Analysis of Triglycerides by Gas Chromatography/Chemical Ionization Mass Spectrometry Takeshi Murata Analytical Application Laboratory, Kyoto Laboratory, Shimadzu Seisakusho Lid., Nakagyo-ku, Kyoto, Japan
Peanut oil samples (having triglycerides with up to 60 carbon atoms) and rapeseed oil and mustard oil samples (both containing triglycerides with up to 62 carbon atoms) were analyzed by GC-CI-MS, and the type and distribution of triglycerides (TG) were determined. The [MH RCOOH]' Ion group showed that the major component of the CB2TG was the 18-22-22 type, and that the 18-22-22 type contained a CI(NH,)-mass chrolargest quantity of 18:l-22:l-22:l. matography provided detailed information on the TG structures. I n the mass chromatography of C54TG, which is recorded as a single peak on the total ion chromatogram, the six MC peaks, m / e 908 (0), m / e 906 ( l ) , m / e 904 (2), m / e 902 902 (3), m / e 900 (4), and m / e 898 (5), which are QM+ Ions, gave different retention times. I n the mass chromatography of triglycerides in milk fat, the existence of odd-carbon-number triglycerides was confirmed. The major component of C53TG was 18-18-17, that of C5, TG was 18-18-15, and those of C49TG were 18-18-13 and 18-16-15.
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T h e satisfactory results obtained in analysis of triglycerides (TG) by gas chromatography-electron impact mass spectrometry (GC-EI-MS) ( I ) and direct inlet probe introduction-chemical ionization mass spectrometry (DI-CI-MS) (2) were reported elsewhere. Our method is different from the method of Bugaut ( 3 )or Bezard et al. ( 4 , 5 )who used preparative gas chromatography. I n our GC-EI-MS method ( I ) , the mass spectra for the GC peaks that are separated on the basis of the number of carbon atoms are obtained, and then the types of fatty acids composing a T G are determined from the [M - RCOO]+, [RCO 128]+, [RCO 74]+, and RCO' ions, or from the M+ ion. T h e compositions and the types of T G can be determined by finding three fatty acids of which the total of the carbon numbers agrees with t h e carbon number of the TG. In DI-CI-MS using ammonia as the reagent gas (Z),the (M + NH4)+ion was recorded as t h e QM' ion and as the major ions, and it was possible t o determine qualitatively and quantitatively, from the QM+ ions, the number of carbon atoms of TG, the number of double bonds, and whether a T G is odd-carbon-cumber type or even-carbon-number type. This method, however, was not satisfactory in the following points. In the case of T G larger than 54 in the number of carbon atoms, C58, c 6 0 , and C62, for example, the GC-EI-MS method could not provide enough molecular ions t o be informative. Though t h e method could determine oddcarbon-number T G by means of DI-CI-MS, i t could not determine the type of TG, or study the behavior of T G having odd-carbon-number fatty acids. I n order to characterize T G in more details, we tried mass chromatography (MC), which is a combinai;ion of a GC-CI-MS a n d a computer, a n d obtained satisfactory results.
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EXPERIMENTAL Samples. Tristearin, tripalmitin, and trilaurin were purchased from Applied Science Laboratories, Inc. The milk fat had a purity
better than 98%. The peanut oil, mustard oil, and rapeseed oil were purchased from a commercial source. Triglycerides were extracted with ethyl ether, and stored below 5 "C. Instrumentation. The equipment was the Shimadzu-LKB 9ooo gas chromatograph-mass spectrometer combined system with a electron impact/chemical ionization duel source. The gas chromatographic conditions were as follows. The column was 0.35 m X 3 mm id., glass with 1%OV-1 on Chromosorb W, 80-100 mesh. The chromatograph was programmed from 220-335 "C at 4 "C/min and kept isothermal at 335 "C until the last peak was recorded. The sample injection port temperature was maintained at 350 "C. The carrier gas was helium, 45 mL/min. A separator was set at 350 "C. A total ion collector was used as the detector for GC-MS. The mass spectrometric conditions for CI-MS were as follows. The ion source temperature was held at 230 "C during the CI-MS runs. The mass spectra were all obtained at 500 eV of electron energy, 500 MAof emission current, and 3.5 kV of accelerating voltage. Ammonia was used as the reagent gas; the pressure in the ion source was adjusted to 0.9 Torr. The mass spectrometric conditions for EI-MS were as follows. The ion source temperature was held at 310 "C. The mass spectra were all obtained at 20 eV of electron energy, 60 HAof trap current, and 3.5 kV of accelerating voltage. The data processing system included a GCMS-PAC 300 DG consisting of an OKITAC 4300 minicomputer with 12K core, a typewritter, an incremental plotter, a magnetic disk, and an interface to the GC-MS.
