ACKNOWLEDGMENT
As stated before, in t h e case of analysis of standard samples, dehydrated ion peaks are recorded at intensities of a few per cent t o ten per cent, b u t in the case of analysis of multicomponent fatty acid samples, such as those extracted from natural oils, dehydrated ions are rarely detected except those of the main components. And, if isobutane is used as reagent gas, other fragment ion peaks are small enough to be neglected. T h e method of CI-MS permits analysis of fatty acids without esterification and provides satisfactory separation without using capillary columns. T h e only disadvantage is that it cannot distinguish branched compounds from nalkanes. This disadvantage, however, may be small enough t o be compensated by the advantages.
We thank S. Onishi for the help in experiments.
LITERATURE CITED (1) (2) (3) (4)
R. Ryhage and E. Stenhagen, Ark. Kemi, 15, 291 (1960) M. S. Munson. Anal. Chem., 43 (13), 28A (1971). F. H . Field, Accounts Chem. Res., 1, 42 (1968). , R. G. Ackman, J. Amer. Oil Chem. SOC.,43, 483 (1966).
RECEIVEDfor review June, 1974. Accepted November 25, 1974. Partially reported a t the Japanese Conference on the Biochemistry of Lipids, 1974.
Chemical Ionization-Mass Spectrometry. II. Application to Analysis of Sterol Esters Takeshi Murata, Seiji Takahashi, and Tsunezo Takeda Analytical Application Laboratory, Kyoto Laboratory, Shimadzu Seisakusho Ltd., Nakagyo-ku, Kyoto, Japan
Concerning the gas chromatography of sterol ester, Kuksis ( I , 2 ) performed direct gas chromatographic analysis of cholesterol esters in blood in 1964 and then in 1967 (3, 4 ) , he succeeded in analysis of fatty acid methyl esters, cholesterols, cholesterol esters, steryl esters, and triglycerides in total lipids extracted with chloroform-methanol mixture. Swell ( 5 ) fed rats with food containing labeled cholesterol esters and extracted the cholesterol esters from the blood serum, the liver, and the kidneys, and measured them by gas-liquid radio chromatography. N. Ikekawa et al. ( 6 ) succeeded in simultaneous analysis of cholesterols and cholesterol esters in blood serum, using a very short column (0.75-m X 4-mm i.d.) of 1.5% OV-17, the column temperature being programmed from 100 t o 280 “C a t 4 ‘C/min. In mass spectrometry (MS) or gas chromatography-mass spectrometry (GC-MS) of sterol esters, the fragment ions produced through ionization by electron impact (EI) can be obtained with the dehydrated ion peak as the base peak, but they are not informative on fatty acid composition ( 7 ) . In chemical ionization-mass spectrometry (CI-MS) (8, 9 ) described in our former report “Chemical Ionization Mass Spectrometry, Application to Analysis of Fatty Acids” ( I O ) , the high intensities of quasi-molecular ions and the low intensities of fragment ions, which are advantages of CI-MS, were utilized in analysis of sterol esters. T h e method permits detection of sterols, determination of their types, and determination of the type and the degree of unsaturation of the fatty acids by the use of the information obtained from the quasi-molecular ions of the fatty acids present in the sterol esters. Thus, it is possible to identify sterol esters. Another great advantage of CI-MS is that it is not necessary to separate sterol esters by gas chromatography and the sample can be directly introduced into the MS. This simplifies the whole procedure.
