase Extractionand Reversed-Phase High-Perfor-mance Liquid ChromatographicTechnique for Isolation and Esti-marion of Platelet Activating Fac-tor in Plasma, Ibid. 382:89-98(1986).

11. Salari. H., Comparative Study ofSolid-Phase and Liquid-PhaseExtraction Techniques for Isola-tion of Phospholipids from Plas-ma. [bid. 419: 103-1 II (1987).

12. Coene J., E. Van den Eeckhcut, P.Herdewijn and P. Sandra, GasChromatographic Determination

of Alkyl Iysophospholipids AfterSolid-Phase Extraction from CellCulture Media, Ibid. 612:21-26(1993).

13. Hcrack, J., and R.E. Majors, Per-spectives from the Leading Edgein Solid-Phase Extraction, LOGC11(2P4. 76. 79-80. 82. 84. 86.88.90(1993).

14. Mills, M.S., E.M Thurman andM.J. Pedersen, Application ofMixed-Mode. Solid-PhaseExtraction in Environmental andClinical Chemistry, J. Chro-matogr, 629:22-21 (1993).

15. Majors, R.E., New Chromatogra-phy Columns and Accessories atthe 1995 Pittsburgh Conference,Part II. LC/GC. 13(4):290,292-293. 295-296. 298-301(1995).

16. Majors, R.E. , New Chromatogra-phy Columns and Accessories atthe 1996 Pittsburgh Conference,Pan II, Ibid. 14(4):278,280,282-289.292-293 (1996).

17. Majors, R.E., New Approaches toSample Preparation, Ibid.13(2),82. 84. 87-88. 90. 92. 94(1995).

18. Barker, S.A., A.R. Long and M.E.Hines II, Disruption and Fraction-ation of Biological Materials byMatrix Solid-Phase Dispersion. J.Chromatogr. 629:23-34 (1993).

19. Blunk, H.-C .. and H. Steinhart.Separation of Phospholipids inBovine Tissue with DisposableSilica Gel Extraction Columns, Z.Lebensm. Unters. Forsch.190,123-125 (1990).

20. Hedegaard, E., and B. Jensen,Nunc-scale Densitometric Quan-titation of Phospholipids, J. Chro-rnatogr. 225:450--454 (1981).

21. Bocckino, S.B .• P.E Blackmoreand J.H. Exton, Stimulation of1,2-Diacylglycerol Accumulationin Hepatocytes by Vasopressin.Epinephrine. and Angiotensin U,J. BioI. Chern.260(26),14201-14207 (1985).

22. Yandrasitz, J.R., G. Berry and S.Segal, High-Performance LiquidChromatography of Phospho-lipids with UV Detection: Opti-mization of Separations on Silica,J. Chromatogr. 225:319-328(1981).

23. Juuneda. P., and G. Rocquelin.Rapid and Convenient Separationof Phospholipids and Nonphos-phorus Lipids from Rat HeartUsing Silica Cartridges, Lipids20,<U}..41(1985).

24. Sebedlo, J.L.. Ch. Septier and A.Grandgirard, Fractionation ofCommercial Frying Oil SamplesUsing Sep-Pak Cartridges, J..Am. Oil Chern. Soc.63,1541-1543 (1986).

25. Roemen. Th.H.M" and GJ. vander Vusse, G. J. , Application ofSilica Gel Column Chromatogra-phy in the Assessment of Non-Esterified Farry Acids and Phos-phoglycerides in Myocardial lis-sue, J. Chrornatogr. 344:304-308(1985).

26. Hamilton, J.G., and K. Cornai,Separation of Neutral Lipids andFree Fatty Acids by High-Perfor-mance Liquid ChromatographyUsing Low Wavelength Uuravio-let Detection, 1. Lipid Res.25(10), 1142-1148 (1984).

27. Hamilton, J.G., and K. Com ai,Rapid Separation of NeutralLipids. Free Fatty Acids andPolar Lipids Using Prepacked Sil-ica Sep-Pak Columns, Lipids23:1146-1149 (1988).

28. Ingalls, S.T., M.S. Kriaris, Y. Xu,D.W. DeWuf, Ke-Y Tsemg andC.L. Hoppel, Method for Isola-tion of Non-Esterified Fatty Acidsand Several Other Classes ofPlasma Lipids by Column Chro-matography on Silica Gel, J.Chromatogr. 619:9-19 (1993).

29. Janero, D.R., and C. Burghardt,Solid-Phase Extraction on SilicaCartridges as an Aid to Platelet-Activating Factor Enrichment andAnalysis, Ibid. 526: 11-24 (1990).

30. Przybylski. R., and N.A.M.Eskin, Two Simplified Approach-es to the Analysis of AerealLipids. Food Chern. 51:231-235(1994).

31. Caboni, M.R., S. Menoua and G.Lercker, High-Performance liq-uid Chromatography Separationand Light-Scattering Detection ofPhospholipids from Cooked Beef,J. Chromatogr. A. 683:59-65(1994).

(continued on page 1102 )

INFORM, Vol. 7. no. 10 (October 1996)

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INSTRUMENTATION

(cominuedfrom page 1/00)32. Grav. H.J .. O.K. Asiedu and R.K.

Berge. Gas ChromatographicMeasurement of 3· and 4-ThiaFatty Acids Incorporated intoVarious Classes of Rat LiverLipids During Feeding Expert-ments. J. Chromalogr. B.658, 1-10 (1994)

33. Prasad, M.R .• R.M. Jones, H.S.Young. L.B. Kaplinsky and O.K.Das. Analysis of Tissue-FreeFatty Acids Isolated by Amino-propyl Bonded-Phase Columns. 1.Chromarcgr. 428:221-228(1988).