RESULTS A N D D I S C U S S I O N Analysis of T r i g l y c e r i d e s up t o 62 in the N u m b e r of C a r b o n Atoms. T h e samples were peanut oil, rapeseed oil, and mustard oil, which contain triglycerides up to 62 in t h e number of carbon atoms. Figure 1 shows the gas chromatogram of mustard oil. It was necessary to raise the column temperature as high as 335 "C. Mustard oil gave peaks u p to c62, peanut oil up t o Cso, and rapeseed oil u p to Ce2. In GC-CI-MS, the CI mass spectra may differ with the reagent gas used. Such is the case with T G samples. When isobutane is used, no QM+ ions are recorded, and the [MH - RC02H]+ ion is recorded as the base peak. When ammonia is used as the reagent gas, the [M NHl]+ ion is recorded on the QM+ ion, and its intensity is about 20 times higher compared with that given by GC-EI-MS. As for the fragment ions, only the [MH - R C 0 2 H ] ion is observed, and no fragment ions are recorded in the region of smaller mass numbers. Figure 2 shows the ammonia-CI mass spectra of c58, cso, and c62 T G of mustard oil. The [ M H - RC02H]+ ion is recorded as the base peak. T h e QM+ ion is intense enough to be used for structure study. This [MH - RC02H]+ion was used to determine the fatty acid compositions of T G groups ranging from 50 to 62 in the number of carbon atoms and to determine the distribution of the triglyceride groups and the T G types in peanut oil, rapeseed oil, and mustard oil. Table I ( I ) shows the distribution of 17 types (6 groups) of triglycerides of peanut oil, ranging from 50 to 60 in the number of carbon atoms. Table I1 ( I ) shows the distribution of 40 types (7 groups) of T G of mustard oil, and Table I11 ( I ) t h a t of 38 types (7 groups) of rapeseed oil. Mustard oil and rapeseed oil are both rich in erucic acid, and their main components are 18-22-22 and 20-20-22 types. But Table I1 and Table I11 show a considerable difference.
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ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
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Table I. Distribution of Triglyceride Groups and Triglyceride Type in Peanut Oil C,, 14-16-20 8 C,, 16-18-22 14-18-18 1 2 16-20-20 16-16-18 80 18-18-20 C,, 16-16-20 9 C,, 16-20-22 18-18-22 16-18-18 91 18-20-20 C, 16-16-22 5 16-18-20 6 C6, 16-22-22 18-18-18 89 18-20-22 20-20-20
Table 11. Distribution of Triglyceride Groups and Triglyceride Types in Mustard Oil C,, 10-16-24 3 C, 14-16-26 2 10-18-22 2 14-18-24 6 10-10-20 2 14-20-22 3 12-16-22 4 16-16-24 5 12-18-20 3 16-18-22 25 14-16-20 9 16-20-20 14 18-18-20 44 14-18-18 1 6 16-16-18 5 9 16-16-26 2 c 58 14-14-24 2 16-18-24 5 14-16-22 2 16-20-22 8 14-18-20 11 18-18-22 45 16-16-20 6 18-20-20 40 16-18-18 79 C, 16-18-26 9 C, 14-14-26 3 18-18-24 16 3 18-20-22 44 14-16-24 14-18-22 6 20-20-20 31 14-20-20 2 16-16-22 2 c62 18-18-26 18-20-24 8 16-18-20 1 7 18-22-22 64 18-18-18 67 20-20-22 25
20 13 67 12 47 41 6 62 32
