Mass spectrometry of some deuterated 1,3-distearins

Chem. Phys. Lipids 4 (1970) 47-59 ~) North-Holland Publ. Co., Amsterdam.

MASS SPECTROMETRY

OF SOME DEUTERATED 1,3-DISTEARINS

A. MORRISON, M. D. BARRATT and R. ANEJA Unilever Research Laboratory Colworth/ Welwyn, The Frythe, Welwyn, Herts., U.K. The mass spectra of 1,3-distearins -dl,-d4 and-d5 with the deuterium incorporated into the glyceryl moiety, have been investigated in order to clarify the electron-impact induced fragmentation of 1,3-distearin itself. High resolution mass spectrometry has been utilised to give accurate masses of fragment ions and consequently molecular formulae. Where applicable, metastable peaks have been correlated with proposed mechanisms. It has been found that the loss of water from the molecular ion is electron-impact induced and that the hydrogen atoms eliminated arise from the hydroxyl group and the stearoyl chains. There is no appreciable scrambling of deuterium between the chains and the glyceryl head. Many of the fragment ions with molecular weights greater than 200 a.m.u. probably have cyclic structures and most retain the glyceryl residue intact. Introduction In spite o f the intense interest in the physical chemistry o f oils a n d fats, glycerides have received little attention f r o m mass spectroscopists. J o h n s o n and H o l m a n 1) have d e m o n s t r a t e d that 1- and 2-monoglycerides can be identified by the mass spectra o f their trimethylsilyl ethers. The same derivatives have been utilised for the identification %3) o f 1,2- and 1,3-diglycerides and isomeric mixed acid 1,2-diglycerides. Barber et al. ~) have examined triglycerides and shown that the fatty acid chains could be identified easily by mass spectrometry. T h e y also distinguished between the positional isomers I-oleo distearin and 2-oleo distearin. The mass spectra o f triglycerides were also recorded a n d discussed by Sprecher et al.5). Ryhage, Stenhagen and c o - w o r k e r s 6, v) used d e u t e r i u m labelling to determine the origins o f m a n y o f the hydrogen a t o m s in the fragment ions o f fatty acid methyl esters. These workers successfully d e u t e r a t e d several positions in the h y d r o c a r b o n chain and by analysis o f the mass spectra o f the resulting deutero c o m p o u n d s were able to show that expulsion o f h y d r o c a r b o n fragments from the chain occurred. They also showed that the base peak, m/e 74, o f long-chain methyl esters was f o r m e d by M c L a f f e r t y r e a r r a n g e m e n t with a hydrogen a t o m extracted from C4. A n a l o g o u s f r a g m e n t a t i o n s would be expected in the mass spectra o f diglycerides. In this p a p e r the e l e c t r o n - i m p a c t induced f r a g m e n t a t i o n o f 1,3-distearin is discussed by c o m p a r i s o n o f its mass spectrum with the spectra o f several 47

48

A. MORRISON, M. D. BARRATT AND R. ANEJA

deuterated analogues, the deuterium being incorporated selectively into the glyceryl residues of the molecules. To facilitate further the interpretation of the spectra, accurate masses of fragment ions were determined by high resolution mass spectrometry.

Experimental The mass spectra were recorded on an AEI MS9 mass spectrometer, using the direct insertion probe with ion chamber temperature 150° above ambient.

i H2OCOR CHOH

I CH2OCOR

Cr03

i H2OCOR

Glacial Acetic Acid

C = 0 I CH2OCOR

R = CI7 H35 Tf

[ aBD4

C2HsOD/NEt3

i

H2OCOR

CDOH

i

I

CH2OCOR

D2OCOR

C=O

I

CD2QCOR 1~7d I -n-T

N a BD4

i

D2OCOR

CHOH

i

~D2OCOR

D2OCOR

CDOH

I

c D2OCOR V

d4

v[ Fig. 1.

d5

MASS SPECTROMETRY OF SOME DEUTERATED ]~3-DISTEARINS

49

The electron voltage was 70 eV and the trap current 100 ~A. High resolution measurements were made by the normal peak-matching procedure. The various deuterated 1,3-distearins were prepared as shown in the scheme (fig. 1). Crystalline 1,3-distearin was oxidised to the ketone (11) using chromium trioxide in glacial acetic acid. Oeuteration of the ketone (II) was achieved by the method of Djerassi et al.8). The ketone (ll) was refluxed in ethanolfree chloroform with ethanol-dr and a trace of triethylamine. The deuterated ketone (Ill) was reduced with sodium borohydride to give 1,3-distearin -d4 (V) and with sodium borodeuteride to give 1,3-distearin-d5 (Vl). 1,3-distearin-d~ (IV) was obtained by reducing the ketone (I|) with sodium borodeuteride.

