HPLC J. Lipid Res. 1983 Kaduce 1398 403

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an d a Model 42 0 system controller . Lipids were de-tected using a Beckman 1 55 variable wavelength de-tec tor fitted with a 20-p1, l- cm optical pat h cell. Forphospholipid chromatography, the wavelength of thedet ector was set at 202 nm. Phospholipid separation wasachieved with a Beckman 4.6 X 250 mm column packedwith 5 pm Ultrasphere-Si. A guard column of 4.6 X 45mm packed with silica gel was used in conjunction withthe analytical column. For routine phospholipid sepa-rations, the lipid extract was applied to the column in10 pl of chloroform-diethyl eth er 1:2 (v/v) at a con-centr ation of abo ut 1 mg/ml, and t he solvent systemconsisted of acetonitrile-methanol-sulfuric acid100:3:0.05 (v/v/v). T h e solvent mixture was deliveredby the pump at a flow rat e of 1 ml/min, which produc eda pump pressure of about 1500 PSI. When in contin-uous use over a period of several days, the column iswashed each night with a solvent mixture of acetoni-trile-methanol-sulfuric acid 100:3:0.1 (v/v/v) at a flowrate of 0.1 ml/min. Once every 2 months, the entire

system is purged with 300 ml of methanol.

Tissue lipids

Bovine aorti c endotheli al cells, passage 14, were in-cubated with [l-'4C]oleic acid for 120 min (13). Afterwashing, the cellular lipids were extracted with chlo-roform-methanol 2: 1 (v/v) ( 1 4). Phospholipid classeswere then separated by either HPLC or TLC on silicagel H plates (Analtech, Inc., Newark, DE) impreg natedwith 1 % boric acid. T h e TL C solvent system contain edchloroform-methanol-ammonium hydroxide-water120:75:2.6 (v/v) (15). Additional radioisotope incor-poration experiments were done with ['H]arachidonic

acid to check the adequacy of the HPL C separation. I nthese experi ments an aliquot of each fraction collectedfro m the H PLC column was subjected to TL C in orderto determine its radiopurity. Radioactivity was mea-sured using 4 ml of Budget Solve (Research ProductsInt ., Moun t Prospect, IL) in a Packard 24 20 liquid scin-tillation spectr ometer . A 226Ra exter nal standa rd wasused in order to monitor and correct for quenching. T omeasure phospholipid content , the lipid fractions weredried u nder Nz , digested with perchloric acid for 20min at 27OoC, and assayed fo r ph osphor us (16).

Transesterification and gas-liquid chromatography

The lipids contained in the fractions separated byHPLC were transesterified to form fatty acid methylesters directly in the eluting solvent mixture. To eachfraction 2 ml of 12% BF3 in methanol was added (1 7)and the mixture was incubated at 100C for 2 hr. Afterthe fatty acid methyl esters were remov ed by extractionwith hep tane, analysis by gas-liquid chr omat ogr aphy(GLC) was per for med with a Hewlett -Packard Model

5700 chromatograph equipped with a 2 mm X 1.9 mglass column packed with 10% SP2330 on 100 /20 0mesh Chromo sorb W-AW (Supelco, Inc.) (1 3).

RESULTS

Effects of mobile phase composition

Preliminary studies demonst rated tha t th e main phos-pholipid classes can be separated on an Ultrasphere-Sicolumn with a mobile phase containing acetonitrile,methanol, and sulfuric acid. Optimum separation wasobtained with a 100:3:0.05 (v/v/v) mixt ure . Even smallchanges from this composition were found to have amarked effect on th e phospholipid reten tion times and ,therefore, the resulting separation. For example, PIeluted with the neutral lipids when the mixture waschanged to 100:5:0.1. T h e reten tion times of all of thephospholipids increased when the methanol content wasreduc ed, with PI being affected most. When t he solvent

ratio was 100:1:0.1, PI eluted together with PE.Changes in the sulfuric acid content of the mobile

phase produced little effect on the retention time of PI.As the sulfuric acid con tent was reduced, however, th eretent ion times of PC, PE, and PS increased, and b road-ening of all of the absorbance peaks occurred. Whensulfuric acid was omitted , PC, PE, and PS did not e lutefrom the column.

