Anal. Chem. 1994,66, 3977-3982
Liquid Chromatography/Mass Spectrometry of Phospholipids Using Electrospray Ionization Hee-Yong Klm,’J Tao-Chin L. Wang,* and Yee-Chung Mat Section of Mass Spectrometry, Laboratory of Membrane Biochemistry and Biophysics, NIAAA, and Section of Analytical Biochemistry, NIMH, NIH, 9000 Rockville Pike, Bethesda, Maryland 20892
An improved technique for phospholipid molecular species analysis was developed using high-performance liquid chromatography/mass spectrometry with the electrospray interface. Using the 0.5%ammonium hydroxide in a water-methanolhexane mixture and a C-18 column, complex mixtures of phospholipid molecular species were separated and detected mainly as protonated or natriated molecular species. The response was linear over 2 orders of magnitude, allowing quantification of each molecular species. In comparison to the existing LC/MS techniques, marked improvement in sensitivity was observed. The present quantification limit is approximately 0.5 pmol before split (5 fmol after 1/100 split). The relative responses were more dependent on the head group identity rather than fatty acyl compositionwithin a phospholipid class. In general, phosphatidylcholine (PC) species are most sensitively detectedfollowed by phosphatidylethanolamine (PE) species. The sensitivity of phosphatidylserine (PS) in the positive ion mode is approximately 20 times less in comparison to PC under our condition. Phospholipids are one of the major constituents in the cell membrane bilayer. Membrane phospholipids are a complex mixture of molecular species containing a variety of fatty acyl and head groupcompositions (Scheme 1). It is widely accepted that chemical and physical properties of cell membranes are largely dependent on the phospholipid composition.1 Maintenance of tightly regulated membrane environment appears to be important for many membrane functions including transport and endocytosis2J as well as activity of membrane bound enzyme^.^ In addition, specific pools of phospholipids serve as reservoirs for polyunsaturated fatty acids that can be metabolized to various biologically important mediatoms Therefore, altering the phospholipid profile may bring about significant biological consequences. Analysis of phospholipid molecular species often requires laborious procedures including separation by column,6 argentation thin-layer (TLC)7 or high-performance liquid chromatography (HPLC).8 Identification of separated comt Section of Mass Spectrometry. *Section of Analytical Biochemistry. (1) Stubbs, C. D.; Smith, A. D. Biochim. Biophys. Acta 1984, 779, 89-137. (2) Van Der Neut-Kok, E. C. M.; De Gier, J.; Middelbcck, E. J.; Van Deenen, L. L. M. Biochim. Biophys. Acta 1974, 322, 97-103. (3) Sandermann, H., Jr. Biochim. Biophys. Acta 1978, 515, 209-237. (4) Imaoka, S.; Imai, Y.; Shimada, T.; Funae,Y. Biochemistry 1992, 31, 60636069. ( 5 ) Lapsata, M.; Kaiser, S. L.; Capriotti, A. M. J. Biol. Chem. 1988,263,32663273. (6) Arvidson, G. A. E. J. Chromatogr. 1975, 103, 201-204. (7) Arvidson, G. A. E. J. Lipid Res. 1965, 6, 574-577. (8) Patton, G. M.; Fasulo, J. M.; Robins, S. J. J. Lipid Res. 1982, 23, 190-196.
This artlcle not subject to U S . Copyright. Published 1994 by the American Chemical Society
pounds must then be made by retention behavior on TLC, HPLC, or gas chromatography with known standards or by mass spectrometric analysis typically using fast atom bombardment9J0 or laser desorptionll techniques. A fast and convenient phospholipid analysis method has been developed using thermospray liquid chromatography/mass spectrometry (LC/MS)l2-l4 and has been employed for the phospholipid analysis of various biological m i ~ t u r e s . ~Although ~J~ on-line HPLC/MS capability at the conventional HPLC flow rate and the characteristic fragmentation allowed efficient qualitative and semiquantitative analyses of the complex mixture, quantitative analysis was not straight forward due to the widely varied response factors for individual molecular species and the narrow range of linearity.17J8 In addition, the technique is not sensitive enough for many metabolic studies, which require the detection of phospholipid molecular species at low or sub picomole levels. Electrospray ionization produces molecular ion clusters from various biomolecules.19-21 Analysis of phospholipids by electrospray ionization mass spectrometry has been explored previously for platelet activating factors22 and phosphatidylethanolamine (PE)23using the positive and negative ion mode, respectively. However, LC/MS capability as well as the quantitative aspects of the technique have not been fully investigated for these compounds. In this paper, we demonstrate that electrospray mass spectrometry coupled to the HPLC using a splitter provides a quantitative technique for the analysis of phospholipidsfrom complex biological mixtures with remarkable sensitivity.