C56
I c54 I
1
i i IC
3c
20
____
S
290
50 m l n
40
~300
320
310
330 335
'c
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Figure 1. Gas chromatogram of mustard oil triglyceride detected by total ion chromatogram. The carbon numbers are written on the peaks
CI-MS has the advantage that the QM+ ions are intense enough to provide useful information. I t is possible to determine the number of double bonds that exist in the T G molecules from the mass numbers in the group of the same carbon number and from the ion intensities. In the spectrum for the CS2T G group in mustard oil, shown in Figure 2, for example, the m / e 1014 ion is the most intense. This mass number indicates that there are three double bonds in the molecule. (If all the fatty acids are saturated, the molecular weight will be m / e 1020). The second most intense is the m / e 1010 ion lndicating the existence of five double 101:
bonds. Combination of the information on the degree of unsaturation and the information on the [MN - RC02H]+ion group may not be able to provide quantitative data but can provide some data on the degree of unsaturation, which may be useful to presume the distribution of fatty acids. I t can be concluded from Table I1 that the Cs2 T G of mustard oil contains 64% of 18-22-22, 25% of 2&20-22,8% of 18-20-24, and 5% of 18-18-26. Let's make further consideration using the information on the intensities of the QM' ion and [MH - RCO,H]+ ion group. Concerning the 18-22-22 type, which is the major component, the QM' ion is recorded at m / e 1014, which indicates the existence of three double bonds. T h e [MH - RCOOH]+ is recorded a t high intensities a t m / e 715 and m l e 659. This shows that much [MH - Cis"]' and [MH - C201=]+ ions are contained. There are 24 possible combinations for the 18-22-22 type with varied degree of unsaturation, 18:l-22:l-22:l being the largest quantity component and
~
659
-
c
c58 456 53.
-
633
2 d
63i 57:
d
Y
,A 1
1
I
1
655
c60 c
i.
5
631
-1
687
631
58.
i
r
I
5'7
h i0c
1
6551
c6 2
, c
i
1
."
hC3
bi.
68'
i15
I
Figure 2. Ammonia chemical ionization mass spectra of CS8,Ce0,CB2of mustard oil 2210
ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
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>
Table IV. Difference of Retention Times on Mass Chromatograms of QM' Ions of C,, C , , , C,,,C,, Triglycerides, and Difference of Retention Times on Mass Chromatograms of RCO' Ion of the C,, Group (the numerals show the number of double bonds in the molecule) TG Degree of unsaturation 2 1 0 4 3 5 c, 3 2 1 c52 3 2 1 0 c50 3 c,, 2 1 0 FA Degree of unsaturation Cn 1 2 0 3 --* retention time
850(1!
c50
81813)
A
820(2!
A-
822 (1)
a
8U(0)
CIS
790(3) 792 ( 2 )
a
79111) 796(0)
0I
'
I
1
:
&
I
I
SCAN NO
Flgure 3. Ammonia chemical ionization mass chromatograms of QM' ion of Cd8,C50, C5*,C54TG (The numerals show the mass numbers, and the parenthesized numerals the number of double bonds.)
