SHORT COMMUNICATIONS Subnanogram Detection of t-Butyldimethylsilyl Fatty Acid Esters by Mass Fragmentography ABSTRACT
The mass spectra of t-butyldimethylsilyl fatty acid esters all display a pronounced (M-C4H9) + ion. The proportion of the total ionization carried by this fragment, particularly for saturated and mono-, di-, and tri-unsaturated acid derivatives, facilitates their qualitative analysis at the subanogram level by mass fragmentography.
We report here a study of the mass spectral properties of t-butyldimethylsilyl (t-BDMS) esters, whose characteristics facilitate specific analysis of fatty acids at very low levels. EXPERIMENTAL PROCEDURES
Fatty acids were all purchasea Irom Applied Science Laboratories, Inc. (State College, PA), except Phytanic acid which was donated by A. Poulos (Adelaide Childrens Hospital, South Australia). t-Butyldimethylsilyl esters of referINTRODUCTION ence acids were prepared by adding 100/11 of a Gas c h r o m a t o g r a p h y - m a s s spectrometry N,N-dimethylformamide solution (10 ml) con(GC-MS) is a technique widely used for the taining 2 mmole imidazole and 1 mmole tidentification of fatty acids in biological mix- butyldimethylsilyl chloride to 500/ag acid. This tures, usually as their methyl esters (1,2). These procedure was reported originally by Corey and derivatives, however, are not considered satis- Venkateswarlu (3) for the formation of t-BDMS factory for unequivocal identification or sensi- ethers as hydroxyl protecting groups. After tive detection purposes, as the mass spectra ob- heating the reaction mixture for 15 min in 60 C tained are typically dominated by intense ions bath, brine was added, and the derivative was at low mass, characteristic of the ester moiety extracted into ether prior to GC-MS analysis. rather than the structure of the acid itself. GC-MS was carried out with an AEI MS-30 mass spectrometer, equipped with a multipeak monitor, and interfaced to a Pye 104 gas chromatograph using a single stage dimethyl silicone membrane separator. All spectra were determined at 25 eV. The gas chromatographic column was a glass coil (1 m x 2 mm internal diameter (ID) packed with 1% OV-225:Silar ~, 10C (9/1) on 100-120 mesh support. The helium flow was 30 ml/min and the column temFIG. 1. Mass spectrum of linoleic acid t-BDMS perature was ! 80 C. ester. TABLE I Retention Indices (I) and Major Mass Spectral Fragments of t - BDMS Esters
t-BDMS ester Palmitie
Phytanic Oleic
Elaidic Linoleic
Linolenic cis- 5-Eicosenoic Arachidonic
I 2430 2535 2620 2625 2655 2700 2805 2855
Mass Fragments a 313 b 369 b 339 h 339 b
(100),131(7),129(6),117(14),75(40). (100),201(11),145(10),143(6),117(16). (100,131(I1),129(3),75(26),73(6). (100),131(8),129(2),75(18).
335 b (100),131 (20),95(28),79(21),75(75). 367 b (100,171(35),157(16),117(40),75(40). 361 b (23),131 (23),129(35),93(40),91(40),81(35) 79(60),75 (100),73(65),67(45).
aExpressed as m/e value with relative intensity in parenthesis.
b(M-C4H9) +ion. 714
SHORT COMMUNICATIONS
715
640pg
6
II
--C~
/CH 3 M--57
/0 \sI/CH 3 ~C:; 9
O---Si / ""CH3
c(c%
"X'C) . I
\CH
3
I
SCHEMEI.
b "_./CH3 .___//~
R
r/
C\
/:~'\
O
CH 3
Tr
638pg
E 0 a
R=HorCH
3
T
STRUCTURE II.
R.~/~
~0~ C\o
/CH~ 1
Si
m / e 283
--.,.CH 3
fir STRUCTURE III.
RESULTS AND DISCUSSION All the acids investigated formed the respective t-BDMS esters in quantitative yield as estimated by thin layer chromatography (TLC), gas liquid chromatography (GLC), and, in one case, isolation. The t-BDMS esters show good gas chromatographic properties, but expectedly have appreciably longer retention times than the corresponding methyl esters (Table I). The base peak in the electron impact spectrum of nearly all the t-BDMS esters studied (Table I and Fig. 1), which is postulated to have the resonance stabilized cyclic Structure I (Scheme I), is formed by fission of a t-butyl radical from the molecular ion. Ions at m/e 129 and 131 are characteristic of all the acids recorded, except phytanic, in which the 2 ions are shifted to m/e 143 and 145. The probable structures of these ions are thus Structures II and III. It is apparent from the data in Table I that as the degree of unsaturation increases, the t-BDMS ester moiety is less able to direct the fragmentational mode of the molecule, and accordingly, the proportion of low mol wt hydrocarbon ions increases. A comparison of the t-BDMS and methyl ester (1) spectra of arachidonic acid shows many common ions, e.g., m/e 67, 79, 91, 93, 105, and 150. The
rn/e339 i
I
I
I
I
0
1
2
3
4
rain.