EXPERIMENTAL SECTION All phospholipid standards were obtained from Avanti Polar Lipids (Alabaster, AL). Deuterium-labeled standards and (9) Matsubara, T.; Hayashi, A. Prog. Lipid Res. 1991, 30, 301-322. (10) Kayganichi, K. A.; Murphy, R. C. AMI. Chem. 1992,64, 2965-2971. (11) Whal,M.C.;Kim,H.S.;Wood,T.D.;Guan,S.;Marshall,A.G.Anol.Chem. 1993.65, 3669-3676. (12) Kim. H. Y.;Salem, N., Jr. AMI. Chem. 1986, 58, 9-14. (13) Kim, H. Y.; Salem, N., Jr. Anal. Chem. 1987, 59, 722-726. (14) Kim,H. Y.;Yergey, J.A.;Salem,N., Jr. J. Chromatogr. 1987,394,155-170. (15) Salem, N., Jr.; Hullin, F.; Yoffe, A.; Karanian, J. W.; Kim, H. Y. In Advances in Prostaglandin and Leukotriene Research; Samuelsson, B.,Wong, P. Y. K., Sun, F. F.,Eds.; Raven Press: New York, 1988; pp 618-622. (16) Kim, H. Y.; Edsall, L.; Ma,Y. C. Submitted to Lipids. (17) Kim, H. Y.; Salem, N., Jr. Prog. Lipid Res. 1993, 32, 221-245. (18) Ma, Y. C.; Kim, H. Y. Proceedings in 41th ASMS Conference on Mass Spectrometry and Allied Topics; 1993; pp 520a-520b. (19) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984,88, 44514459. (20) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-7 1. (21) Mann, M. Org. Mass Spectrom. 1990, 25, 575-587. (22) Weintraub, S. T.; Pinckard, R. N.; Hail, M. Rapid Commun. 1991,5, 309311. (23) Chan, S.; Reinhold, V. The Proceedings in 41th ASMS Conference on Mass Spectrometry and Allied Topics; 1993; pp 1060a-1060b.
Analytical Chemistry, Vol. 66, No. 22, November 15, 1994 3977
Scheme 4. Chemical Structures of Varlous Phospholipid Classes
Phospholipids
CHZ-O-RI
-
~
CH-0 R2
I
F
)
CHZ-O-Y-O-R~ OW) R i , Rz =
Fatty Acyl Chain
R3 =
-H
Phosphatidic Acid (PA)
-CHz-CHz-NHz
Phosphatidylethanolamine(PE)
+
-CHZ-CHZ-N(CH~)~
Phosphatidylcholine(PC)
-CHz-CH(CO0H)-NHz
Phosphatidylserine(PS) Phosphatidylinositol (PI)
Sphingomyelin
9
t
CH3(CH2)1z-CH=CH-CH-CH-CHZ-O-!~'-O-CHZ-CH~)~ I 1 01 1 OH NH RC=O
polyunsaturated phospholipids were custom synthesized by the same company. All the solvents were HPLC grade purchased from EM Scientific. 18:0, 20:4-PI and 18:O-SM were isolated from bovine liver PI and bovine brain SM by HPLC using an Ultrasphere-ODS column (3 pm, 4.6 mm X 7.5 cm) andmethanol/hexane/O. 1 M ammonium acetate (500: 25:25).13 A Hewlett-Packard 5989 mass spectrometer with H P electrospray source was used for this study. Samples were injected onto a C-18 HPLC column (EM Scientific, 5 pm, 2.1 mm i.d. X 15 cm) and separated using the mobile phase consisting of 0.5% ammonium hydroxide in water/methanol/ hexane changing linearly from 12:88:0 to 0:88:12 in 17 min after holding at the initial composition for 3 min. At the end of each run, the HPLC column was washed with 0.1 M ammonium acetate at 0.5 mL/min for 5 min. All the separation was done at the room temperature (20-25 "C); care must be taken since operating the column at a higher temperature destroys the column irreversibly. Under this condition, we have not observed any unusual deterioration of chromatographic performance. The column flow rate was 0.5 mL/min, and the effluent was split 1:lOO using a commercial splitter (LC Packings) to deliver 5 pL/min to the electrospray needle. The capillary, end plate, and cylinder voltages were set at -4500, -3500, and -3000 V, respectively. The capillary exit voltage was normally set at 200 V and raised up to 300 V for the detection of diglyceride ions. The nitrogen drying gas flow was approximately 3 L/min, and the temperature was 220 " C . The standards were calibrated by phosphorous assay or GC analysis in the presence of an internal standard after transmethylation. Phosphorus Assay. The phosphorous assay was performed according to the reported procedure24with a slight modifica-