Table 111. Distribution of Triglyceride Triglyceride Types in Rapeseed Oil 10-20-20 8 c58 12-18-20 12 14-16-20 11 14-18-18 24 16-16-18 45 12-20-20 8 C5, 14-18-20 13 16-16-20 19 16-18-18 60 c60 14-18-22 6 c, 14-20-20 4 16-16-22 5 16-18-20 15 18-18-18 70 26' 14-16-26 3 56 14-18-24 3 14-20-22 3 16-16-24 4 16-18-22 28 16-20-20 15 18-18-20 44
Groups and 14-18-26 14-20-24 14-22-22 16-16-26 16-18-24 16-20-22 18-18-22 18-20-20 16-18-26 16-20-24 16-22-22 18-18-24 18-20-22 20-20-20 18-18-26 18-20-24 18-22-22 20-20-22
4 3 4 4 8 10 42 25 7 5 10 10 43 23 7 8 65 20
18:2-22:l-22:l being the next largest. Also, there are 17 possible combinations for the 20-20-22 type, 2O:l-2O:l-22:l being the major component. We also successfully determined the possible combinations for the 18-22-22 type and the 20-20-22 type of the CB2group, with varied degrees of unsaturation and those of other number of carbon atoms taken into consideration. It may be concluded that, compared with GC-EI-MS, GC-CI-MS can provide more information, because of its higher intensity of the QM' ion, making it possible to make more precise and more detailed study of molecular structures. S t r u c t u r e S t u d y of T r i g l y c e r i d e s by C I ( N H 3 ) Mass C h r o m a t o g r a p h y . Mass chromatography is a technique to measure the mass spectra of a desired range of a gas chromatogram at 5 6 second intervals, store them on a disc of
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Flgure 4. Electron impact ionization mass chromatograms of m l e 267 (18:0), m l e 2 6 5 (18:1), m / e 2 6 3 (18:2), and m l e 261 (18:3), which are RCO' ion of Cz0 54 TG in milk fat
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a computer, and then take out the MC peaks of a desired mass number (mle)range. We applied this technique to analysis of triglycerides. Figure 3 shows the mass chromatograms of the C48, C50, C j p , and CS4T G in milk fat. On the total ion chromatogram, the four T G groups are recorded as four peaks, one peak for one group. On the mass chromatogram, the Cj4 group gives peaks at mle 908 (0), mle 906 (1)mle 904 (2),mle 902 (3), mle 900 (4)and mle 898 (5). The numerals show the QM' ions, [M NHJ', and the parenthesized numerals show the number of double bonds in the molecules, These six MC peaks have . order of retention slightly different retention times ( t ~ )The times is mle 904 (2),mle 906 (l),mle 908 (O), m /e 900 (4), mle 902 (3),and mle 895 (5). The retention times are related to the number of double bonds in some way (See Table IV). The Cjq T G group gives MC peaks in the order of mle 876 (2),m/e 878 (I),and mle 874 (3). The Cjo T G group gives peaks in the order of mle 848 (2),mle 850 (l),mle 852 ( O ) , and mle 874 (3),and the C4*TG group in the order of mle 820 (2),m/e 822 (I),mle 824 ( 0 ) ) and mle 818 (3). Figures 3 and 4 are compiled in Table IV. In order to clarify why retention times depend on the number of double bonds and why the MC peaks of Cj4TG are eluted in the order of 2-1-0-4-3-5 (the numerals show the number of double bonds), and also why in the case of the TG other than Cj4, the MC peaks are traced in the order of 2-1U3, the EI-MC were recorded at mle 267,mle 265,mle 263,and mle 261,the data being presented in Figure 4. Figure 4 shows the MC profile of the four fatty acids contained in the triglycerides ranging from 30 to 54 in the number of carbon atoms. The peaks are recorded in the order of C18:1,C18:2, C18:0,and C18:3;the retention times depend on the number of double bonds, in this case, too. The retention times of triglycerides of the same number of carbon atoms depend on the degree of unsaturation of the C,, group. The larger variation of tRin Figure 3, compared with Figure
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ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
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2 1 , 7 : :
LqJ v' c
' W h
*:
5 SIC.