FIG. 2. Mass fragmentogram of myristoleic (m/e 283), palmitic (m/e 313) and elaidic (m/e 339) acids as their t-BDMS esters. structural information available from these hydrocarbon ion fragments has been discussed previously (1). Figure 2 shows the results of analysis of 3 fatty acid t-BDMS esters by the technique of mass fragmentography (4,5). The very small amounts detected and the specificity for each individual acid are noteworthy points. Furthermore. by monitoring the (M-C4H9) + ions, acids which differ in mol wt need not be resolved by GLC to be detected. Because the (M-57) peak is usually the most intense, the use of these derivatives facilitates the measurement of the mol wt of trace amounts of u n k n o w n acids with much greater confidence than is possible when using methyl ester derivatives. Work is now in progress to assess the scope LIPIDS, VOL. 10, NO. 11
716
SHORT COMMUNICATIONS REFERENCES
of these derivatives for analysis of biological mixtures.
1.
Myher, J.J., L. Marai, and A. Kuksis, Anal. Biochem. 62:188 (1974). McFadden, W.H., "Techniques of Combined Gas Chromatography/Mass Spectrometry," John Wiley and Sons, New York, NY, 1973, p. 367. Corey, E.J., and A. Venkateswarlu, J. Am. Chem. Soc. 94:6190 (1972). Hammar, C.G., B. Holmstedt, and R. Ryhage, Anal. Biochem. 25:532 (1968). Brooks, C.J.W., and B.S. Middleditch, Clin. Chim. Acta 34:145 (1971).
2. G. PHILLIPOU The Queen Elizabeth Hospital Woodvilte, South Australia 5011 D.A. BIGHAM R.F. SEAMARK Department of Obstetrics and Gynecology University of Adelaide South Australia 5000
3. 4. 5.
[ Received July 7, 1975 ]
Mass Spectrometric Localization of Methyl Branching in Fatty Acids Using Acylpyrrolidines ABSTRACT
must be separated by gas liquid chromatography (GLC), the bleed from which is a disturbing factor in interpreting mass spectra. As part of our investigation of mass spectra of amides of fatty acids (2-4), the spectra of pyrrolidides of methyl branched fatty acids were studied. They were found to reveal readily the location of a methyl branch, and to distinguish between normal, iso, and anteiso structures. In this study, the pyrrolidides were prepared and the mass spectra measured under conditions identical to those previously published for pyrrolidides of unsaturated fatty acids 2,4). Because the fatty acid pyrrolidides contain a nitrogen, it is very easy to deduce their fragmentation patterns, for the nitrogen containing fragments yield even numbered ions, and the odd numbered ions contain only C, H, and O. The mass spectra of fatty acid methyl esters contain mostly odd numbered masses to which
Localization of a methyl branch in a fatty acid molecule by mass spectrometry is facilitated by using the pyrrolidide rather than the methyl ester. Branched fatty acid methyl esters are converted to pyrrolidides and are then analyzed by gas chromatography and mass spectrometry. The diagnostic fragments indicate position of the methyl branch. Mass spectra of methyl esters of normal, methyl branched iso and'~nzeiso fatty acids are rather similar, and the structure must be deduced from very small differences in intensity of diagnostic ions (1). Moreover, the isomers IHormel Fellow 1972-73. Present address: Institute of Medical Biochemistry, University of Gothenburg, S.400 33 Gothenburg 33, Sweden.
10 4
g-oL-MET HYLOCTADECANOYLPYRROLIDINE
113 CZ
i~
I ~
70, g81 C6
Ca
i~H~ji
Ig61
224L
ICQS~
M
CH C~O 224
CI4
SS
C9
I [ tt ?
r
S'0
'
'
'
,
I00
150
,
200
Clz
CI3
.,I, ,L 250I, I,
CI8 CI7
, .],3 ~ L J,m,,6 ,,,,
FIG. 1. Mass spectra of 9-DL-methyloctadecanoyl-pyrrolidine. LIPIDS, VOL. 10, NO. 11
Ci6 Chs
350
The mass spectra of t-butyldimethylsilyl fatty acid esters all display a pronounced (M-C4H9) + ion. The proportion of the total ionization carried by this fragment, particularly for saturated and mono-, di-, and tri-unsaturated acid derivatives, facilitates their qualitative analysis at the subanogram level by mass fragmentography.