tion. This method was based upon the colorimetric measurement of a phosphomolybdenum complex blue formed during reduction of phosphomolybdic acid in the presence of sulfite. Organic solvents in samples were evaporated under nitrogen, and 1 mLof28,3%H2SO4wasadded. Themixturewas heated at 160 "C overnight. After cooling, 100 p L of 30% H202 was added, and the sample mixtures were vortexed. The mixture was further heated at 160 "C for 2 h. After cooling, 1.61 mL of water, 0.5 mL of reagent mixture containing 0.5 mg of l-amino-2-naphthol-4-sulfonic acid (ANSA, Sigma, St. Louis, MO), 13.6 mg of NaHS03, and 1 mg of Na2S03, and 1 mL of 2.5% (w/w) ammonium molybdate in water were added. The mixture was vortexed and heated in a boiling water bath for 15 min. Absorbance was read at 820 nm after cooling. All the reagents for the phosphorous assay were obtained from Sigma Chemical Co. Fatty Acid Analysis. Fatty acid methyl esters were prepared according to the procedure by Morrison and Smith25in the presence of an internal standard tricosanoic acid (23:O). They were separated using Hewlett-Packard 5890 gas chromatograph equipped with a J&W DB-FFAP capillary column (30 m X 0.25 mm id., with a 0.25 pm film thickness) using hydrogen gas as the carrier gas (55 cm/s). The oven temperature was programmed from 130 to 175 "C at 4 "C/ min and then raised to 240 "C at 30 "C/min. The injector and detector temperatures were set at 240 0C.26 Incorporation of Docosahexaenoic Acid (22:6n3) into C-6 Glioma Cells. Rat glial tumor C-6 cells (American Type Culture Collection, Rockville, MD) were cultured in the medium (DMEM) containing 10% fetal bovine serum (Gibco, Grand Island, NY), penicillin (100 U/mL), and streptomycin (100 pg/mL). Cells were grown at 37 "C in a humidified atmosphere of air containing 5% C02 and were subcultured
(24) Nelson, G. J. In Blood lipids and lipoproteins: quantitation, composition and metabolism; Nelson, G. J., Ed.; Wiley-Interscience: New York, 1972.
(25) Morrison, W. R.; Smith, L. M. J . Lipid Res. 1961, 5, 600-608. (26) Kim, H. Y.; Salem, N., Jr. J . Lipid Res. 1990, 31, 2285-2289.
3970
Analytical Chemistty, Vol. 66, No. 22, November 15, 1994
Abundance (M+Na)+ 20000.
18:0, 20:4-PI (mw=886)
50000
45000
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8
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m/z
m/z
Flgure 1. Positive ion electrospray mass spectra of phosphatidylinositol(PI), phosphatldyicholine(PC), phosphatidylserine (PS), and sphingomyelin (SM). Approximately 40-80 pmol (400-800 fmol after 1/100 split) of standards was introduced directly into the mass spectrometer by the flow injection technique at the flow rate of 0.5 mL/min using - the mixture of methanol/hexane/water (96:Q:l).The capillary exit voltage was set at 200 v.
every 4-5 days. For modification of the glial cell fatty acyl composition, cells were subcultured in the medium containing 100 pM 22:6n3. After 24 and 48 h, cells were harvested and extracted according to the procedure by Bligh and Dyer.*' The lipid extracts were dried under the nitrogen flow, redissolved in chloroform, and injected directly into an HPLC column for electrospray MS analysis. Aliquots of the lipid extract were transmethylated in the presence of 23:O as an internal standard for GC analysis.