Phospholipid separations

Fig. 1 shows the separation of a mixture of PC, PE,PI, PS, LPC, SPH, and cardiolipin (CL) with th e mosteffective mix tur e of acetonitrile-methanol-sulfuric acid

100:3:0.05 delivered by the pump at a f low rate of 1ml/min. T h e first large absorbance peak is du e to thepresence of chloroform and diethyl ether which elutein the solvent front and absorb at 202 nm. Under theseconditions, triacylglycerol, cholesterol, cholesteryl es-ter, and fatty acid standards eluted with the solventfront. CL also eluted in the solvent front and was notseparated from the neutral lipids. Baseline separationwas obtained fo r th e ot her phospholipid classes, the or-der of elution being PI, PS, PE, PC, LPC, and SPH. Asmall, unidentified absor bance peak appear ed betweenPE and PC. All components elute d in 40 min.

A separation of the phospholipids extr acted fr om cul-tures of bovine aortic endothelial cells is shown in Fig.2. The lipids were extracted from a confluent mono-layer cultur e containing 30 0 pg o f cell protein dissolvedin chloroform-diethyl eth er 1:2, and applied to theHPLC column. Peaks correspon ding to PI, PS, PE, PC,LPC, and SPH were detected by absorbance at 20 2 nm.A small unidentified absorbance peak was noted be-tween PI and PS, and t w o smal unidentified absorbance

Journal of Lipid Research Volume 24, 1983 Notes on Methodology 1399


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peaks occurred between PE and PC. Smaller amountsof lipid were chromatographed in routine analyses ofthe endothelial cells, corresponding to 90 to 100 pg ofcell protein. Under these conditions, neither LPC norSPH appeared on the chromatograms. Th e PC fractionin Fig. 2 appears to include two absorbance peaks. Onlya single PC peak was observed, however, in the routineassays where smaller amounts of lipid extract were chro-matographed. Furthermore, experiments with radio-active endothelial cell lipid extracts indicated that morethan 95% of the radioactivity contained in the entirePC fraction migrated with a PC standard on TLC.

Radioactivity was incorporated into the lipids of bo-vine aortic endothelial cultures by incubation of the cellsfor 120 min with [l-' 4C]oleic acid. Th e lipids were ex-tracted from the cells and chromatographed either bythis HPLC procedure or by TLC (15). Of the radio-activity applied to the Ultrasphere-Si column, 97.4

2.5 (mean SE, n = 3) was recovered in the eluentcollected over 4 min. Table 1 shows the distribution

of radioactivity in the various fractions eluted from theHPLC column and, for comparison, the distribution ofradioactivity in the fractions separated by TLC. Al-though there were small differences in several of thefractions, the percentage distributions obtained by theHPLC separation were, in general, quite similar to thoseobtained by TLC separation.

Additional radioisotope incorporation experimentswere done to check the purity of the fractions separated

0.4

0.3

0

E(D

cr

Qq

2 0 2

0.1

0 -

0.15

Q 0.10

9E(D

u

2 0.05

-

-

-

-

1

PC

0 10

iLPC

2 0 30 40Time min)

Fig. 1. Separation of a phospholipid standard mixture. The mobilephase consisted of acetonitrile-methanol-sulfuric acid 100:3:0.05(v/v/v) at a flow rate of 1.0 ml/min. Absorbance was measured at202 nm

10 2 0 30 4 0 50Time (min)Fig. 2. Separation of bovine aortic endothelial cell lipids. Th e materialchromatographed was extracted from a cultur e containing 300 r g ofcell protein. The conditions were the same as indicated in Fig. 1.

by HPLC. T he endothelial cultures were incubated for16 hr with ['Hlarachidonic acid. Th e distribution ofradioactivity as dete rmined by HPLC was 37% as PC,29% PE, 21%, PI , 2.2% PS, 7.2% as neutral lipids, and3.5% in the remaining fractions. Each of the eluted frac-tions then was subjected to T LC with the corresponding

standard added, and the percentage of radioactivity thatco-chromatographed with the standard was determined.Th e recoveries were: PC, 96 1%; PE, 84 1%; PI,89 1 ; PS, 89 8% ; and neutral lipids, 93 2%(mean SE, n = 3).

TABLE 1. Comparison of the distribution of lipid radioactivityfollowing separations by HPLC and TLC

Radioactivity

Lipid Fraction HPLC TLC

9

PC 64.0 0 7 60.5 0.6PE 7.8 0.2 10.9 0.7

6.3 0.6.2 0.2S1.4 0.2.7 0.1I

17.6 0.5eutral lipids 18.9 0.6Others 4.4 k 0.5 2.9 0.2

Monolayer cultures of confluent bovine aortic endothelial cells wereincubated with [I-'4C]oleic acid for 120 min. Each value is the mean

SE of the data obtained f r o m three separate cultures.