We report here a study of the mass spectral properties of t-butyldimethylsilyl (t-BDMS) esters, whose characteristics facilitate specific analysis of fatty acids at very low levels. EXPERIMENTAL PROCEDURES
Fatty acids were all purchasea Irom Applied Science Laboratories, Inc. (State College, PA), except Phytanic acid which was donated by A. Poulos (Adelaide Childrens Hospital, South Australia). t-Butyldimethylsilyl esters of referINTRODUCTION ence acids were prepared by adding 100/11 of a Gas c h r o m a t o g r a p h y - m a s s spectrometry N,N-dimethylformamide solution (10 ml) con(GC-MS) is a technique widely used for the taining 2 mmole imidazole and 1 mmole tidentification of fatty acids in biological mix- butyldimethylsilyl chloride to 500/ag acid. This tures, usually as their methyl esters (1,2). These procedure was reported originally by Corey and derivatives, however, are not considered satis- Venkateswarlu (3) for the formation of t-BDMS factory for unequivocal identification or sensi- ethers as hydroxyl protecting groups. After tive detection purposes, as the mass spectra ob- heating the reaction mixture for 15 min in 60 C tained are typically dominated by intense ions bath, brine was added, and the derivative was at low mass, characteristic of the ester moiety extracted into ether prior to GC-MS analysis. rather than the structure of the acid itself. GC-MS was carried out with an AEI MS-30 mass spectrometer, equipped with a multipeak monitor, and interfaced to a Pye 104 gas chromatograph using a single stage dimethyl silicone membrane separator. All spectra were determined at 25 eV. The gas chromatographic column was a glass coil (1 m x 2 mm internal diameter (ID) packed with 1% OV-225:Silar ~, 10C (9/1) on 100-120 mesh support. The helium flow was 30 ml/min and the column temFIG. 1. Mass spectrum of linoleic acid t-BDMS perature was ! 80 C. ester. TABLE I Retention Indices (I) and Major Mass Spectral Fragments of t - BDMS Esters
t-BDMS ester Palmitie
Phytanic Oleic
Elaidic Linoleic
Linolenic cis- 5-Eicosenoic Arachidonic
I 2430 2535 2620 2625 2655 2700 2805 2855
Mass Fragments a 313 b 369 b 339 h 339 b
(100),131(7),129(6),117(14),75(40). (100),201(11),145(10),143(6),117(16). (100,131(I1),129(3),75(26),73(6). (100),131(8),129(2),75(18).
335 b (100),131 (20),95(28),79(21),75(75). 367 b (100,171(35),157(16),117(40),75(40). 361 b (23),131 (23),129(35),93(40),91(40),81(35) 79(60),75 (100),73(65),67(45).
aExpressed as m/e value with relative intensity in parenthesis.
b(M-C4H9) +ion. 714
SHORT COMMUNICATIONS
715
640pg
6
II
--C~
/CH 3 M--57
/0 \sI/CH 3 ~C:; 9
O---Si / ""CH3
c(c%
"X'C) . I
\CH
3
I
SCHEMEI.
b "_./CH3 .___//~
R
r/
C\
/:~'\
O
CH 3
Tr
638pg
E 0 a
R=HorCH
3
T
STRUCTURE II.
R.~/~
~0~ C\o
/CH~ 1
Si
m / e 283
--.,.CH 3
fir STRUCTURE III.
RESULTS AND DISCUSSION All the acids investigated formed the respective t-BDMS esters in quantitative yield as estimated by thin layer chromatography (TLC), gas liquid chromatography (GLC), and, in one case, isolation. The t-BDMS esters show good gas chromatographic properties, but expectedly have appreciably longer retention times than the corresponding methyl esters (Table I). The base peak in the electron impact spectrum of nearly all the t-BDMS esters studied (Table I and Fig. 1), which is postulated to have the resonance stabilized cyclic Structure I (Scheme I), is formed by fission of a t-butyl radical from the molecular ion. Ions at m/e 129 and 131 are characteristic of all the acids recorded, except phytanic, in which the 2 ions are shifted to m/e 143 and 145. The probable structures of these ions are thus Structures II and III. It is apparent from the data in Table I that as the degree of unsaturation increases, the t-BDMS ester moiety is less able to direct the fragmentational mode of the molecule, and accordingly, the proportion of low mol wt hydrocarbon ions increases. A comparison of the t-BDMS and methyl ester (1) spectra of arachidonic acid shows many common ions, e.g., m/e 67, 79, 91, 93, 105, and 150. The
rn/e339 i
I
I
I
I
0
1
2
3
4
rain.
