MASS SPECTRA OF FATTY ACID PYRROLIDIDES Part 1. Intrductin, Saturated and Branched-Chain Acids Intrductin t Pyrrlidide Derivatives Fatty acid pyrrlidine derivatives r a O pyrrlidides r acyl-pyrrlidines were the first C f the nitrgen-cntaining derivatives t be a recgnized as ffering new pssibilities fr the structural analysis f fatty acids by mass spectrmetry. Indeed, they may have been described t sn t be f immediate practical value, as the packed GC clumns then available were nt stable enugh t high temperatures and affrded insufficient reslutin t handle fatty acid pyrrlidides satisfactrily. Fllwing their discvery (Vetter et al., 1971), there was a series f papers frm Andersn and Hlman describing results with mdel cmpunds, fllwed by many applicatins frm thers t natural samples (see ur web page with a literature survey f the tpic). When 3-pyridylcarbinl ( piclinyl ) ester and dimethylxazline (DMOX) derivatives were described a decade later, capillary clumns f fused silica with thermally stable phases and high reslutin tgether with a new generatin f simple bench-tp mass spectrmeters had just becme available. The new derivatives caught the eye f analysts, and pyrrlidides were relatively neglected, perhaps unjustly. netheless, they have been used extensively by a dedicated band f analysts, especially thse interested in marine lipids. Hwever, althugh a great deal f tabulated mass spectral data has been published in the literature, relatively few spectra have been represented pictrially. I have used the ld adage that a picture is wrth a thusand wrds elsewhere in these web pages and this philsphy cntinues here. Thugh it is nw smewhat ut f date, a useful review f mass spectrmetry f pyrrlidides has been published by Anderssn (1978). Cmparisn with DMOX derivatives. By an interesting and useful cincidence, the pyrrlidide f a given fatty acid has exactly the same mlecular weight as the crrespnding DMOX derivative, in spite f the very different structures. As with DMOX derivatives, the base peak is usually the McLafferty in at m/z =, with the in at m/z = als being very abundant. At least fr the cmmn range f fatty acids, the fragmentatin mechanisms fr acyl-pyrrlidines are essentially the same as fr DMOX derivatives and indeed were wrked ur first fr the frmer. In analysing mass spectra f pyrrlidines, we are ften lking fr exactly the same diagnstic ins as fr the crrespnding DMOX derivatives. One imprtant ptential advantage is the fact that pyrrlidides lack the methyl grups attached t the ring that can hinder the interpretatin f spectra frm DMOX derivatives (Hamiltn and Christie, 00). My present subjective impressin is that DMOX derivatives may be better than pyrrlidides fr lcating duble bnds. Pyrrlidides are certainly better when functinal grups, e.g. methyl branches r ring structures, are near the terminal end f the mlecule. They are als better fr sensitive fatty acids such as epxides. W.W. Christie lipidlibrary.acs.rg 1
Pyrrlidide derivatives are prepared under much milder cnditins than DMOX derivatives, directly frm methyl esters, and are mre stable chemically (see ur web-pages n Preparatin f derivatives fr mass spectrmetry ). Ins in the high mass range that are required fr diagnstic purpses tend t be f lw abundance in the mass spectra f pyrrlidides (and with DMOX derivatives smetimes), but it shuld be remembered that this is nly a relative feature. The cnventin in mass spectrmetry is t set the mst abundant in (base peak) at 0, with thers adjusted in relatin t this. Is the base peak at an especially high abundance in the mass spectra f pyrrlidides, r are the remaining peaks at lw abundance? It is nt difficult with mdern mass spectrmetry sftware t magnify minr ins, and prvided that this is clearly indicated, there shuld be n bjectin. Gas Chrmatgraphy. Pyrrlidides elute frm GC clumns at smewhat higher temperatures than DMOX derivatives, but this is n lnger a disadvantage, because f the wider availability f plar statinary phases that have greater thermal stability. Indeed, the authr has bserved that sme astnishingly gd separatins f ismeric pyrrlidides. Fr example, pyrrlidides