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Transesterification for GLC

GLC analysis of the lipids contained in the solventmix tur e of acetonitrile-methanol-sulfu ric acid100:3:0.05 revealed that transesterification can beachieved by directly adding BF,-methanol (1 7), withoutfirst drying the sample. An initial experiment was donewith [ 1 '4C]dipalmitoyl phosphatidylcholine dissolved in

5 ml of this solvent mixture in ord er t o determine thetime course of transesterification. After 2 ml of 12%BF3 in methanol was added, the mix ture was incubatedat 100C for various times and the percentage conver-sion of radioactivity into fatty acid methyl esters wasdetermined. There was a 55% conversion in 30 min,75% in 45 min, 88% in 1 hr, and 94 in 2 hr or 3 hr.Additional studies indicated that there was no appre-ciable difference in either the percentage or rate of con-version to fatty acid methyl esters when the amount ofBF3 in methanol added to 5 ml of the HPLC elutionmixture was between 1 and 3 ml.

Table 2 shows the recovery of the various fatty acidscontained in soybean PC following separation by HPLCand methylation in the eluting solvent mixture. Thesamples separated by HPLC were methylated by adding2 ml of 12% BF3 in methanol to ml of the elutingsolvent mixture an d incubating at 100C for 2 hr. Thefatty acid composition of the PC fraction separated byHPLC was almost identical to that of correspondingaliquots of the soybean PC preparation that were notsubjected to HPLC. Further, there was no appreciablechange in the fatty acid composition when the PC frac-tion was stored for 3 days at 4C in the eluting solventmixture before it was methylated. Additional studies

revealed that a considerable loss of polyunsaturatedfatty acids occurred if the eluting solvent was evapo-rated before the BF3-methanol was added. This losscould be prevented if the mixture was neutralized withmethanolic NaOH prior to drying (data not shown).

Lipid phosphorus assay

Aliquots of soybean PC containing 16 nmol of lipidphosphorus were injected into the HPLC column andthe PC fraction was collected. After the eluting solventmixture was evaporated under N 2 the lipid residue wasdigested with perchloric acid. The recovery of lipid

phosphorus was 15.5-

0.5 nmol (mean SE n 4),accounting for 97% of the material injected into thecolumn.

DISCUSSION

This HPLC procedure offers many advantages forthe routine separation of the main phospholipids usuallycontained in animal tissues. Several of these ar e due to

TABLE 2. Effect of HPLC separation on the fatty acidcomposition of soybean PC

Composition

PC Fraction Collected following HPLC

Methylated afterMethylated Immediately Storag e for 3

Days in HP LCFatty Acid Soybean PC Solven t Mixture Solvent Mixture

in HPLC Eluting

R

16:O 12.1 f 0.5 12.5 k 0 .2 13.3 0.14.1 0.1 4.6 0.18:O 4.0 0.1

8.8 0.1.5 f 0.18:l 8.4 f 0.118:2 67.6 0.7 66.4 f 0.2 65.2 k 0.2

7.3 0.3.0 0.18:3 7.8 0.30.8 f 0.1thers 0.1 0.1 0.5 0.2

a Mean ? SE of three determinations.

the fact that the elution is done under isocratic condi-tions. Since only one pump is required, the HPLC

equipment is less expensive than that needed for gra-dient separations. In addition, processing of multiplesamples is speeded up because the column does not haveto be recycled. The isocratic elution also avoids exces-sive baseline absorbance changes, simplifying interpre-tation of the chromatogram. These advantages also ap-ply to the isocratic separations for phospholipids devel-oped by Gross and Sobel (12) and Chen and Kou (10).However, the former method does not resolve PS andPE (12), and the latter procedure does not provide agood separation between PI and the neutral lipids thatelute in the solvent front. Furthermore, the fact thatphosphoric acid, which is contained in the mobile phaseof Chen and Kou (1 0), is not used eliminates a potentialsource of error in subsequent phospholipid assays. Fi-nally, the very small water content of the solvent systemallows preparat ion of fat ty acid methyl esters by transes-terification without first drying the eluted phospho-lipids.