Materials and methods Chromium trioxide and sodium borohydride were obtained from British Drug Houses Ltd., sodium borodeuteride from Koch-Light Ltd. and ethanol -dj (99~i~) from Fluka A.G., Basle. Microanalysis was carried out by Pascher, Bonn: melting points were determined using a Kofler hot-stage and are uncorrected.

2-Dehydro-l,3-distearin (H) 1,3-distearin (1 g) was added to powdered chromium trioxide (l g) in glacial acetic acid (100 ml). The mixture was stirred at room temperature for four hours, lsopropanol (1 ml) was added to the mixture and stirring was continued until the colour of the mixture changed from olive-green to deep blue-green. The reaction mixture was poured into water (100 ml) and extracted into ether (250 ml). The ether layer was washed several times with water and then with 501~ saturated sodium bicarbonate solution until neutral, dried over magnesium sulphate and evaporated to dryness. Two recrystallisations from petroleum ether (40-6ff ~) gave material, pure by thinlayer chromatography, 0.66 g (66~,, of theory), m.p. 85.5-86.5°9), v max. 1740 cm ~ (KBr disc), N.M.R. spectrum r 5.24 (4H, singlet), - - C H 2 - - C O CH 2 . Analysis found : C,75.0Uo ; H,11.6~o; m.w. 622 (mass spectrometry) C 3 9 H 7 4 0 5 requires' C,75 2°J,/,• H,12.0')~,, m.w. 622.

2-Dehydro- l,3-distearin-d 4 (Ill) The ketone (II) (100 rag) was refluxed with ethanol-free chloroform (20 ml), ethanol-d r (2 ml) and triethylamine (1 drop) for twenty-four hours. The solvents were then evaporated under reduced pressure and the process was repeated. Recrystallisation from petroleum ether (40-60 °) gave the deuterated ketone (80 rag, 80~o of theory) m.p. 84.5-85.5 °. The N.M.R.

50

A. MORRISON. M . D . BARRATT AND R. ANEJA

spectrum exhibited no resonance at z 5.24. The molecular weight determined by mass spectrometry was 626. The deuterium content, also measured by mass spectrometry S), was 87~o of theory calculated for C39H70D405.

1,3-Distearin-d 5 The ketone-d4 ( l i d (100 mg) was added to ethanol-free chloroform (25 ml) with ethanol-dl (5 ml). Excess sodium borodeuteride (30 rag) was added with stirring. The reaction was followed by thin-layer c h r o m a t o g r a p h y until reduction o f the ketone was complete. Thin layer c h r o m a t o g r a p h y showed the product to be a mixture of 1,2- and 1,3-distearins. The reaction mixture was washed with water (25 ml) and the chloroform layer dried over magnesium sulphate. After filtration and evaporation o f the solvent, slow crystallisation from petroleum ether (40-60 °) gave 1,3-distearin-d 5 pure by thin layer c h r o m a t o g r a p h y (60 rag, 60j°~o o f theory), m.p. 77.5-79 °, v max. 1710 and 1730 cm -1 (KBr disc). The deuterium content was 75~o o f theory calculated for C39H71D505. The dl-(IV) and d4-(V ) distearins were prepared in a similar manner as indicated in the scheme (fig. I). Deuterium contents were 85~o and 75Yo o f the theory, calculated for C39H75DO5 and C39H72D405 respectively. Deuteroxy 1,3-distearin was prepared by exchanging the hydroxylic proton of 1,3-distearin with deuterium from ethanol-d~. Deuterium incorporation was 62~,, of theory, (measured by mass spectrometry) calculated for C39HvsDOs, but exchange would have occurred within the spectrometer source prior to electron bombardment. An attempt was made to exchange the hydroxylic proton of 1,3-distearin for deuterium by the addition of deuterium oxide to the sample on the probe. Only very slight exchange occurred. Results and discussion