f 5-, 7-, 9-, 11- and 13-18:1 fatty acids were all reslved t the baseline n a 25 m clumn f Supelcwax TM, with the 5-ismer actually eluting befre the 18:0 derivative (Christie, 02), as illustrated in the figure belw. Detectr respnse (TIC) 16:0 9-16:1 11-16:1 im-16:0 aim-16:0 4-17:1 17:0 8-17:1 5-18:1 7-18:1 9-18:1 11-18:1 13-18:1 18:0 9,12-18:2 34 36 38 42 44 Time (min.) The pyrrlidine miety can change the selectivity f the separatin appreciably, and I have bserved reversals in the rder f elutin f certain plyunsaturated cmpnents in the frm f the pyrrlidides as cmpared t the methyl esters. A cmprehensive study f the gas chrmatgraphic prperties f pyrrlidides might prve illuminating. Summary. Pyrrlides are very similar in their mass spectrmetric prperties t DMOX derivatives f fatty acids with advantages in sme circumstances and disadvantages in thers. They are a useful alternative t DMOX derivatives and cmplement 3-pyridylcarbinl esters in studies f fatty acid structure. As with ther sectins n this website, the fllwing accunt is a subjective ne that nly uses thse mass spectra that were available frm my persnal researches. In this sectin, spectra f the cmmn range f fatty acids are described. Spectra f mre unusual fatty acids are described (tgether with ther derivatives in the Miscellaneus sectin f these dcuments. Mechanistic studies f mass spectra f pyrrlidides f nrmal saturated fatty acids (labelled with stable istpes) have been published by Anderssn et al. (1975, 1982). W.W. Christie lipidlibrary.acs.rg 2
Straight-Chain Saturated Fatty Acids The mass spectrum f palmityl pyrrlidine (16:0) is illustrated first - x 4 9 98 1 168 196 238 2 0 1 1 1 1 0 2 2 2 2 0 m/z As mentined earlier, the base peak is the McLafferty rearrangement in at m/z =, a 2 while that at m/z = is als relatively O abundant (as with DMOX derivatives). It was necessary t magnify the ins in the high mass regin at least fur fld t C visualize the significant ins sufficiently. 238 Fllwing the mlecular in at m/z = 9 (dd numbered as with all nitrgencntaining a derivatives), the ins in the high mass range are unifrmly 14 amu apart (and even numbered) fr lss f successive methylene grups, i.e. at m/z =, 2,,, 238, etc. As a further example, the mass spectrum f the pyrrlidide f tetracsanate (24:0) is - 71 98 1 x 168 196 2 322 3 364 378 6 421 0 1 0 2 0 3 0 m/z - in which all the significant ins in the high mass range are 14 amu apart as expected, althugh it was nw necessary t magnify the ins in the higher mass regin ten-fld t see them clearly. W.W. Christie lipidlibrary.acs.rg 3
The mass spectrum f the pyrrlidide f hexanate (6:0) - 85 98 1 169 0 1 1 1 1 1 1 1 m/z Pyrrlidides seem t be especially useful fr shrter-chain fatty acids, because f the cnvenience f the methd f preparatin. We have mass spectra n file fr pyrrlidides f many mre saturated fatty acids, including sme labelled with stable istpes, and these can be fund in the Archive sectin f ur web pages (withut interpretatin). Mnmethyl-Branched Fatty Acids The mass spectra f pyrrlidide derivatives f fatty acids with anteis- and is-methyl branches, in cntrast t thse f DMOX derivatives, are distinctive and can be used fr characterizatin purpses (see ur web pages n Mass spectrmetry f DMOX derivatives. Part 1. Saturated and branched fatty acids ). Few if any f the fllwing spectra have been published frmally elsewhere. The first paper t be published n the tpic was by Anderssn and Hlman (1975), wh demnstrated the ease f distinguishing is and anteis-ismers in particular. T illustrate the effect f the psitin f the branch pints n fragmentatin, it has been necessary t chse examples f fatty acids f differing chain-length, because f the limited range f material available t us frm natural surces. The mass spectrum f the pyrrlidide f 13-methyl-tetradecanate (is ismer) 2 98 1 168 196 2 2 295 0 1 1 1 1 0 2 2 2 2 m/z The methyl branch is easily lcated by the gap f 28 amu between m/z = and 2 fr lss f carbn 13 with its methyl grup. In this and mst f the fllwing spectra, all the ins in the higher mass range have been magnified five fld t simplify the interpretatin. W.W. Christie lipidlibrary.acs.rg 4