While the present method resolves CL from the o the rphospholipids, it does not separate CL from any neutrallipids contained in the sample. This should not repre-sent a serious difficulty, however, for CL usually is nota major phospholipid component in biological speci-mens. Another potential problem may be encountered

if the sample contains disaturated phosphoglycerides.Silica columns often separate the disaturated compo-nents of a phospholipid class from molecular speciescontaining unsaturated acyl groups (5, 12). Since de-tection is by ultraviolet absorbance, disaturated phos-phoglycerides would not appear on the chromatogramif they eluted separately. Although this must be takeninto account in studies with surfactant or liposomes, itis not an important consideration in most metabolic ap-



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plications because t he phospholipid classes usually con-tain few disaturated species. For example, Gross andSobel (12) have shown that at least 95% of the phos-pholipids contained in rabbit mycoardium elute froma silica column in ultraviolet absorbing peaks. In addi-tion, Mahadevappa an d Hol ub (18) have shown thatonly 1.6% of the total diacyl phospholipid species inhuman platelets is disaturated.

Several oth er possible sources of e rr or should be rec-ognized by those contemplating use of this procedure.Since the HPLC system utilizes an ultraviolet absor-bance detector, the peak areas obtained from the chro-matogram tracing are not a measure of the weight ormolar amounts of lipid. To obtain quantitative data,some type of chemical assay of the eluted fractions isrequired. Careful attention must also be given to thecomposition of the mobile phase because even smallchanges in the methanol or sulfuric acid content canhave an important effect on the phospholipid separa-tion. Because of this, w e prepare the mobile phase

freshly each day. Furthermore, lipid samples are in-jected in a chloroform-diethyl ether mixture in orderto avoid introducing an excess of methanol into thesystem. Finally, the method is not suitable for the re-covery of acid-labile phospholipids such as plasmalogensbecause the mobile phase contains sulfuric acid. Exper-iments with the plasmalogen form of PE indicate thatmost of it is converted to the lyso derivative duringchromatography and that lyso PE elutes with PC in thissystem.

Sphingomyelin (SPH) was detected in the standa rdmixtur e and when large am ounts of endothelial cell lip-ids were separated. In routine studies where smalleramounts of endothelial cell lipids were analyzed, how-ever, SPH was not detect ed. Gross and Sobel (12) alsodid not detect SPH by ultraviolet absorbance in chro-matograms of rabbit myocardial lipids. One possibilityis that t he amo unt of S PH pre sent in these tissues is toosmall to detect unless large amounts of lipid are chr o-matographed because the fatty amide group containsrelatively little unsaturation. Alternatively, SPH may belabile in the acidic mobile phase that was employed, sothat some SPH remains only when large amounts arepresent initially. Since SPH recovery was not measured,the possibility that this HPLC pro cedu re is not suitable

for use with SPH cannot be excluded.Even though the present HPLC procedure is rapid,it is still much more time-consuming than a one-dimen-sional TLC separation in which a number of samplescan be analyzed in a single chromatogram. There are,however, several important advantages of this HPLCmethod over TLC. One is the separation of PI and PS.While PI and PS can be resolved by one-dimensionalTLC (1 9), this often causes a loss of resolution of other

phospholipids. Good separation of all phospholipids isobtained by two-dimensional TL C (20), but only on esample can be ru n o n each chr omatog ram, only a smallamount of lipid can be separated, and positive identi-fication is sometimes difficult in those cases where stan-dards cannot be added to th e same chromatogram. An-oth er problem with phospholipid separation by T L C isthe possible loss of polyunsatura ted fatty acids as a resultof staining (21). Since HPLC avoids such problems, aprocedure such as the one that we have developed ap-pears to be a preferable approach for some metabolicapplications.IB

This study was supported by research grant AM 28516 fro mthe National Institute of Arthritis, Diabetes, Digestive andKidney Diseases and Arteriosclerosis Specialized Center ofResearch grant, H L 14230, from the National Heart , Lung ,and Blood Institute, National Institutes of Health.

Manuscript received 15 December 1982 and in reuvisedform 7 April 1983

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1. Jungalwala, F. B., R. J. Turel, J. E. Evans, and R. H.McCluer. 1975. Sensitive analysis of eth anolamine - andserine-containing phosphoglycerides by high perfor-mance liquid chromatography. Biochem.J 145: 5 17-526.