The mass spectrum of 1,3-distearin* is tabulated (table 1) with intensities recorded as a percentage o f that o f the base peak. Table 2 indicates the shift of peak positions in the spectra o f the variously-deuterated diglycerides. Table 3 contains high resolution data for 1,3-distearin. The mass spectrum o f 1,3-distearin at 70 eV may be considered in three parts: The high mass end above 500 a.m.u, where ions occur due to the loss * The mass spectra of 1,3- and 1,2-distearins are virtually identical.") It is possible that thermal isomerisation occurs in the mass spectrometer source and the mass spectrum recorded is that of an equilibrium mixture of distearins. However, for mechanistic purposes the structure of 1,3-distearin has been used here, and in fact close examination of the postulated fragmentation of 1,2-distearin reveals that in the majority of cases, the intermediates and products would be identical. In the instances where this is not the case a note has been made.

MASS SPECTROMETRY OF SOME DEUTERATED 1,3-DISTEARINS

51

of small neutral fragments from the molecular ion, the intermediate region about 200-500 a.m.u, where the bulk of interesting fragment ions are found, and finally, the low-mass region below 200 a.m.u. The last mentioned region TABLE l Mass spectrum of 1,3-distearin The intensities are calculated as a percentage of the intensity of the base peak m/e

Intensity

m/e

Intensity

m/e

40 41 42 43 44 45 51 53 54 55 56 57 58 59 60 61 66 67 68 69 70 71 72 73 74 75 77 79 80 81 82 83 84 85 86 87 88 91 93 94 95

2.0 55.0 15.5 100.0 7.0 4.0 1.0 4.0 6.5 54.0 16.0 74.0 5.5 2.5 8.0 4.5 1.0 9.0 6.0 32.5 9.5 34.0 2.0 9.0 9.0 4.0 1.0 2.5 2.0 10.0 5.0 22.0 18.5 20.0 2.0 5.5 1.0 1.0 2.0 1.5 10.5

96 97 98 99 100 lOl 102 107 108 109 I lO 1l I I 12 113 114 115 I 16 117 121 123 124 125 126 127 129 130 134 135 139 140 141 143 146 149 153 154 157 158 168 169 171

4.0 15.0 22.5 4.5 2.0 2.5 1.5 1.5 1.O 6.0 2.0 7.0 9.5 2.5 1.0 3.0 9.0 6.0 2.5 2.5 1.0 3.0 2.5 1.0 12.5 2.0 4.5 2.0 1.5 1.0 1.0 1.5 1.0 1.0 1.0 2.0 1.0 1.0 1.0 1.0 3.0

181 185 196 199 210 213 224 227 241 253 264 265 266 267 268 269 283 284 285 286 298 299 325 327 328 339 340 341 342 343 359 360 382 383 395 400 593 606 607 608

Note, m/e 624 and m/e 563 have intensities less than 1 ~o.

Intensity 1.0 2.5 2.0 I.O 1.5 1.5 1.0 1.5 2.0 1.5 1.0 2.0 5.0 45.0 9.5 2.0 1.0 3.0 4.0 1.0 1.5 1.5 1.0 11.5 2.5 3.0 9.5 36.0 8.5 1.5 7.0 2.0 4.5 2.0 2.0 1.0 1.0 12.0 6.5 2.0

Ca9H7605 C39H740,1 C88H7304 C36H6704 C23H4405 C24H4304 C23H4204 C21H480-1 C21H410',~ C21H4oO3 C~0Hu~O3 C~sH~Oe C~sHz.~O

Molecular formula of undeuterated ion

624 606 593 563 400 395 382 359 341 340 327 284 267

HeO CH:~O C,~H00 CI~H3'z CI~H;~30 C16H340 CI~H330 CasHa.~O2 ClsH:~O2 C1.H3702

Undeuterated

328 285 267

625 606 593 563 401 395 382 360 342 C1~H.3702

HOD CH2DO C3HsDO C16H3,2 C~sHz32DO C16H:~aDO ClsHa30 C16H~hO'z

Deuteroxyl

625 607 H20 594 CH~O 564 C.~H.~O 401 Ct6H32 396 CasH330 383 C16H340 360 CIsH3aO 342 CIsH3502 341 C1sH',~602 328 C~oHaTO~ 284 267