Similarly, it may be concluded that the major component of the m / e 906 (1)ion is [18:1-18:(r18:0], that of the m l e 908 (0) ion is [18:1-18:O-18:0], and that of the m / e 900 (4) ion is The major [ 18:l-18:3-18:0] rather than [18:1-18:l-18:2]. component of the m / e 902 (3) ion is [18:3-18:O-18:0] rather than [ 18:l-18:2-18:0] or [ 18:l-18:l-18:1]. We did not investigate the possibility of 18:l-18:l-18:l or 18:l-18:2-18:0, because their QM' ion has a long retention time. The major component of the m / e 898 ( 5 ) ion is 18:3-182-18:O. The QM+ ion ratios are 30% for m / e 904 (2) ion, 28% for m l e 906 (1) ion, 24% for m l e 902 (3) ion, 9% for m l e 908 (0)ion, 9% for m / e 900 (4) ion, and 2 % for m l e 898 (5) ion. The ions mle 904, 906, and 902 total more than 80%. T h e major components of the C j P group, which is the combination of 18-18-16, are [ 18:l-18: 1-16:0], [ 18:l-18:O-16:0], a n d [18:1-18:2-16:Ol. The major components of the CS0group, which is the combination of 18-16-16, are [18:1-16:O-16:0], [18:2-16:(r16:0], and [18:3-16:C&16:0], which are almost equal in quantity. Qualitative Determination of Odd-Carbon-Number Triglycerides by CI(NH3)Mass Chromatography and E1 Mass Chromatography. When triglycerides are analyzed by gas chromatography, the peaks will be eluted in the order of the numbers of carbon atoms. It is quite difficult, however, to separate all the peaks completely: baseline resolution is quite difficult to obtain. It is said that this unsatisfactory separation is not so much due to the bleeding of t h e liquid phase from the column as to the existence of odd-carbonnumber TG. This assertion has not so far been proved. Figure 5 shows the EI-MC of the m l e 239 ion in C16:0, mle 237 ion in C16:1, m l e 225 ion in C15:O and mle 71 ion in C4:O contained in the TG ranging from 30 to 54 in the number of carbon atoms. The m / e 225 ion, which is the C15:O odd-carbon-number fatty acid, is traced as a valley a t the position of the peak for C4:O ion, which is C16:O or C16:l even-carbon-number fatty acid. This suggests the existence of odd-carbon-number TG. In order to confirm the existence of odd-carbon-number fatty acids, the MC of the m l e 237, 225, and 71 ions of Ca6
I
'9
1
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n
f
'I"
r,
'~d".
h l 8
5
'A ,
j,
I
"
&
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-1
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Figure 5. Electron impact ionization mass chromatograms of m / e 239 (16:O) m l e 237 (16:1), m / e 225 (15:0),and m l e 71 (4:0),which are RCO ions of C30 - 5 4 TG of milk fat 4, may be accounted for by the synergetic influence of the three fatty acids. The fewer the CI8 groups, the smaller the variation in retention time: the variation of Cj4 T G which has three CISgroups is larger compared with CS2,Cj0, and Cd8 T G which have fewer CISgroups. i n the study of the MC of other fatty acids, we found that the retention time variations are not so large as t h a t of the CIB group, except short-chain fatty acids such as C4, C6, and C8, of which variations are of the same level as CIS group. That the retention times of T G with same number of carbon atoms differ with the number of carbon atoms suggests that, though it is common to take a mass spectrum at the top of a GC peak, the MC results w ill differ depending on this timing, and that, to obtain a high reliability, it is necessary to calculate the quantities of QM+ ions, fragment ions, and other ions from CI-MC or EI-MC. T h e difference of retention times on MC of the QM+ ions or RCO' ions of the TG having a same number of carbon atoms can be effectively used for structure study. Concerning the C, group, for example, the m / e 904 (2) ion, which has the shortest t ~has , two double bonds. This suggests 1%-18:l-18:O and 18:2-18:O-18-0 as the possible combinations of the three fatty acids. And it may be concluded from Table IV that 18:l-18:l-18:O is the major component.
, 2 39
ClBO
732C16'1
Figure 6 . Electron impact ionization mass chromatograms of RCO' ions of C36,Ca7,C3* TG, and chemical ionization mass spectra of C36, C37, c36
TG
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ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
c50 ( 8 3 4 )
TIC
537
565 579
593 607
I
l
I
~
0
l
52
l
/
I l 1
100
I
l
l
I
I
l
15C
SCAN NO
Flgure 7. Identification of TG types of C48,C49,Cs0,C5,, C5*, CS3,CS4 TG in milk fat by chemical ionization mass chromatography usin m l e 607, 593, 579, 565, and 537 ions, which are [MH - RCO,H]' ions