2. Jungalwala, F. B., J. E. Evans, and R. H. McCluer. 1976.High-performance liquid chromatography of phosphati-dylcholine and sphingomyelin with direct detect ion in th eregion of 200 nm. Biochem.J 155: 55-60.

3. McCluer, R. H ., and F. B. Jungalwala. 1976. High-per-formance liquid chromat ographic analysis of glycosphin-golipids and phospholipids. In Current Trends in Sphin-golipidosis and Allied Disorders. B. W. Volk and L.Schneck, editors. Plenum Press, New York. 533-554.

4. Hax, W. M. A., and W. S. M. Geurts Van Kessel. 1977.High-performance liquid chromatographic separationand photometric detection of phospholipids. J Chroma-togr. 142: 735-741.

5. Geur ts Van Kessel, W. S. M., W. M . A. Hax, R. A. Demel,and J. DeGier. 1977. High perform ance liquid chro-matographic separation and direct ultraviolent detectionof phospholipids. Biochim. Biophys. Acta. 486: 524-530.

6. Porter, N. A., R. A. Wolf, and J. R. Nixon. 1979. Sep-aration a nd purification of lecithins by high press ure liq-uid chromatography. Lipids. 14: 20-24.

7. Jungalwala, F. B., V. Hayssen, J . M . Pasquini, and R. H.McCluer. 1979. Separat ion of molecular species of sphin-gomyelin by reversed-phase high performance liquidchromatography. J Lipid Res. 20: 579-587.

8. Hanson, V. L., J. Y. Park, T. W . Osbor n, and R. M. Kiral.198 1. High-per formance liquid chromato graphic analysisof egg yo lk phospholipids. J. Chromatogr. 205: 393-400.

9. Patton, G. M., J. M. Fasulo, and S. J . Robins. 1982. Sep-aration of phospholipids and individual molecular speciesof phospholipids by hig h-perfor mance liquid chromatog -raphy. J. Lipid Res. 23: 190-196.

10. Chen, S. S. and A. Y. Kou. 1982. Improved procedurefor the separation of phospholipids by high-performanceliquid chromatography. J Chromatogr. 227: 25-3 1

11. Yandrasitz, J. R., G. Berry, and S. Segal. 1981. High-

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performance liquid chromatography of phospholipidswith UV detection: optimization of separations on silica.J. Chromatogr. 225: 3 19-328.

12. Gross, R. W., and B. E. Sobel. 1980. Isocratic high-per-formance liquid chromatographic separation of phos-phoglycerides an d lysophosphoglycerides. J . Chromatogr.

13 . Kaduce, T. L., A. A . Spec tor, and R. B. Bar. 1982. Lin-oleic acid metabolism and prostaglandin prod ucti on bycultured bovine pulmonary artery endothelial cells. Ar-teriosclerosis. 2: 380-389.

14. Folch, J., M. Lees, and G. H. Sloane Stanley. 1957. Asimple method for the isolation and purification of totallipids from animal tissues. J Biol. Chem. 226: 497-509.

15. Fine, J . B., and H. Sprecher. 1982. Unidimensional thin-layer ch romatogr aphy of phospholipids on boric acid-im-pregnated plates. J. Lipid Res. 23: 660-663.

16. Chalvardjian, A. , and E. Rudnicki. 1970 . Determi nation

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of lipid phosphorus in the nanomolar range. Ana l. Bwchem.

17. Morrison, W . R., and L. M . Smith. 19 64. Preparation offatty acid methyl esters and dimethylacetals from lipidswith boron fluoride-methanol. J Lipid Res. 5: 600-608.

18. Mahadevappa, V. G., and B. J. Holub. 1982. Th e molec-ular species composition of individual diacyl phospholip-ids in human platelets. Biochim. Biophys. Acta 713: 73-79.

19. Touchstone, J. C., J. C. Chen, and K. M. Beaver. 1980.Improved separation of phospholipids in thin-layer chro-matography. Lipids 15: 61-62.

20. Evans, R. W., M. Kates, M. Ginz burg, and B. Z. Ginsburg.1982. Lipid composition of halotolerant algae, dunaliellap a m a lerche and cunaliella tertiolecta. Biochim. Biophys. Acta712: 186-195.

21. Nichaman, M. Z., C. C. Sweeley, N. M . Oldham, andR . E. Olson. 1963. Changes in fatty acid composition du r-ing preparative thin-layer chromatography. J. Lipid Res.

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HPLC J. Lipid Res. 1983 Kaduce 1398 403