d~

628 610 H20 595 CHD20 567 CaHgO 404 C16Ha2 399 C16HauO 386 C1~H'.~40 363 ClsH.~30 345 ClsH3~O2 344 C18Ha~02 329 C19Ha~D202 284 267

d.l

TABLE 2 Mass at which ion appears and molecular formulae of lost fragments

629 611 596 568 405 400 387 364 346 345 330 284 267

H20 CHD20 CaHgO C16Ha2 C15H330 C16H:340 ClsH330 C~sH3502 C18H36Oz C19H3~D20~

d5

MASS SPECTROMETRY OF SOME DEUTERATED

1,3-DISTEARINS

53

TABLE3 Mass measurements and molecular formulae of some ions in the mass spectrum of 1,3-distearin

Molecular formula of ion

Calculatedm a s s

Measuredm a s s

Error.ppm

C:~6H670,i C~3H4405 C~t H4:30,~

563.5039 400.3189 395.3161 382.3083 359.3161 341.3056 327.2900 129.0552 129.0915 II 6.0473

563.50215 400.3187 395.3160 382.3088 359.3160 341.3055 327.2900 129.0551 129.0911 116.0471

4.0 0.5 0.3 1.7 0.3 0.3 1.0 4.0 2.0

C23H4204

C',1H430~ C21HuO3 C2oH3~,O:~ C~;H!~O:~ (90 °~o) C7H1:302 (10 '~o) C:~HsO:l

is of little interest as most of the ions arise from the hydrocarbon chains of the molecule and is only therefore characteristic of such chains. A dozen or so ions have been selected for examination from the middle and high molecular weight regions on the basis of their being either diagnostically important or lending themselves to at least partial interpretation. The molecular ions of all the compounds studied, although of low intensity, were easily identified. The most abundant ion (m/e 606) at the high-mass end of the spectrum of 1,3-distearin is that due to the loss of water from the molecular ion. If this elimination were a 1,2-thermal dehydration1°), deuterium would be lost from 1,3-distearin d~. However, ilo loss of deuterium is observed, the peak occurring at m/e 61 l, M H20. Metastable peaks at m/e 588.5 (m/e 624-~-606) in the spectrum of 1,3-distearin and at m/e 593.5 (m/e 629~61 l) in that of the d5 compound, confirm the electronic nature of the elimination. The deuteroxy compound loses HOD from the parent ion (m/e 625) giving a peak at m/e 606, indicating, as expected, that the deuteroxyl-deuterium is involved in the elimination. The remaining proton must, therefore, originate from the hydrocarbon chain. The two fundamentally different but, nevertheless, plausible fragmentation pathways (figs. 2a and b) may be distinguished by ~So labelling of the hydroxyl or ester oxygens. The common product ion, although odd-electron, is resonance-stabilised, which explains the high abundance of role 606; its structure is analogous to the intermediates in the fragmentation of ethylene ketalsH). A peak due to the loss of the elements of methoxyl appears at role 593 in the mass spectrum of 1,3-distearin. Examination of the spectra of the deuterated compounds indicates that the hydrogens lost originate from the methylene groups of the glyceryl residue and the hydroxyl group. Thus,

54

A. MORRISON,M.D. BARRATTAND R. ANE,IA

with the methine group of the glyceryl head deuterated, only loss of CH30 is observed, but in the d, and d5 compounds the peaks appear at role 595 and 596 respectively (loss of CH D20). The spectrum of the deuteroxy corn-

i

HC

H 0COCl7H3s

C H2OCOCITH3s

I c, S.JC ,

--I-9 -- H

H-C-O-H

z ONco/CH(CH2)IsCH 3

m/¢624

O~ICI'~C (CH2)15CH3

C39H7605

m/¢ 024

C39 H76 05

CH2 OCOCI7H35

~5

~CH 2

ixC - - O /

./ CH(CH2)I5CH 3

C H20COClTH3s

(

CH2OCOCI7H35

"CH(CH2)mCHa

2 15¢H3

CH2OCOCI7H35

CH2Oc0c17 H35

~.)--O c~ (CH2)~scH3

CHLCH2 15CH3

m/e

606

C 3 9 H 7 4 O4 Fig. 2a

MASS SPECTROMETRY OF SOMEDEUTERATED [,3-DISTEARINS

C H2 0COC17H35

!

