SPHINGOLIPIDS. of synthetic ceramides. Gas-liquid chromatography-mass spectrometry. GL C-Mass Spectrometry

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1 Gs-liquid chromtogrphy-mss spectrometry of synthetic cermides BENGT SAMUELSSON nd KARN SAMUELSSON Deprtment of Medicl Chemistry, Royl Veterinry College; Deprtment of Neurology, Krolinsk Sjukhuset; nd Lbortory of Mss Spectrometry, Krolinsk nstitutet, Stockholm, Sweden ABSTRACT Two series of cermides with either sphingosine (sphing-4-enine) or sphingnine s bse nd with one of the sturted ftty cids C16, CS, (220, CZZ, (224, CZ6, or oleic cid were nlyed s the 1,3-di-O-trimethylsilyl ether derivtives by gs chromtogrphy-mss spectrometry. The frgments formed on electron impct cn be divided into three min groups, nmely moleculr weight frgments, long-chin bse frgments, nd ftty cid frgments. The m/e vlues of these frgments cn be used to determine unequivoclly the structures of the long-chin bse nd ftty cid of cermide derived from sphingolipid. SUPPLEMENTARY KEY ORDS sphingosine. sphingnine trimethylsilyl ethers SPHNGOLPDS hve been subjected to extensive nlysis with respect to the structures of the LCB (1-7) nd of the ftty cids (7-11) obtined on hydrolysis. However, the structures of the individul moleculr species of cermides resulting from the combintion of vrious ftty cids nd LCB hve not been determined. Cermides cn be derived from sphingomyelins nd glycosphingolipids by enymtic nd chemicl methods (12-14), nd studies in our lbortories hve shown tht cermides with different LCB nd ftty cid compositions cn be nlyed by GLC nd lso by GLC-mss spectrometry (15). These experiments hve been extended in the present study, which is concerned with nlysis of synthetic cermides s TMS ether derivtives by GLC-mss spectrometry. This method hs been found to provide n efficient tool for determining the structures of cermides. Abbrevitions: LCB, long-chin bses; GLC, gs-liquid chromtogrphy; TMS, trimethylsilyl; TLC, thin-lyer chromtogrphy. MATERALS AND METHODS The cermides were prepred with either DL-sphingnine or DL-sphingosine (~~-sphing-knine) obtined from Miles Lbortories, nc., Elkhrt, nd. n both series cermides with the ftty cids 16 : 0, 18 : 0, 18 : 1, 20:0, 22:0, 24:0, 26:0, nd perdeuterted 18:O (the lst-nmed kindly supplied by Professor Einr Stenhgen) were prepred by direct coupling, using crbodiimide to ctivte the crboxylic cid. The procedure will be described elsewhere. The cermides were purified by silicic cid chromtogrphy nd the purityws estblished by TLC nd GLC.2 GL C-Mss Spectrometry The cermides were converted into TMS derivtives by tretment of 200 pg with 100 pl of pyridine, of hexmethyl disilne, nd 10 p1 of trimethyl chlorosilne t room temperture for 20 min (2). An LKB gs chromtogrph-mss spectrometer, model 9000, ws used. The electron energy ws 22.5 ev nd the trp current 120 rmp. The column consisted of coiled glss column (3 mm.d.) with pcking of 1% OV-1 (nonpolr silicone phse) on mesh Gs- Chrom Q (Applied Science Lbortories nc., Stte College, P.). The column ws conditioned t 350 C for 24 hr. The column temperture ws 275 C, nd the flsh heter nd seprtor tempertures were bout 300 C. A gs-liquid chromtogrm of TMS-N-oleoyl sphingnine is shown in Fig. 1. The nomenclture for sphingolipid bses is bsed on recommendtions of the Commission on Biochemicl Nomenclture of UPAC nd UB (see 1967 J. Lipid Res. 8: 523). Ftty cids re denoted by chin length: No. of double bonds. Smuelsson, R., nd K. Smuelsson. To be published. JOURNAL OF LPD RESEARCH VOLUME 10,