EXPERIMENTAL Materials. Cholesterol caprilate (C8-ester), cholesterol palmitate (Cis-ester), and cholesterol stearate (Cls-ester) were purchased from Applied Science Laboratories Inc. Cholesterol esters were extracted from ordinary human blood, egg yolk, and silkworm eggs (presented by Kyoto University of Industrial Arts and Textile Fibers, Kyoto, Japan). The C8-ester, (218-ester, and Cis-ester were examined for purity by gas chromatography, using an OV-1 (1%)column. All of these esters were recorded as a single peak. Preparation of Cholesterol Esters. The total lipids were extracted with chloroform-methanol mixture as described by Folch et al. ( I 1 ). The extract was chromatographed on a thin layer plate of silica gel G “Merck,” the development being done with petroleum ether-ethyl ether-acetic acid (82:18:1) mixture (12). A standard sample of CIS ester was run on the same plate for reference. The bands were visualized by exposure to iodine vapor. After the iodine had been evaporated, the sterol ester bands were scraped off and transferred to a centrifuge tube. Some ethyl ether was added as the extraction solvent. Then the mixture was agitated twice and centrifuged. The supernatant liquid was decanted into another tube and dried. The product was introduced into CI-MS. Instrumentation. The equipment used was: Shimadzu GC4BM gas chromatograph, Shimadzu-LKB 9000 gas chromatograph-mass spectrometer combined system, and Shimadzu-LKB 9000 gas chromatograph-mass spectrometer combined system attached with a chemical ionization source. The gas chromatographic conditions were as follows. The column was a 0.35-m X 3-mm i.d. glass column packed with 1%OV-1 on Chromosorb W 80-100 mesh. The column temperature was programmed from 200 to 320 “C at 4 “C/min. A flame ionization detector was used as the detector for GC. Sample introduction to the mass spectrometer was made by the direct sample introducing unit. The reagent gas was isobutane. As for the method of sample introduction t o EI-MS, a sample was put in a glass cell at the tip of the operation rod, and the cell was introduced while being cooled with water. Then the sample was evaporated by heat. In the case of CI-MS, since the operation rod was not cooled with water, the sample was put in a glass cell and introduced into the ionization source which is kept at 200 OC. The mass spectra were recorded, watching the ionization current indicated on the recorder.
A N A L Y T I C A L CHEMISTRY, VOL. 47, NO. 3, M A R C H 1975
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In the CI-MS analysis of standard samples of cholesterol palmitate and cholesterol stearate using isobutane as reagent gas, the cholesterol palmitate gave the dehydrated peak of cholesterol a t rnle 369 and the (M 1)+ ion peak of palmitic acid a t m l e 257, while the cholesterol stearate 1)+ ion peak of stearic acid a t rnle 285. gave the (M Other fragment peaks are barely recorded. When isobutane or methane is used as reagent gas, the quasi-molecular ions of cholesterol palmitate and cholesterol stearate are not recorded a t rnle 625 and 653 a t all. When ammonia is used as reagent gas, cholesterol stearate gives the base peak ( = 100%) a t rnle 386, a fragment peak of 31% a t rnle 369, the (M NH4)+ peak of 8% a t m l e 670, and the (M H ) + peak of 0.3%; cholesterol caprilate gives the base peak (= 100%)a t m / e 386, a fragment peak of 27% a t rnle 369 and the (M NH4)+ peak of 13% ( 1 3 ) .Ammonia has the disadvantages when compared to isobutane that the sensitivity is lower, that good separation of unsaturated components cannot be expected, and that the reproducibility is low. The mechanism of ionization in the CI source may be deduced, from the fragmentation using ammonia or isobutane as reagent gas, to be as shown in Figure 1. In chemical ionization using isobutane gas as reagent gas, sterol esters are protonated into quasi-molecular ions, MH+, but they are too unstable to be recorded as peaks, and the dehydrated peak of cholesterol ( m l e 369) and various protonated fatty acids are recorded. Figure 2 shows the CI-MS spectrum of a mixture sample of cholesterol caprylate, cholesterol palmitate, and choles-
+
+
Figure 1. The mechanism of chemical ionization of sterol ester
The mass spectrometric conditions for EI-MS were as follows. The ion source temperature was held at 210 "C during the EI-MS runs. The mass spectra were all obtained at 70 eV of electron energy, 3.5 kV of accelerating voltage, and 60 p A of trap current. The scan speed was 6. The mass spectrometric conditions for CI-MS were as follows. The ion source temperature was held at 200 "C during the CI-MS runs. The mass spectra were all obtained at 500 eV of electron energy, 3.5 kV of accelerating voltage, and 500 pA of emission current. The scan speed was 6. The pressure in ionization source was 0.5-1 Torr.