RESULTS AND DISCUSSION Electrospray Mass Spectra of Phospholipids. Figure 1 shows the typical spectra obtained from various phospholipids using the electrospray technique in the positive ion mode. Protonated and natriated molecules were detected as the major peaks for 18:0,20:4-phosphatidylinosotol(PI), 18:0,20:4- and 18:0,22:6-phosphatidylserine(PS), 18:0,22:6-phosphatidylcholine (PC), and 18:O-sphingomyelin (SM). Phosphatidylethanolamine (data not shown) also produced (M + H)+ as the major ion. Formation of sodium adducts was most prominent with acidic phospholipids. For neutral phospholipids, PC, PE, and SM, protonated molecular species were the major ions observed in the spectra. The spectra were obtained with capillary exit voltage set at 200 V where the intensities of molecular ions were near maximum. At this voltage, PI showed the substantial fragmentation as indicated by the appearance of the diglyceride ion at m f z 627. The diglyceride ion resulted from loss of the polar head group. The capillary exit voltage not only affected the intensity of the molecular ion but also induced diacylglyceryl fragments (27) Bligh, E. G.;Dyer, W. J. Can. J. Biochem. Physiol. 1959, 37, 911-917.
o.ooe+o
A 100
200
300
DO ( m i l 651) MHt(rrVz792)
400
Capillary Exit Potential (V)
Flgure 2. Effect of capillary exit vottage on digiyceride ion production. Approximately 125 pmol of 18:0,22:6-PE was injected by the flow injection technique as in Figure 1.
as is shown for 18:0,22:6-PE in Figure 2. At around 200 V, MH+ intensity was maximum. As the capillary exit voltage increased from 200 to 300 V, intensity of the diglyceride fragment increased and peaked at around 300 V. Similar results were observed for PI, PA, and PS, although the extent of fragmentation was higher for these species than for PE. Unlike these phospholipids, phosphatidylcholines did not fragment to diglyceride fragments even at the maximum capillary exit voltage of 399 V (Figure 3). Instead, the phosphocholine fragment was detected at m f z 184. A slight difference in fragmentation extent was observed between monoene and polyunsaturated species within a given class, but much larger differences were observed between various classes of phospholipids. The ease of fragmentation was in the order of phosphatidylinositiol, phosphatidic acid, and PS followed by PE. AnalyticalChemistry, Vol. 66, No. 22, November 15, 1994
3979
a Abundance 18:0, 22:6-PE (mw=791) 18:0, 20:4-PE (mw=767) 4000 3500 3000
DG
(M+H)+
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-
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200
300
I
I
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I I
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m/z Flgure 3. Positive ion electrospray spectra of 18:0,22:6- and 18:0,20:4-PE (a) and 18:0,22:6-PC (b) obtained with the capillaryexit vottage set at 300 and 399 V, respectively. Abundance m/Z 7 8 8
j
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a
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100
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18:0,18:l-PC (pmoles) Flgure 5. Response curve constructed for 18:0,18:1-PC.
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Figure 4, Ion chromatograms obtained from 1.8 pmol of 18:0,18: 1-Pc (a) and 1.35 pmol of 18:0,20:4-~c(b). Samples were introduced by flow injection as described in Figure 1, and molecular ions were selectively monitored with the dwell time set at 500 ms.
