MASS SPECTRA OF HALOGEATED FATTY ACIDS As with my other documents on mass spectrometry, this is a subjective account that details only those fatty acids relevant to this topic which have been encountered during our research activities here and for which we have spectra available for illustration purposes. Spectra of methyl esters, 3-pyridylcarbinol ( picolinyl ) esters, DMOX derivatives and pyrrolidides are all described here (when available), but I will only describe key diagnostic ions as general features of each type of derivative are described elsewhere on this website. Only a few of the spectra have been published elsewhere, and references to prior publications are cited if known. The fluoro fatty acids were perfectly stable and were easily derivatized. The others can lose the halogen atom readily unless care is taken during derivatization. Fluoro Fatty Acids One natural seed oil contains several fatty acids with a fluorine atom on the terminal carbon, i.e. Dichapetalum toxicarum. We re-investigated this oil using 3-pyridylcarbinol ester and DMOX derivatives and, in addition to obtaining novel mass spectrometric information, we found several hitherto unknown minor fatty acid components (Christie et al., 1998). We also have some mass spectra from several synthetic fluoro-fatty acids kindly donated by Dr David O Hagan of St Andrews University, and details of some of these have been published (Hamilton and Christie, 00). They provided important evidence for some mechanistic concepts, but the aim of these pages is simply to act as a practical guide. Methyl esters give distinctive spectra, but information on more isomers would be required to give definitive interpretations. The mass spectrum of methyl 18-fluoro-octadecanoate follows 74 87 CH 3 OOC CH 2 F 97 111 129 143 199 316 228 242 253 264 285 296 0 1 1 1 1 0 2 2 2 2 0 In the high mass range, there is an ion at = 296, which presumably represents loss of HF, and ions for loss of the methanol moiety from the carboxyl group ( = 285), and for the McLafferty ion ( = 74) can be recognized. Without further information, there is no feature that locates the fluorine atom on its specific position. Further comment would be merely speculation. W.W. Christie lipidlibrary.aocs.org 1
Similarly, in the mass spectrum of methyl 18-fluoro-octadec-9-enoate, there is an ion at = 294, which presumably represents loss of HF, together with the expected ions for the loss of methanol ([M-31/32] +, = 283, and [M-HF-32] +, = 262). CH 3 OOC CH 2 F 69 81 74 96 1 123 137 163 178 198 213 2 262 283 294 314 0 1 1 1 1 0 2 2 2 2 0 Mass spectrum of methyl 2-fluoro-octadecanoate CH 3 OOC F 43 57 5 87 127 147 161 199 213 227 185 241 0 1 1 1 1 0 2 2 2 2 0 2 269 316 The base peak is the McLafferty ion at =, which confirms that the fluorine atom is on position 2. Abundant ions at = 147 and 161 indicate preferential cleavages between C-6 and C-7, and C-7 and C-8, of each of the derivatives. Furthermore, losses of 47 ( = 269) and 61 ( = 2) amu from the molecular ion indicate the expulsion of fluorinated ethyl and propyl radicals, while other losses of 75, 89, 3 and 117 amu represent the expulsion of 4-, 5-, 6- and 7- carbon fragments that must contain a CHF group together with an additional hydrogen atom. 3-Pyridylcarbinol esters: As expected, these give much more informative mass spectra, locating the fluorine atom and other structural features. W.W. Christie lipidlibrary.aocs.org 2
The mass spectrum of 3-pyridylcarbinyl 18-fluoro-octadecanoate is - After the molecular ion, there are gaps of amu to = 373 (loss of HF) and one of 33 amu to 3 (loss of the terminal -CH 2 F). Thereafter, there are regular gaps of 14 amu for loss of successive methylene groups. Mass spectrum of 3-pyridylcarbinyl 18-fluoro-oleate - 8 288 2 CH 2 F 358 2 274 391 69 133 1 2 316 3 344 371 358 0 1 1 1 1 0 2 2 2 2 0 3 3 3 This is very similar to the spectrum of 3-pyridylcarbinyl oleate, and the position of the double bond is determined as described in the section of this website on 3-pyridylcarbinol esters of monoenes. However, in the high mass range, there are again diagnostic gaps of amu to = 371 (loss of HF just discernable) and one of 33 amu to 358 (loss of the terminal -CH 2 F). W.W. Christie lipidlibrary.aocs.org 3