2 Minutes FG. 1. Gs-liquid chromtogrphy of 1,3-di-O-trimethylsilyl- N-oleoyl sphingnine. Column: 1% OV-1 on Gs-Chrom Q; column temperture: 275 "C. RESULTS AND DSCUSSON The mss spectrum of the TMS derivtive of N-steroyl sphingosine will first be considered in detil (Fig. 2, see lso Tble 1). The moleculr weight is indicted by ions t m/e 694 (M-l5), m/e 619 (M-90), nd m/e 606 (M-103) formed by elimintion of methyl group, trimethyl silnol, nd the terminl -CHrO-Si(CH,) 3, respectively. Elimintions involving the ftty cid residue produce ions t m/e 426 [M-(6 + l)] nd m/e 336 [M-(b )] by loss of steroyl mide nd, in the ltter cse, lso trimethyl silnol. Loss of steroyl mide plus CHS(CH~)~~ [M-(b e)] gives n ion t m/e 243; clevge between C-2 nd C-3 with chrge retention on the min sphingosine frgment results in n ion t m/e. The sme clevge but with chrge retention on the other prt of the molecule gives rise to n ion ppering t m/e 398 (M-). Another frgmenttion involving loss of the min prt of the sphingosine molecule gives n ion t m/e 471 [M-(-73)], which is tenttively scribed to clevge between C-2 nd C-3 nd trnsfer of the TMS group to the remining frgment. An ion ppering t m/e 247 is lso tenttively interpreted to be formed by this rection nd, in ddition,,%clevge of the steroyl residue with trnsfer of hydrogen from the y- crbon tom, i.e. [M-( )]; the dditionl elimintion of trimethyl silnol gives rise to n ion t m/e 157. n order to obtin experimentl evidence for the frgmenttions proposed bove, we prepred the corresponding derivtive of N-perdeuterosteroyl sphingosine nd subjected it to GLC-mss spectrometry. The mss spectrum is shown in Fig. 3. The frgments (M-15), (M-90), nd (M-103) now ppered t m/e vlues 35 units higher thn those of the corresponding nondeuterted derivtive. This is lso seen for other frgments tht retin the ftty cid residue, i.e. (M-) nd [M-(-73)]. However, frgments formed by elimintion of the ftty cid, [M-(6 + l)], [M-( )], nd [M-(b e)] hve the sme m/e vlues s in the nondeuterted derivtive. Evidence for of the steroyl residue with trnsfer of hydrogen from the y-crbon tom to the crbonyl oxygen in the formtion of frgments [M-( )] nd [M-( )] ws lso obtined. The rection involves retention of three hydrogens originting in the ftty cid; it ws ccordingly found tht the frgments mentioned bove pper t m/e 250 nd 160 insted of 247 nd 157, respectively. t is evident from the results described bove tht the moleculr weight of cermide cn be determined from the frgments M-15, M-90, nd M-103, nd the nture of the LCB from the frgments formed by elimintion of the cylmide, with or without trimethyl silnol, nd by clevge between C-2 nd C-3. n the exmple discussed bove these frgments pper t m/e 426, 336, nd 31 1, respectively. The structure of the ftty cid moiety cn be deduced from the frgments formed by clevge between C-2 nd C-3 with or without trnsfer of TMS group. The resulting ions pper t m/e 471 [M-(-73)] nd m/e 398 (M-), respectively f! FG. 2. Mss spectrum of 1,3-di-O-trimethylsilyl-N-steroyl sphingosine. 42 JOURNAL OF LPD RESEARCH VOLUME 10, 1969