34. Nurmela, K.V.V., and L.T. Sala-ma, Quantitative Analysis of TOby High-Performance LiquidChromatography Using Non-Lin-ear Gradient Elution and FlameIonization Detection. Ibid.435,,39-,48 (1988).

35. de Jong, C., K. Palma and R.Neeter, Sample PreparationBefore Capillary Gas-Chromato-graphic Estimation of Free FattyAcids in Fermented Dairy Prod-ucts, Netherlands Milk & Dairy J.48,,5,-,56 (1994).

36. Wilson. R.. R.J. Henderson. I.C.Burkow and J.R. Sargent, TheEnrichment of n-S Polyunsaturat-ed Fatty Acids Using Amino-propyl Solid Phase ExtractionColumns, Lipids 28:51-54(1993).

37. Prieto, J.A., A. Ebri and C. Col-lar, Optimized Separation of Non-polar and Polar Lipid Classesfrom Wheat Flour by Solid-PhaseExtraction, J. Am. Oil Chern.Soc. 69,387-391 (1992).

38. Pempkowiak, J., CI8 Reversed-Phase Trace Enrichment of Short-and Long-Chain (C2·C8-C20)

SCORES 100% IN SPECIAliZED TECHNICALSERVICES FOR CONSUMER PIIOOUCTS.

Boston • AtlantaContact Ttida Baresst, TEL (617) 328-7600 FAX(617) 77Q.<1)57

For information circle '118

Fatty Acids from Dilute AqueousSolutions and Sea Water, J. Chro-rnatogr, 258:93-102 (1983).

39. Figlewicz, D.A., C.E. Nolan, l.N.Singh and EB. Jungalwala, Pre-Packed Reverse Phase Columnsfor Isolation of Complex LipidsSynthesized from RadioactivePrecursors, J. Lipid Res.26,140-144 (1985).

40. Styrene. S. and G. Glad. AcylExchange Between Oleoyl-Coeand Phosphatidylcholine inMicrosomes of Developing SoyaBean Cotyledons and Its Role inFatty Acid Desaturation, Lipids16,298-305(1981).

41. Stymne, S .• A.K. Stoban and G.Glad, The Role of the Acyl-CoAPool in the Synthesis of Polyun-saturated 18-Carbon Fatty Acidsand Triacylglycerol Production inthe Microsomes of DevelopingSafflower Seeds, Biochim. Hie-phys. Acta. 752: 198-208 (1983).

42. Stymne, S., and A.K. Stobart,Involvement of Acyl ExchangeBetween Acyl-CoA and Phos-phatidylcholine in the Remod-elling of Phosphatidylcholine inMicrosomal Preparations of RatLung, Ibid. 837:239-250 (1985).

43. Molaparast-Saless. E. E. Shrago,T.L. Spennetta, S. Douatetlo.L.M. Kneeland, S.H. Nellis andA.J. Liedtke, Determination ofIndividual Long-Chain FattyAcyl-CoA Esters in Heart andSkeletal Muscle, Lipids2N90-492 (1988).

44. Deutsch, J., E. Grange, S.1.Rapoport and A.D. Purdon, Isola-tion and Quantitation of Long-Chain Acyl Coenzyme A Estersin Brain Tissue by Solid-PhaseExtraction, Anal. Biochem.2B21-323 (1994).

45. Morris, L.J., Separation of Lipidsby Silver Ion Chromatography, J.Lipid Res. 7:717-732 (1966).

46. Analysis of Triglycerides, C.Litchfield, Academic Press, NewYork, 1972, 355 pp.

47. Christie, W.W., Silver Ion Chro-matography Using Solid-PhaseExtraction Columns Packed witha Bonded-Sulfonic Acid Phase, J.Lipid Res. 30: 147 [-1473 (1989).

48. Ulbenh, E, and E. Achs, Argenta.

INFORM. Vol. 7. no. 10 (October 1996)

1103

lion Chromatography of FattyAcid Methyl Esters Using Silver-Loaded Solid-Phase ExtractionColumns. J. Chromatogr.504,202-206 (1990).

49. Hoving. E.B .• FAJ. Muskiet andW. W. Christie. Separation ofCholesterol Esters by Silver IonChromatography Using High-Per-formance Liquid Chromatogra-phy or Solid-Phase ExtractionColumns Packed with a BondedSulfonic Acid Phase, Ibid.565,102-110 (1991).

50. Augustyn. O.P.H .. D. Ferreira andJ.L. Kock. DifferentiationBetween Yeast Species. andStrains Within a Species. by Cel-lular Fatty Acid Analysis. 4. Sac-charomyces sensu stricto. Hense-niaspora. Saccharomycodes andWickerhamiella, System. App!.Microbial. 14:324-334 (1991).

Bonded Silica Phases from Solid-Phase Extraction Columns. Cor-relation of Surface Chemistrywith Chromatographic Behavior,Ibid. 542:205-220 (1991).

55. Junk, G.A., M.J. Avery and J.J.Richard, Interferences in Solid-Phase Extraction Using C 18 Bond-ed Porous Silica Cartridges. Anal.Chern. 60: 1347-1350 (1988).

56. Crowley, X.w., V. Murugaiah, A.Nairn and R.W. Giese, Maskingas a Mechanism for EvaporativeLoss of Trace Analyte, Especiallyafter Solid-Phase Extraction, J.Chromatogr. A., 699:395-402(1995).

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51. Augustyn, O.P.H., J.L.F. Kockand D. Ferreira, DifferentiationBetween Yeast Species, andStrains Within a Species, by Cel-lular Fatty Acid Analysis. 5. AFeasible Technique? Ibid.15:105-115 (1992).

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54. Shreedhara Murthy, R.S., and L.J.Crane, Characterization of Cyano

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INFORM. Vol. 7. no. 10 (October 1996)