Mass spectrum of 3-pyridylcarbinyl 18-fluoro-linoleate - 2 0 CH 2 F 8 274 67 389 121 1 2 2 274 314 328 0 342 369 0 1 1 1 1 0 2 2 2 2 0 3 3 3 As anticipated, this is similar to the spectrum of 3-pyridylcarbinyl linoleate, and the position of the double bonds are determined as described in the section of this website on 3-pyridylcarbinol esters of dienes. However, in the high mass range, there is again a diagnostic gap of amu to = 369 (loss of HF). The expected gap of 33 amu to 356 (loss of the terminal -CH 2 F is not clearly seen, but successive gaps of 14 amu are apparent so the fluorine atom must be on the terminal carbon. The mass spectrum of synthetic 3-pyridylcarbinyl 2-fluoro-octadecanoate 8 169 F 169 393 224 238 294 266 2 336 322 3 364 57 65 332 196 378 134 0 1 1 1 1 0 2 2 2 2 0 3 3 3 3 The McLafferty ion is shifted from = to 169 as expected and indeed all ions from here upwards are increased by 18 amu in comparison to those in the spectrum of 3-pyridylcarbinyl stearate. DMOX derivatives of fatty acids with terminal fluorine atoms are much less useful (this might not be true of more centrally-placed fluorine atoms). W.W. Christie lipidlibrary.aocs.org 4
As an example, the mass spectra of the DMOX derivatives of 18-fluoro-octadecanoate and of 18-fluoro-oleate 83 113 126 O CH 2 F 1 168 196 224 252 2 294 312 3 326 3 0 1 1 1 1 0 2 2 2 2 0 3 3 113 126 O 8 196 CH 2F 72 81 1 236 2 222 168 8 196 264 296 3 324 338 353 0 1 1 1 1 0 2 2 2 2 0 3 3 For the first of these from the molecular ion, there is a gap of 15 amu to = 3, representing loss of a methyl group, not of -CH 2 F. We now know that the explanation is a facile loss of a methyl group from the ring structure that confounds the picture (see Hamilton and Christie, 00). Similarly for 18-fluoro-oleate, the pattern is repeated; a loss of 15 amu from the molecular ion to = 338 confuses the picture at the terminal carbon. The position of the double bond is determined as described in the section of this website on DMOX derivatives of monoenes. Mass spectrum of the DMOX derivative of synthetic 2-fluoro-octadecanoate 131 O 144 F 131 72 87 113 144 158 186 0 0 1 1 1 1 0 2 2 2 2 0 3 3 214 242 256 2 294 8 326 3 3 W.W. Christie lipidlibrary.aocs.org 5
The McLafferty ion at = 131 has been shifted upwards by 18 amu (from 113) as expected, and most other ions are 18 amu higher in comparison to the non-fluorinated analogue. Pyrrolidine derivatives of fatty acids with terminal fluorine atoms are best compared with DMOX derivatives, and the mass spectrum of the pyrrolidine derivative of 18-fluoro-linoleate is 113 8 248 OC CH 2 F 196 236 318 126 72 79 1 168 222 196 236 331 351 8 248 276 2 318 0 1 1 1 1 0 2 2 2 2 0 3 3 Although the ions in the high mass range are less abundant than in the spectrum of the DMOX derivative (at least relative to the base peak), the diagnostic ions for the double bonds are clearly delineated (in the same place as with DMOX derivatives). Following the molecular ion, there is a gap of amu to = 331 (loss of HF) and of 33 amu to = 318 (very small) for loss of CH 2 F. There is no confusing ion at [M-15] + as with the DMOX derivative, because there are no methyl groups on the ring. Chloro Fatty Acids We only have spectra available of 3-pyridylcarbinol esters of a few omega-chloro fatty acids, produced by microbial fermentation of chlorohydrocarbons. Other chlorinated fatty acids have been found in marine samples, but we do not have access to these. Our single attempt to prepare DMOX derivatives by the usual method of heating the free acid with 2-amino-2-methyl-1-propanol at 1ºC caused elimination of the chlorine atom. The 3-pyridylcarbinol esters were prepared by hydrolyzing to the free acid, and proceeding via the imidazolide route (see the section on Preparation of derivatives for mass spectrometry ). The mass spectrum of 3-pyridylcarbinyl 16-chloro-hexadecanoate - CH 2 Cl 8 346 65 2 248 262 2 4 133 1 332 0 1 1 1 1 0 2 2 2 2 0 3 3 3 381 W.W. Christie lipidlibrary.aocs.org 6