Flow Injection Analysis of Phospholipids. When phospholipids were injected by flow injection using a methanol/ 3980
Analytical Chemistty, Vol. 66,No. 22, November 15, 1994
ammonium acetate or methanol/water/hexane mixture as the carrier solvent system at 0.5 mL/min with 1/100 split, reproducible responses were observed without significant peak broadening. Shown in Figure 4 are selected ion monitoring traces obtained by injecting 1.8 pmol of 18:0,18:1-PC and 1.35 pmol of 18:0,20:4-PC using the mixture of methanol/ hexane/water (96:3: 1) without column separation. Under the conditions employed, the peak width at the half height was less than 10 s. The response appeared to be slightly dependent on molecular species. For example, 18:0,18:1-PC was about 1.1 times more sensitive than 18:0,20:4-PC, and 18:0,20:4-PC has a similar sensitivity as 18:0,22:6-PC. It appeared that the nature of the phosphohead group affected the sensitivity much more than the degree of unsaturation. Phosphatidylcholine exibits the greatest molar responsivity followed by phosphatidylethanolamine. For all 18:0,18:1,18: 0,20:4, and 18:0,22:6 species, the sensitivity of PE and PS by monitoring MH+ was and 2o times less than phsophatidylcholine, respectively. Under the present conditions, the detection limit for p c is in the 0.5-1 pmol (5-10 fmol after 1/ 100 split) range. As shown for 18:0,18:1-PC in Figure 5, the response was linear in the 4-200 pmol range; above 200 pmol, the increase in the response according to the sample size decreased. In the presenceof a deuterium-labeled
18:O, 22:6-PE (M+H)+
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300
400
500
Figure 6. Response curve of 18:0,18:1-PC obtained in the presence of 45 pmol of 18:0435,18:1-PC as an Internal standard.
looooo
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(M+Na)+ 18:0, 22:6-PS
internal standard, the linear response range was extended to
858
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81 2
Fbure 7. Ion chromatogramsobtained from standards(83pmol each) by selectively monitoring molecular Ions with the 200 ms dwell time. The capille,,,exitvo~w was set at 200 The separation was achieved using C-18 column (5 wm, 2.1 mm X 15 cml and the mobile phase consisting 0.5 o/o ammonium hydroxide in water/methanol/hexane changing from 12:88:0 to 0:88: 12 in 17 mln after holding at the initial solvent composition for 3 min. i / i O O of the HPLC flow 10.5 mL/min) was introduced to the mass spectrometer.
H p L C / m ifPbo&oKpids. In conjunction with reversedphase HPLC, separation and detection of various molecular species wereachievedusing0.5 mL/minand 1/100split. Using 0.5% ammonium hydroxide in the water/methanol/hexane gradient changing linearly from 12:88:0 to 0:88:12 in 17 min after holding at the initial composition for 3 min, theseparation of various phospholipids was achieved as shown in the Figure 7. Under this condition, PS eluted first followed by PI, phosphatidic acid (PA), PE, and PC in a successive manner for phospholipids containing a given fatty acyl composition. The chromatographic peak width at the half height was generally in the 18-20 s range. For PS, sodium adducts were monitored instead of protonated molecules. Protonated molecule also appeared as is shown for 18:0,20:4-PS in the channel of 18:0,18:1-PS ( m / z 812). Similarly, in the 18:0, 20:4 ion channels, sodium adducts of the 18:0,18:1 species were also detected. In the ion channel monitoring m / z 834, 18:0,20:4-PSas (M +Na)+, 18:0,18:1-PSas(M-H+ 2Na)+, and 18:0,22:6-PC were detected. The chromatogram obtained from 83 pmol of each phospholipid molecular species in this figure indicated that the sensitivity was more dependent on the type of phospho head group than the fatty acyl composition within a class. In general, monitoring molecular ions by electrospray technique is approximately 20-50 times more sensitive than the existing methodology using thermospray LC/MS, which generates diglyceride fragments as the major ions.12J7 Incorporation of 22:6n3 into C-6 Glioma Cells. This technique was applied to monitor the incorporation of 22:6n3 into phospholipids and subsequent remodeling processes. Docosahexaenoic acid (22:6n3) is highly enriched in neuronal membranes and has been suggested to be essential for proper neuronal functions.** In order to understand its enrichment
processes, incorporation of 22:6n3 into the C-6 glioma cells, a cell line of neural origin, was investigated as a model system. Without supplementation, glioma cells contain very low level (2-3% of total lipid) 22:6n3.29 After supplementation with 100 pM 22:6n3, its composition was rapidly elevated to 15% in 24 h and was slightly increased further in the next 24 h. As mentioned above, with high capillary exit voltage, fragmentation can be induced for PS, PI, PA, and PE. Therefore, in order to minimize the number of ions monitored, diglyceryl fragments were monitored for 22:6n3-containing phospholipid species except for PC. For PC, protonated molecular species were monitored selectively. Using these conditions, the intensity ratio of diglyceryl fragments of PS and PE and the protonated PC molecule containing a given fatty acyl composition was 1:3:15. Upon comparison of this ratio to the intensity ratio obtained from the protonated molecules (1 :4:20), it was obvious that phosphatidylserine fragmented better than PE. Phosphatidylserine and phosphatidic acid fragmented to a similar extent. After 24 h of incubation with 100 pM 22:6n3, incorporation of 22:6n3 into PA, PI, PS, and PC was observed (Figure 8a). Incorporation into PA was most prominent when considering the response factors associated to each phospholipid class. In comparison to PC and PE, PS and PI also incorporated 22:6 readily after 24 h of incubation. For these classes, incorporation was
(28) Sa1em.N. Jr. InNewprotectivero/es/orse/ectivenutrients;Spiller,G.,Scala, J., Eds.; Alan R Liss: New York, 1989; pp 109-228.