3 Support for the proposed frgmenttions cn lso be found in the mss spectrum of the TMS derivtive of N-oleoyl sphingosine, which is shown in Fig. 4. Compred with the steroyl nlogue, the moleculr weight frgments hve m/e vlues which re two units lower, i.e. m/e 692, 617, nd 604, wheres the LCB frgments re the sme, i.e. m/e 426, 336, nd. n ccord with these dt, the ftty cid frgments hve m/e vlues (396 nd 469) tht re two units lower thn corresponding frgments of the steroyl nlogue. Some of the frgmenttions described bove hve lso been observed by Gver nd Sweeley, who chrcteried N-cetyl derivtives of sphingosine nd sphingnine (1 6). The mss spectrum of the TMS derivtive of N- steroyl sphingnine is shown in Fig. 5. The moleculr weight is gin obtined from the elimintions M-15, Z u f w 100 y D m /, (157+3) 160 (M-d) M-d) (M-(b*1*90)) L lu (M-) 433 (M-(-73)) 469 FCiiTd1 CH3(CH2)12 CHZCH CH FH-CH2OTMS M = 707 A.. TMs0b{&CD2)~ FG. 3. Mss spectrum of 1,3-di-O-trimethylsilyl-N-perdeuterosteoyl sphingosine. CD3 Mw=744 (M-103) (M-90) (M-15) :M-d) / Mw-711 (M-(-73)) 471 FG (M-) 398 (M-lO3) 608 (M-90) (M-15) 621, 696 L - k m/e Mss spectrum of 1,3-di-O-trimethylsilyl-N-steroyl sphingnine. SMuELssoN AND SAMUELSSON GLC-MS of Cermides 43

4 100 w y 75 0 = m 50 w? + 4 E TABLE 1 SOME MPORTANT FRAGMENTS BY MASS SPECTROMETRY mol wt-795 )*/, FOR STRUCTURE DETERMNATON OF CERAMDES Moleculr eight LCB Ftty Acid Sphingnine Sphingosine Frgments Frgments Frgments Derivtives Derivtives M-15 M-90 M-(b 1) M-(b ) M- M-( + 89) M-(b ) M-(b e) M-103 M-d M-(-73) For explntion of portions -d of the molecule, see upper right-hnd corner of Fig. 2; e is CH~(CHZ)Z from sphingnine. mol wt :o mol wt=739 18:O \ (M-d) //260 6g2,? /, /./ ! 2 1, ( 1./ y !212fO 384 1/42 1,/ 309 (M-) D soo m/e FG. 6. Mss spectrum of 1,3-di-O-trimethylsilyl-N-oleoyl sphingnine. 1 FATTY ACD 22: 0 mol wt=71 16:O mol wt.683 om/. 100 */ / f7 32r( 370 2!21270 SPHNGANNE CERAMDES 1 608, , JOURNAL OF LPD RESEARCH VOLUME 10, 1969

5 SPHNGOSNE CERAMDES 26:O mol wt :O mol wt :o mol wt=765 20:o mol wt.737 1CQ% 0 7. FG. 8. M-d) M-(b*l)) lm (M-(b*l+gO)) M-l-73)) ,/ (M-(b+l*e)l :r4, M-90, nd M-103, which give rise to ions ppering t m/e 696, 621, nd 608, respectively, nformtion on the structure of the LCB is provided by M-d (clevge between C-2 nd C-3) ppering t m/e, wheres chrge retention on the other prt of the molecule gives frgment (m/e 398) which cn be used to determine the structure of the ftty cid residue. Other ftty cid frgments re formed by dditionl elimintion of trimethyl silyloxy group [M-( + 89)] or cpture of the TMS group [M-(-73)] nd pper t m/e 309 nd 471, respectively. An ion tht is bsent from the mss spectrum of the sphingosine cermide derivtives but tht is reltively prominent in the sphingnine series ppers t m/e 217. This ion most probbly consists of C-1 to C-3 of the LCB from which cyl mide hs been eliminted. The mss spectrum of the oleoyl nlogue of the sphingnine cermide derivtive is shown in Fig. 6. The moleculr ion t m/e 709 is reltively prominent nd the moleculr weight of the compound cn lso be found from the frgments M-15, M-90, M-103, nd M-( ). The structure of the LCB is obtined from M-d 426 lm-103j 718 \,7:1 (M-901 M-15) y L2 14i0 95 \, #/ iy>58 3:s \, 4;2L!7 \,,/ 1 ~ % L q6 3!7,/ 409 \.,/ 1398 Mss spectrometric dt for TMS derivtives of sphingnine cermides. ppering t m/e nd the sphingnine nture of the LCB from frgments ppering t m/e 217. The ftty cid frgments, Le. M-, M-(-73), nd M-( + 89), pper t m/e 396, 469, nd 307 nd show conclusively tht monounsturted