RESULTS AND DISCUSSION Prior to the study of the spectra of sterol esters, it is necessary to know how cholesterol and fatty acids are presented as spectra in CI-MS. As we have reported (IO), the peaks recorded in analysis of fatty acids are mostly those of quasi-molecular ions (QM+). As for cholesterol, the dehydrated ion peak, (M 1) - HzO, is recorded a t rnle 369 as the base peak, and the only fragment peaks are (M - 1)+ ion with an intensity of 8% a t m l e 385 and ( M l)+ ion with an intensity of 7% a t rnle 387 using isobutane as reagent gas. No other fragment peaks are recorded.
+
+
+
% 100
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I
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+
+
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I.
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,
350
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Ll
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Figure 2. CI-MS spectrum of a mixture sample of cholesterol caprylate (&-ester), cholesterol palmitate (Cls-ester) and cholesterol stearate (Cis-ester) (top), the ELMS spectrum of Cs-ester (middle),and the El-MS spectrum of a mixture sample of Cle-ester and Cls-ester (bottom) 578
ANALYTICAL C H E M I S T R Y , VOL. 47, NO. 3 , M A R C H 1975
As for the reagent gas, we used isobutane because it gives fewer dehydrated ion peaks and fragment ion peaks than methane. T h a t the dehydrated peak of sterol and the quasi-molecular ion peak of the conjugated fatty acid are recorded ensures ease in identification of sterol esters. Besides, the quasi-molecular ion peak provides information on the degree of unsaturation. In the case of unsaturated fatty acids, since the mass decreases by 2 amu, the isotopic ion of the QM+ 2 ion may seem to cause errors, but this can be reasonably neglected so far as quantitation is done from the peak heights, because the QM+ 2 ion peak is 2-3% as high as that of QM+ ion peak (10). Analysis of Various S t e r o l E s t e r s by CI-MS. Figure 3 shows a gas chromatogram of sterol esters in blood serum. Concerning the cholesterol esters, the peaks, 41, 43,45, and 47 are, respectively, identified as the cholesterol esters of (2x4, C16, CIS, and C20 (3, 4 ) . Figure 4 shows the CI-MS spectrum of the same sample. C14:o is recorded a t rnle 229, C14:1 a t rnle 221, C16:o a t rnle 251, CIS:^ a t rnle 255, Cls:o a t mle 285, C18:1 a t mle 283, Cls:2 a t mle 281, C18:3 a t mle 279, C2o:o a t rnle 313, and C20:1 a t mle 311. These data show that the sample contains cholesterol esters having these fatty acids. I t is clear that CI-MS is superior to GC in resolution, especially in resolution of unsaturated compounds. Concerning the quantitativeness, the same conclusion as that about fatty acids ( 1 0 ) may be drawn. We can see that there is a good agreement between the data in Figures 3 and 4, in the relative peak areas of C14,CIS,C18, and Czo. At the bottom of Figure 4 is shown the EI-MS spectrum of the same sample. Figure 5 shows the CI-MS spectrum of the cholesterol esters in egg yolk. C10:o is detected a t mle 173, C l ~ : oa t mle 201, c14:o a t mle 229, c16:o a t mle 257, cl6:l a t mle 255, C18:o a t mle 285, C18:1 a t mle 283, C18:2 a t mle 281, C18:3 a t mle 279 and C19:o a t mle 299, showing that the sample contains the cholesterol esters having those fatty acids in the molecules. Figure 6 shows the CI-MS spectrum of sterol esters extracted from the eggs of silkworms. The analysis was conducted to study the change in sterol ester composition in a stage of eggs before hatching.