Following the molecular ion at = 381, there is a gap of 35 amu to = 346 for loss of the terminal chlorine atom. Thereafter, there is a regular series of ions 14 amu apart for cleavage at successive methylene groups. Mass spectrum of 3-pyridylcarbinyl 16-chloro-hexadec-9-enoate - 2 CH 2 Cl 67 8 133 178 62 0 1 1 1 1 0 2 2 2 2 0 3 3 2 288 274 2 316 344 379 Again, there is a gap of 35 amu to = 344 for loss of the terminal chlorine atom. Thereafter, there is at first a regular series of ions 14 amu apart for cleavage at successive methylene groups. The position of the double bond is determined as described in the section of this website on 3- pyridylcarbinol esters of monoenes. Bromo Fatty Acids Our first attempts to prepare derivatives of bromo fatty acids (produced by microbial fermentation of bromohydrocarbons) failed when our transesterification and hydrolysis procedures caused elimination of bromine with formation of ethoxyl or methoxy fatty acids (see the section of this website on Mass spectra of of epoxy, furanoid and alkoxy fatty acids. ). However, we obtained some synthetic 2-bromo-octadecanoic acid and successfully derivatized this as described below. Other brominated fatty acids have been found in marine samples, but we do not have access to these. Methyl 2-bromo-octadecanoate was prepared by reacting the free acid with trimethylsilyldiazomethane. Its mass spectrum is - 87 CH 3 OOC Br 269 152/4 97 113 143 185 194 227 277 348 3 0 1 1 1 1 0 2 2 2 2 0 3 3 W.W. Christie lipidlibrary.aocs.org 7
The spectrum is complex, but the expected double molecular ion at = 348 and 3 can be seen (bromine exists as two isotopes in approximately equal amounts of atomic mass 79 and 81). However, the key diagnostic feature is the fact that the McLafferty ion forms a doublet of ions at = 152 and 154, instead of at 74. The ion at = 269 is for the loss of the bromine atom. 3-Pyridylcarbinyl 2-bromo-octadecanoate was prepared from the free acid via the imidazolide route. Its mass spectrum, which follows, is surprisingly unhelpful. 242/244 Br 346 65 8 2 178 6 242 244 276 2 328 3 396 425/7 0 1 0 2 0 3 0 The small molecular ion is a doublet ( = 425/7), but the base peak at = 346 reflects loss of a bromine atom. The doublet of ions at = 242/244 presumably results from cleavage between carbons 3 and 4, and there are a few small ions in the higher mass region that are doublets so presumably contain bromine. DMOX derivative of 2-bromo-octadecanoate. This was prepared successfully by our relatively mild two-step method (see the section of this website on Preparation of derivatives for mass spectrometry ). Its spectrum follows. O 191/3 Br 8 72 96 113 126 152 191/3 4/6 246/8 2/2 2 0 1 1 1 1 0 2 2 2 2 0 3 3 3 346 372/4 387/9 The molecular ion is a small doublet at = 387/9 and there is also a small doublet at = 372/4 for loss of a methyl group (probably from the ring system). Again the key diagnostic feature that locates the bromine on carbon 2 is the fact that the McLafferty ion forms a doublet at = 191/3. The base peak at = 8 reflects loss of bromine from the molecule. Further mass spectra of halogenated fatty acids in the form of the various derivatives are available, but without interpretation, in the Archive Sections of these web pages. W.W. Christie lipidlibrary.aocs.org 8
References o o Christie, W.W., Hamilton, J.T.G. and Harper, D.B. Mass spectrometry of fluorinated fatty acids in the seed oil of Dichapetalum toxicarium. Chem. Phys. Lipids, 97, 41-47 (1998). Hamilton, J.T.G. and Christie, W.W. Mechanisms for ion formation during the electron impact-mass spectrometry of picolinyl ester and 4,4-dimethyloxazoline derivatives of fatty acids. Chem. Phys. Lipids, 5, 93-4 (00). William W. Christie James Hutton Institute (and Mylnefield Lipid Analysis), Invergowrie, Dundee (DD2 5DA), Scotland Last updated: April 5 th, 12 W.W. Christie lipidlibrary.aocs.org 9