CS cid is present in the cermide. n order to provide dt for the identifiction of cermides of nturl origin nd to fcilitte the interprettion of frgmenttion processes, two series of cermides hve been prepred, one with sphingosine s bse nd the other with sphingnine s bse. n both cses the ftty cid residues re sturted norml cids rnging from CB to Cie. The results hve been summried in Figs. 7 nd 8 nd Tble 1, where the frgments tht re importnt for structure determintion of cermides re summried. t is evident from Fig. 7 tht in the sphingnine derivtives the LCB frgments M-(b + 1) nd M-d pper t the sme m/e vlue in ll members of the series, wheres the ftty cid frgments M-, M-(- 73), nd M-( + 89) re shifted 28 units between neighboring members of the series. This is lso true for SAMUELSSON AND SAMUELSSON GLC-MS of Cermides 45

6 frgments ppering t m/e vlues 451, 468, nd 493 in the homologue nd the moleculr weight frgments M-15, M-90, nd M-103. Three ions tht pper t m/e 217 [M-( c)], 232, nd 260 re the sme for ll of the homologues of the sphingnine series. They re bsent from the sphingosine series nd might be useful for the identifiction of sphingnine derivtives, lthough dditionl studies with LCB of different chin lengths re required before definitive conclusions cn be drwn. The sphingosine derivtives (Fig. 8) show similr groups of frgments. Thus the LCB frgments M-(6 + l), M-( ), nd M-d pper t the sme m/e vlue in ll of the homologues. The ftty cid frgments M- nd M-(-73) nd the moleculr weight frgments pper t m/e vlues tht differ by 28 units between homologues. This is lso seen for the frgments ppering t m/e 451,468, nd 493 in the Cs homologue, nd since the sme ions re present in the sphingosine nd sphingnine series they cn probbly be referred to s ftty cid frgments lthough no structures hve s yet been ssigned. Two ions ppering t m/e 243 nd 258 in ll of the sphingosine homologues re bsent from the sphingnine series nd might be importnt for the identifiction of the former series. An ion t m/e 247 is present in ll of the homologues of both the sphingosine nd sphingnine series. t is evident from the dt presented tht mss spectrometry cn be used to determine the structure of the ftty cid residue nd the LCB in cermides. n combintion with gs chromtogrphy it provides n efficient method for identifiction of moleculr species of sphingoiipids. The uthors re indebted to Dr. R. Ryhge for providing fcilities for mss spectrometry. Mnuscript received 78 June 7968; ccepted 76 August REFERENCES 1. Sweeley, C. C., nd E. A. Mosctelli J. Lipid Res. 1: Gver, R. C., nd C. C. Sweeley J. Amer. Oil Chem. SOL. 42: Krlsson, K. A., nd G. A. L. Holm Act Chem. Scnd. 19: Krlsson, K. A Act Chem. Scnd. 21: Crter, H. E., nd R. C. Gver Biochem. Biophys. Res. Commun. 29: Polito, A. J., T. Akit, nd C. C. Sweeley Biochemistry. 7: Crter, H. E., P. Johnson, nd E. J. eber Annu. Rev. Biochem. 34: 109. Sweeley, C. C J. Lipid Res. 4: 402. Kishimoto, Y., nd N. S. Rdin J. Lipid Res. 4: 437. O Brien, J. S., nd G. Rouser J. Lipid Res. 5: 339. Stllberg-Stenhgen, S., nd L. Svennerholm J. Lipid Res. 6: 146. Sribney, M Biochim. Biophys. Act. 125: Renkonen, J. Amer. OilChem. SOC. 42: 298. Crter, H. E., J. A. Rothfus, nd R. Gigg J. Lipid Res. 2: 228. Smuelsson, B., nd K. Smuelsson Biochim. Biophys. Act. 164: 121. Gver, R. C., nd C. C. Sweeley J. Amer. Chem. SOL. 88: JOURNAL OF LPD RESEARCH VOLUME 10, 1969