(29) Thomas, S. E.; Byers, D.M.; Palmer, F.B. St. C.; Spcng, M. W.; Cook, H. W. Biochim. Biophys. Acta 1990, 1044, 349-356.
Ana&timIChemistry, Vol. 66, No. 22, November 15, 1994
3981
m/z
a Abundance
24 Hours PI
I PE
1S:Q, 22:6
1 PE
18:0, 22:6
65 1
16:0, 22:6
623
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Time (min)
Time (min)
Flgure 8. Ion chromatograms of 22:6n3 containing phospholipids obtained after incubatlon of C-6 glioma cells with 100 pM 22:6n3 for 24 (a) and 48 h (b). The HPLC condition was the same as in Figure 6. The capillary exit voltage was set at 300 V. Sometimes the random base line drift was observed as shown in the chromatogram of the 24-h sample.
primarily into 18:O-containing species while PC was mainly incorporated into 16:O-containing species. Figure 8b was obtained from the lipid extract obtained after 48 h of incubation. The injected total lipid amount was comparable to that of the 24-h lipid extract. In comparison to the case with 24-h incubation, incorporation of 22:6n3 into both PI and PA decreased after 48 h of incubation. Instead, PE increased considerably and accumulation of PE-plasmalogens (1-0-alkenyl, 2-acyl phosphatidylethanolamine) was noticed. Decrease in 16:0,22:6-PC was also observed. PS did not show significant change. From these results, it was apparent that 22:6n3 is taken up into PA, PI, and PC first, then remodeled to PE, and then to PE-plasmalogens in C-6 glioma cells. PS accumulated during the first 24 hand did not appear to undergo significant remodeling. As indicated in this example, monitoring the lipid profile at the molecular species level is facilitated using the electrospray mass spectrometry in conjunction with separation byreversed-phase HPLC. Phospholipid remodeling processes in biomembranes can be monitored in one picture without complicated sample preparation procedures. This electrospray phospholipid analysis technique may not offer certain conveniences obtainable by the existing thermospray LC/MS technique; that is, producing common diglyceride ions for all
3982
Analytical Chemistry, Vol. 66, No. 22, November 15, 1994
phospholipid classes with a given fatty acyl composition. However, even after 1/ 100 split, the electrospray technique was still about 50 times more sensitive in comparison to the thermospray technique, based upon the response of PC. The sensitivity of the technique may be further improved with a lower split using capillary HPLC or with the recently developed electrospray interface accommodating higher LC flow. The chromatographic integrity was also well maintained. In addition, linear response was obtained for a wider range, indicating that quantification is much simpler than in the thermospray technique. Therefore, we believe that the phospholipid molecular species analysis technique using reversed-phase HPLC/electrospray mass spectrometry will be extremely valuable in many research areas involving lipid biochemistry. ACKNOWLEDGMENT The authors thank Hewlett Packard for loaning their electrospray instrument. Dr. Sanford Markey in the Mental Health Institute is especially appreciated for his generosity, encouragement, and valuable suggestions. Received for review May 26, 1994. Accepted August 9, 1994." e Abstract
published in Aduance ACS Abstracts. September 15, 1994.