C H20CO C 17H35

i

" C =' O

+

•

:o +

~ oH

I I

H c[~,.~H [ I

CH 2

(CH2)IsCHa m/¢ 6 2 4

55

(CH2)IS CHa

C39H76 0 5

CH2 0COCI7 H35

et, c.

CH('-CH2 )15 CH3 m/e

606

C39 H74 04

Fig 2b

pound has a peak at m/e 593, indicating the loss of deuterium. Presumably both hydrogens come from the same methylene group and the elimination is via an e-fission on the 1,2-distearin formed by intramolecular acyl migration. The ion of mass 563 a.m.u, was shown by mass measurement to have the molecular formula C36H6704, representing the formal loss of C~HgO, from the molecular ion. A metastable peak at role 523.0 (role 606-+563) in 1,3distearin and one at m/e 527.0 (role 610--~567) in the d4 compound indicate that this ion arises, at least partially, by the loss of C3H7 (43 a.m.u.) from m/e 606. The retention of deuterium in the corresponding fragment m/e 568 in the ds compound shows that the C3H 7 iS lost from the stearoyl chain. Long-chain esters 6) also lose a fragment, 43 a.m.u., which has been shown by deuterium labelling to originate from the three carbon atoms adjacent to the acyl carbonyl group. In the region of intermediate mass, two groups of ions may be distinguished. Firstly, there are those ions derived from one of the fatty acid

56

A. MORRISON, M. D. BARRATT AND R. ANEJA

residues, and secondly, there are ions usually containing one fatty acid residue as well as all or most of the glyceryl head. The former group includes the diagnostically important acyl ion, Cj 7HasCO +. This ion occurs at m/e 267 in all the compounds studied and there is no significant sign of deuterium incorporation, ft is reasonable to assume, therefore, that little or no scrambling takes place between the glyceryl residue and the fatty acid chains. There is also an ion at m/e 284, corresponding to the carboxylic acid itself C~ 7H35CO2 H +. Again, there is no evidence in the spectra of the carbondeuterated materials to suggest scrambling. However, the deuteroxy compound shows deuterium retention with a peak at m/e 285 and on the addition of deuterium oxide to the probe, 1,3-distearin also shows slight deuterium incorporation at m/e 285. Thus, the extra hydrogen is derived both directly from the hydroxyl hydrogen and from water in the mass spectrometer source after hydrogen exchange. Note, vide supra, that the hydroxyl proton of 1,3-distearin was not easily exchanged with deuterium when deuterium oxide was introduced onto the probe. [n the second group of intermediate mass ions, that at m/e 400, C23H440 5 is probably formed by a McLafferty rearrangement (fig. 3). It is analogous CH2OCOCI7H3s

CH2 0COCI7 H35

I

I

HO.

CH
iHL, H H2

+ CH2=CHCI4 H29

~o/C~c

H2

m/e 400, C23H4405

C39H7605

-H20

CH20COCITH3s

o

~,, CH2

m/, ae2, %3 H42 o4 Fig. 3

MASS SPECTROMETRY OF SOME DEUTERATED 1,3-DISTEARINS

57

to the ion m/e 74 in the mass spectra of fatty acid methyl esters. The deuterated distearins yield corresponding ions which retain deuterium. Loss or water from C23H4¢O5 yields m/e 382, C23H4204, probably by the mechanism illustrated (fig. 3). This fragmentation is supported by the presence of a metastable peak at m/e 364.8 (m/e 400-~382) in the mass spectrum of 1,3distearin. The elimination of the hydroxyl proton is confirmed by the spectrum of the deuteroxy compound which shows that HOD is eliminated. The ion of mass 359, C21H430 4 probably has the structure shown in fig. 4. All the deuterated compounds show corresponding ions which retain deuteri urn. -+ <] H20H +H