FIG. 2. Mass fragmentogram of myristoleic (m/e 283), palmitic (m/e 313) and elaidic (m/e 339) acids as their t-BDMS esters. structural information available from these hydrocarbon ion fragments has been discussed previously (1). Figure 2 shows the results of analysis of 3 fatty acid t-BDMS esters by the technique of mass fragmentography (4,5). The very small amounts detected and the specificity for each individual acid are noteworthy points. Furthermore. by monitoring the (M-C4H9) + ions, acids which differ in mol wt need not be resolved by GLC to be detected. Because the (M-57) peak is usually the most intense, the use of these derivatives facilitates the measurement of the mol wt of trace amounts of u n k n o w n acids with much greater confidence than is possible when using methyl ester derivatives. Work is now in progress to assess the scope LIPIDS, VOL. 10, NO. 11
716
SHORT COMMUNICATIONS REFERENCES
of these derivatives for analysis of biological mixtures.
1.
Myher, J.J., L. Marai, and A. Kuksis, Anal. Biochem. 62:188 (1974). McFadden, W.H., "Techniques of Combined Gas Chromatography/Mass Spectrometry," John Wiley and Sons, New York, NY, 1973, p. 367. Corey, E.J., and A. Venkateswarlu, J. Am. Chem. Soc. 94:6190 (1972). Hammar, C.G., B. Holmstedt, and R. Ryhage, Anal. Biochem. 25:532 (1968). Brooks, C.J.W., and B.S. Middleditch, Clin. Chim. Acta 34:145 (1971).
2. G. PHILLIPOU The Queen Elizabeth Hospital Woodvilte, South Australia 5011 D.A. BIGHAM R.F. SEAMARK Department of Obstetrics and Gynecology University of Adelaide South Australia 5000
3. 4. 5.
[ Received July 7, 1975 ]
Mass Spectrometric Localization of Methyl Branching in Fatty Acids Using Acylpyrrolidines ABSTRACT
must be separated by gas liquid chromatography (GLC), the bleed from which is a disturbing factor in interpreting mass spectra. As part of our investigation of mass spectra of amides of fatty acids (2-4), the spectra of pyrrolidides of methyl branched fatty acids were studied. They were found to reveal readily the location of a methyl branch, and to distinguish between normal, iso, and anteiso structures. In this study, the pyrrolidides were prepared and the mass spectra measured under conditions identical to those previously published for pyrrolidides of unsaturated fatty acids 2,4). Because the fatty acid pyrrolidides contain a nitrogen, it is very easy to deduce their fragmentation patterns, for the nitrogen containing fragments yield even numbered ions, and the odd numbered ions contain only C, H, and O. The mass spectra of fatty acid methyl esters contain mostly odd numbered masses to which
Localization of a methyl branch in a fatty acid molecule by mass spectrometry is facilitated by using the pyrrolidide rather than the methyl ester. Branched fatty acid methyl esters are converted to pyrrolidides and are then analyzed by gas chromatography and mass spectrometry. The diagnostic fragments indicate position of the methyl branch. Mass spectra of methyl esters of normal, methyl branched iso and'~nzeiso fatty acids are rather similar, and the structure must be deduced from very small differences in intensity of diagnostic ions (1). Moreover, the isomers IHormel Fellow 1972-73. Present address: Institute of Medical Biochemistry, University of Gothenburg, S.400 33 Gothenburg 33, Sweden.
10 4
g-oL-MET HYLOCTADECANOYLPYRROLIDINE
113 CZ
i~
I ~
70, g81 C6
Ca
i~H~ji
Ig61
224L
ICQS~
M
CH C~O 224
CI4
SS
C9
I [ tt ?
r
S'0
'
'
'
,
I00
150
,
200
Clz
CI3
.,I, ,L 250I, I,
CI8 CI7
, .],3 ~ L J,m,,6 ,,,,
FIG. 1. Mass spectra of 9-DL-methyloctadecanoyl-pyrrolidine. LIPIDS, VOL. 10, NO. 11
Ci6 Chs
350