+
+
Figure 3. Gas chromatogram of sterol esters in blood serum detected by FID
terol stearate (top), the EI-MS spectrum of cholesterol caprylate (middle), and the EI-MS spectrum of a mixture sample of cholesterol palmitate and cholesterol stearate (bottom). On studying the mass spectrum of sterol ester mixture by CI-MS (top), we find there are recorded, besides the dehydrated ion peaks, the quasi-molecular ion peak for caprylic acid a t rnle 145, that for palmitic acid a t rnle 257, and that for stearic acid a t mle 285. I t is quite easy to conclude that the sample contains these three cholesterol esters. The EI-MS spectrum of the standard sample of Cs-ester (middle), in contrast, has many fragment ion peaks, besides the base peak. The base peak for caprylic acid is recorded a t mle 144 with an intensity of 9%, but the identification is entirely impossible because many fragment ions are recorded having the same mass numbers as the fatty acids with the number of carbon atoms ranging from seven to twenty. (EI-GC-MS will give no better results.) No useful information is available from the EI-MS of '216-ester or (218-ester, which has a greater molecular weight than Cs-ester.
CIS
100-
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% 50 .
CIS' CIS'. c14
100-
%
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50 -
I
.
.
ub.
I50 200 250 5-80 3 50 Figure 4. CI-MS spectrum of the cholesterol esters in blood serum (upper) and El-MS spectrum of the same sample
I
400
A N A L Y T I C A L CHEMISTRY, VOL. 47, NO. 3, M A R C H 1975
579
], Choels;l ClO
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Figure 5. CI-MS spectrum of the cholesterol esters in egg yolk
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369
397 383 395 397
I 400 IOO-
369
Cholesterol- HzO
CIS"
50-
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111111
1
...
I1
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Figure 6. CI-MS spectrum of sterol esters extracted from the eggs of silkworms Conditions: 0.35-m X 3-mm i.d. glass column packed with 1 % OV-1 on chromosorb W 80-100 mesh; 200-320 OC (at 4 OC/min) programmed temperature
It is easy and simple to identify the sterol esters from the fatty acids detected on the spectrum. I t is also easy to determine the types of sterols from the dehydrated ion peaks. The sample contains plant sterols as well as cholesterol, because silkworms live on mulberry leaves. The peak a t mle 369 shows that cholesterol is the main component and those a t mle 383,395, and 397 show the existence of campesterol, stigmasterol, and the sterol ester of /%sitosterol, respectively. The four CI-MS spectra of the four cholesterol ester samples have respective characteristic patterns. The cholesterol ester sample extracted from blood serum contains Cls,l-ester as main component. I t contains Cls:oester, Clez-ester, and Cls,s-ester. The cholesterol ester component of egg yolk contains some C1::o-ester and Clg,o-ester as well as Clas-ester which is the main component. The cholesterol ester sample extracted from silkworm eggs, contains Cls,s-ester as the main component and a variety of cholesterol esters of the to group.
580
ACKNOWLEDGMENT We thank S. Onishi (Scientific and Industrial Instrument Div.) for help with the experiments.
LITERATURE CITED (1) (2) (3) (4)
(5) (6) (7) (8) (9) (10) ( 1 1) (12) (13)
A . Kuksis. Can. J. Biochem., 42, 409 (1964). A. Kuksis, Can. J. Biochem., 42, 419(1964). A. Kuksis, L. Mari, and D. A. Garnall, J. LipidRes., 8, 352 (1967). A . Kuksis. "Lipid Chromatographic Analysis," G. V. Marinetti, Ed., Marcel Dekker, New York, N.Y., 1967, Vol. l,p 312. L. Swell, Proc. SOC.Exp. Biol. Med., 121, 1290 (1966). N. Ikekawa, Jap. J. Exp. Med., 41, (3), 163 (1971). T. Murata, unpublished data. M. S.Munson, Anal. Chem., 43, (13), 28A (1971). F. H. Field, Accounts Chem. Res., 1, 42 (1968). T. Murata, S. Takahashi, and T. Takeda, Anal. Chem., 47,537 (1975). J. Folch, M. Lees, and G. H. Sloane-Stanley, J. Bo/. Chem., 266, 497 (1957). G. Schlierf and P. Wood, J. LipidRes., 6, 317 (1965) T. Murata. unpublished data'.
RECEIVEDfor review June 7, 1974. Accepted November 25, 1974. Partially reported a t the Japanese Conference on the Biochemistry of Lipids, 1974.
A N A L Y T I C A L C H E M I S T R Y , VOL. 47, NO. 3, M A R C H 1975