and CB TG were obtained, between the Scan No. 150 and Scan No. 190 shown in Figure 5. Also, the CI mass spectra of C36, and the peak top of the mle 225 (C15:0), which is C37, were obtained. These spectra are shown in Figure 6. It can be seen from the QM' ion that the C36 and C38 T G are large in quantity and, hence, there is no interference of other components. T h e peak of the CBi T G which is quite small in quantity, is interfered with by the c 3 6 and CS8TG. In the case of CI mass spectra, because of the high intensity of the QM' ion, the mle 670 ion which corresponds to C3i TG can be distinguished from the mle 656 and 684 ions which correspond to c36 and CS8TG, respectively (Figure 6 middle chromatogram). T h e [MH-RCO2H]+ion group, however, gives a rather complicated spectrum due to the interference of C36T G and C38TG. T h e spectrum shows the existence of odd-carbonnumber fatty acids such as Cg, Cg, Cll, C13 CIS, CI;, and C19. Figure 7 shows the CI-mass chromatograms of the [MH RC02H] ion of Cd8,Cd9,Cjo, CS1,Cjz, Cj3, and Cjd T G in milk fat. T h e TIC, which is also shown in Figure 7, shows no odd-carbon-number T G such as Cd9,CS1,or Cj3 TG. The MC,
by contrast, shows clearly how even-carbon-number T G and odd-carbon-number T G overlap each other. On detailed study of the mass chromatograms, we found t h a t the Cj4 T G has a molecular weight of 890 and, also we knew from the MC peaks of the m/c>607 ion that [E-18- 1' ion exists. I t can be concluded from these data that the CS4 T G is of 18-18-18 type. T h e Cj2 T G has a molecular weight of 876 and gives MC peaks of mle 607 and mle 579 ions. Calculation on [MH RCOzH]+ shows that the mle 607 ion corresponds to [18-18-1' ion and mle 579 ion to [B8-16- 1' ion. I t can be concluded that the Cjz T G is of the 18-18-16 type. The Cjo T G has a molecular weight of 834 and gives MC peaks of mle 607 and mle 579. These ions corresponds to [18-16- 1' and [18-18- 1' ions. Thus it can be concluded that the Cjo T G is of 18-16-16 and 18-18-14 types. The C4ET G has a molecular weight of 806 and gives MC peaks of mle 607 and mle 579 ions. These ions correspond to [18-16- 1' and [18-18- 1' ions and suggest the existence of T G of 18-16-14 and 18-18-12 types. As for the odd-carbon-number TG, the Cj3 T G has a molecular weight of 876 and gives the MC peak of mle 593 ion. This ion corresponds to [18-17- 1' ion and, therefore, suggests that the CS3T G is of the 18-18-17 type. The Cjl TG has a molecular weight of 848 and gives MC peaks of mle 593 and mle 565 ions, which indicate the existence of [E-18- 1' ion and [ 18-15 1' ion, and that the Cjl TG is of the 18-1&15 type. The Cd9T G has a molecular weight of 820. T h e MC peaks of mle 607, mle 565, and mle 537 ions show the existence of [ 18-15- ]+, [ 18-18- ]+, and [ 16-15- ]' ions and that the C4gTG is of the 18-18-13 and 18-16-15 types. In Figure 7, only the important types of T G are shown; it is possible to identify trace-quantity TG, through suitable selection of [MH-RCO,H]+ ions. Odd-carbon-number triglycerides can be identified by effective use of EI-MC, CI-MC, and CI-MS.
LITERATURE CITED (1) T. Murata and S.Takahashi, Anal. Chem., 45, 1816 (1973). (2)T. Murata and S.Takahashi, Anal. Chem., 49, 728 (1977). (3) M. Bugaut and J. Bezard, J . Chromatogr. Sci., 8,380 (1970). (4) J. Bezard, M. Bugaut. and G. Clement, J . Am. OilChem. SOC.,48,134
(1971). (5) J. Bezard, Lipids, 8, 9 (1971).
RECEIVED for review July 13, 1977. .Accepted September 13, 1977.
Gas Chromatographic Determination of 6-Hydroxyethylhydrazine in the Presence of Its Synthesis By-products Philip J. Palermo Norwich Pharmacal Company, Division of Morton-Norwich Products, Inc., Norwich, New York
A rapid programmed temperature gas chromatographic analysis using thermal conductivity detection Is presented for water, hydrazine, P-hydroxyethylhydrazine (HEH), and high-boilers, which are the products of the reaction between hydrazine and ethylene oxide. The HEH assay has an accuracy of f1.3% at the 95% tolerance level. The other major reaction products are also identified and quantitated. The
138 15
detection limits of the minor constituents are: water, 0.05 % and hydrazine, 0.1 YO.
For some time a fast and accurate analysis of P-hydroxyethylhydrazine (HEH) and other components within its formation mixture has been lacking. Most laboratories use ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
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