CHOH

CH2 OCOCl7H35 Fig. 4

CH2 ) O

O II - C - CI7H35

+. C H - Obl CH 2 OCOCI7H35 CH2_

. + C 17 H3sC02

I

Jr,

C H2 0COC17 H35

CH - OH CH2 OCOCI7H35

m/¢ 6:)4,

C39 H76 05

m/e Fig. 5a

341,

C21 H41 0 3

58

A. MORRISON, M. D. BARRATT A N D R. ANEJA

--C -- CI7 H35 .(~ CH2 0 ~CH-O-H

II

CI7H35 - C

+0

OH

~

0~C-CI7H35

o/CH2

CI7H3

m/e 624 C39 H7605

m/e 341

C21H4103

Fig. 5b The loss of CI7H35CO2 gives rise to the ion at m/e 341, C21H410 3 and again all the deuterated analogues retain deuterium. The elimination of C I 7H35CO2 is probably assisted by participation of either (i) the hydroxyl group (fig. 5a), or (ii) the ester carbonyl (fig. 5b). The product from the latter is a resonance-stabilised oxonium ion and has a structure and stability consistent with the high intensity of role 341. Analogous carbonyl-assisted eliminations of RCO2 are perhaps responsible for the intense peak at role 359 in 2-deoxy-2-chloro-l,3-distearin lz) and the base peak, role 607 in the mass spectrum of tristearin 4). Although a six-membered ring structure is shown, the alternative five-membered ring structure (from the isomeric 1,2-diglycerides) is not excluded and must be invoked to explain the spectra of mixed acid triglycerides 4, ~2). ~-Cleavage of the molecular ion with charge localisation on the hydroxyl oxygen leads to m/e 327, C20H390 3. The observation that the -d 4 and -d 5 compounds produce ions at role 329 and 330 respectively, which each have two deuterium atoms less than the parent molecules, supports the suggested mechanism for this fragmentation (fig. 6). Finally, the loss of the C15H31 from C30H7404., 111/8 606, gives the evenCH20COCI7H35

t

CH2 0COCI7H35

+.

I

CH,I- OH

p,

CH20COCI7H35

m/e 624,

+

CH = OH +

C39H7605

C:H20COCI7H35

m/e 327, C20H3903 Fig. 6

59

MASS SPECTROMETRY OF SOME DEUTERATED 1,3-DISTEARINS

CH2OCOCI7H35

CH2 OCOCI7H35

+ O-~

4- CI5H31

O CH = CH2

"CH - t ~ 2 - CI5H31

mice 6 0 6 ,

C39H'7404

m/e 395, C24H4304 Fig. 7

e l e c t r o n species

m/e 395,

C 2 4 H 4 3 0 4 (fig. 7)11). T h e c o r r e s p o n d i n g ions origi-

n a t i n g f r o m the c a r b o n - d e u t e r a t e d m a t e r i a l s retain d e u t e r i u m (table l), but the d e u t e r o x y l c o m p o u n d yields an ion f r o m w h i c h it is lost.

Acknowledgements T h e a u t h o r s wish to t h a n k Dr. A. P. D a v i e s for helpful discussions.

References l) C. B. Johnson and R. T. Holman, Lipids 1 (1966) 371 2) M. Barber, J. R. Chapman and W. A. Wolstenholme, J. Mass Spec. and Ion Physics 1 (1968) 98 3) M.G. Horning, G. Casparrini and E. C. Horning, J. Chromatographic Science 7 (1969) 267 4) M. Barber, T. O. Merren and W. Kelly, Tetrahedron Letters (1964) 1063 5) H. W. Sprecher, R. Maier, M. Barber and R. T. Holman, Biochem. 4 (1965) 1856 6) R. Ryhage and E. Stenhagen, Mass spectrometry of organic ions, ed. F. W. McLafferty, Academic Press, New York, 1963, p. 403 7) R. Ryhage and E. Stenhagen, J. Lipid Res. 1 (1960) 382 8) H. Budzikiewicz, C. Djerassi and D. H. Williams, Structure elucidation of natural products by mass spectrometry, Vol. I, Holden-Day Inc., San Francisco, 1964, Chap. 2 9) A. Grun and F. Wittka, Ber. 54 (1921) 273 10) H. Budzikiewicz, C. Djerassi and D. H. Williams, Mass spectrometry of organic compounds, Holden-Day Inc., San Francisco, 1967, p. 98 11 ) of. Reference 9, p. 18 12) R. Aneja and A. Morrison; unpublished