Similarly, in the mass spectrum f the pyrrlidide f 12-methyl-tetradecanate (anteis ismer), the gap f 28 amu is shifted dwnwards as expected t between m/z = 238 and fr lss f carbn 12 and its methyl grup. 238 98 295 1 168 238 2 196 2 0 1 1 1 1 0 2 2 2 2 m/z The mass spectrum f the pyrrlidide f 17-methyl-tetracsanate the diagnstic gap f 28 amu is between m/z = 8 and 336 fr lss f carbn 17 and its methyl grup. 71 98 1 168 238 2 0 1 0 2 0 3 0 8 8 336 336 435 3 364 392 4 m/z The mass spectrum f the pyrrlidide f 14-methyl-heptadecanate - nw the diagnstic gap f 28 amu is shifted t between the ins at m/z = and. 85 98 1 168 2 238 8 337 0 1 1 1 1 0 2 2 2 2 0 3 m/z W.W. Christie lipidlibrary.acs.rg 5
The mass spectrum f the pyrrlidide f 11-methyl-ctadecanate 71 85 1 168 196 2 8 322 351 0 1 1 1 1 0 2 2 2 2 0 3 3 m/z - the diagnstic gap f 28 amu is shifted t between the ins at m/z = and. The mass spectrum f the pyrrlidide f 9-methyl-tetradecanate 196 196 295 168 98 1 238 1 2 0 1 1 1 1 0 2 2 2 2 m/z - as expected the diagnstic gap f 28 amu is between the ins at m/z = 196 and. The mass spectrum f the pyrrlidide f 3-methyl-tetradecanate 1 1 71 85 98 168 2 295 196 0 1 1 1 1 0 2 2 2 2 m/z W.W. Christie lipidlibrary.acs.rg 6
The gap f 27/28 amu fr carbn-3 and its assciated methyl grup is between m/z = and 1, and the usual in at m/z = can nly be seen because f the cnsiderable magnificatin. We have mass spectra n file fr pyrrlidides f many mre branched-chain fatty acids, and these can be fund in the Archive sectin f ur web pages (withut interpretatin). Multi-Branched Fatty Acids An unusual dimethyl-branched fatty acid, 8,-dimethyl-hexadecanate, has been fund in a spnge and the mass spectrum f its pyrrlidine derivative is 71 85 98 1 168 2 8 0 1 1 1 1 0 2 2 2 2 0 3 2 2 337 m/z The methyl grups are lcated by the tw gaps f 28 amu as indicated. The mst cmmn multi-methyl-branched fatty acids encuntered in nature are the isprenid cmpunds derived frm phytl, such as 4,8,12-trimethyltridecanic acid, which is ften encuntered as a minr cmpnent f marine ils, and the mass spectrum f its pyrrlidide is illustrated next. Again, each f the methyl grups is lcated by the gaps f 28 amu as indicated (the last ne is nt clear in this instance). 196 85 98 196 9 238 0 1 1 1 1 0 2 2 2 2 m/z W.W. Christie lipidlibrary.acs.rg 7
Finally, the best knwn f such acids is prbably phytanic (3,7,11,15-tetramethyl-hexadecanic) acid, and the mass spectrum f its pyrrlidide is 1 2 2 3 322 1 98 71 2 3 85 168 365 238 2 322 0 1 1 1 1 0 2 2 2 2 0 3 3 m/z The gaps f 27/28 amu between m/z = and 1, and 2, and 2, and 322 and 3 lcate the methyl branches in psitins 3, 7, 11 and 15, respectively. References Anderssn, B.A. Mass spectrmetry f fatty acid pyrrlidides. Prg. Chem. Fats ther Lipids, 16, 279-8 (1978). Anderssn, B.A. and Hlman, R.T. Mass spectrmetric lcalizatin f methyl branching in fatty acids using acylpyrrlidines. Lipids,, 716-718 (1975). Anderssn, B.A., Dinger, F. and Dinh-guyen,. Mass spectrmetry studies f stable istpe labelled carbxylic acid derivatives. I. rmal chain saturated deuterium and carbn-13 cmpunds. Chem. Scripta, 8, 0-3 (1975). Anderssn, B.A., Dinger, F. and Dinh-guyen, D. Mass spectrmetry studies f stable istpe-labelled carbxylic acid derivatives. IV. Pyrrlidides f vicinal and geminal dideuterated n-ctadecanic acids and the mega trideuterated analgue. Chem. Scripta, 19, 118-121 (1982). Christie, W.W. Unusual gas chrmatgraphic prperties f fatty acid pyrrlidides. Eur. J. Lipid Sci. Technl., 4, 69- (02). Hamiltn, J.T.G. and Christie, W.W. Mechanisms fr in frmatin during the electrn impact-mass spectrmetry f piclinyl ester and 4,4-dimethylxazline derivatives f fatty acids. Chem. Phys. Lipids, 5, 93-4 (00). Vetter, W., Walther, W. and Vecchi, M. Pyrrlidides as derivatives fr structural analysis f aliphatic and alicyclic fatty acids by mass spectrmetry. Helv. Chim. Acta, 54, 1599-15 (1971). William W. Christie James Huttn Institute (and Mylnefield Lipid Analysis), Invergwrie, Dundee (DD2 5DA), Sctland Last updated: vember 6 th, 13 W.W. Christie lipidlibrary.acs.rg 8