suggested carrier suppressed sideband technique. The modulation depth us. modulation frequency corresponds closely to the dashed line in Figure 5 with F 3 db = 20 MHz. A continuously wavelength tunable source having identical time characteristics was also realized by cavity dumping a cw dye laser.
are available that have a resolution of 0.1', the minimum lifetime measureable a t 60 MHz would be -5 nsec. Using a sampling oscilloscope to measure the phase lag between signals resulting from fluorescence and scattered excitation, we have checked the principles of operation with several fluorophors. As a typical example, Rhodamine B in ethanol gave a lag of 45' when f = 51 MHz, yielding a lifetime of 3.1 nsec. This is in excellent agreement with the literature value (8).
ACKNOWLEDGMENT The authors are indebted to H. Merkelo for mentioning that our original cavity-dumped laser could be used in phase fluorimetry, and T. McCain for discussions about rf sidebands.
CONCLUSION I t has been demonstrated that the continuously dumped argon-ion laser is an extremely good source for generating visible frequency optical signals suitable for phase fluorimetry. This is particularly important when considering the fact that this same device is an equally good source for pulse fluorimetry ( 3 ) . Thus, only the driving electronics have to be changed (BNC connection) t o switch from one technique t o the other. Further instrumental improvements will be centered about two areas: extending the frequency range by the use of carrier suppressed sideband modulation, and increasing the wavelengths available for excitation by dumping a CW dye laser (9). Note Added in Revision. Since the submission of the original manuscript, we have successfully extended the modulation frequency range of the instrument by using the
LITERATURE CITED R. D. Spencer and G. Weber, Ann. N. Y. Acad. Sci., 156,361 (1969). A. M. Bonch-Bruevich, lzv. Akad. Nauk SSSR, Ser. Fiz.. 20, 591 (1956). F. E. Lytle and M. S.Kelsey, Anal. Chem.. 46, 855 (1974). F. E. Lytle. Anal. Chem., 46, 545A (1974). J. B. Birks and W. A. Little, Proc. Phys. SOC.,London, Sect, A, 66, 921
(1953).
H. Merkelo e t a / . , Science, 164, 301 (1969). Govindjee et ab, Siophys. J., 12, 809 (1972). I. 8. Berlman, "Fluorescence Spectra of Aromatic Molecules." Academic Press, New York, N.Y.. 1971." C. V. Shank and E. P. Ippen, Appl. Phys. Lett., 24,373 (1974).
RECEIVEDfor review July 29, 1974. Accepted October 28, 1974.
Chemical Ionization-Mass Spectrometry. 1. Analysis of Fatty Acids
Application to
Takeshi Murata, Seiji Takahashi, and Tsunezo Takeda Analytical Application Laboratory, Kyoto Laboratory, Shimadzu Seisakusho Lid., Nakagyo-ku, Kyoto, Japan
The recent remarkable development in the combined technique of gas chromatography and mass spectrometry (GC-MS) has opened a wide field of application in analysis of lipids. A great many reports have been presented about analysis of fatty acids since R. Ryhage (1 ) released the first report in 1960. I t is necessary in the GC-MS of fatty acids, as is generally known, that the carboxyl group be esterified before gas chromatography and that the GC fractions be introduced one after another into the mass spectrometer for identification. GC-MS, however, is now one of the most effective tools for analysis of fatty acids. In the mean time, a new technique called chemical ionization-mass spectrometry (CI-MS) has come to be effectively employed in biochemical, pharmaceutical, clinical, and chemical fields, after it was developed by M. S. Munson ( 2 ) and F. H. Field ( 3 )a few years ago. We have noticed that the technique of CI-MS ensures high rate of quasi-molecular ion formation and low rate of fragment ion formation, and applied this technique to analysis of fatty acids. In mass spectra obtained by a CI source, more than 90-95% of the ions formed are quasi-molecular ions (QM+), and only a few per cent of QM+-H20 ions are detected as fragment ions. This follows that it is possible to determine the type and the degree of unsaturation of the fatty acids from the quasi-molecular ions thus obtained, and that, besides, the use of the CI method eliminates the cumbersome proce-
dures to esterify fatty acids and separate them by GC columns: samples can be directly introduced into the mass spectrometer.
EXPERIMENTAL Materials. Lauric acid (Clz:o), myristic acid (C14:.), palmitic acid (Clc:~),and stearic acid (C1S:o) were purchased from Applied Science Laboratory, Inc. Fatty acids samples of coconut oil, cottonseed oil, peanut oil, linseed oil, rapeseed oil and corn oil, of commercial source, were hydrolyzed, and then fatty acids were extracted with ether. For gas chromatography, the extracts were methyl esterified with diazamethane. 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 2-m X 3-mm i.d. glass coil with 15% diethylene glycol succinate on Shimalite 80-100 mesh: 190 "C isothermal. A flame ionization detector was used as the detector for GC. Sample introductions to the mass spectrometer were made by the direct sample introducing unit. To introduce a sample into an E1 source, the sample was put in a glass cell which is attached to the tip of the operation rod. Being cooled with water, the cell was inserted into the ionization source and the sample was then vaporized by rapid heating. In the case of introduction to a CI source, since the operation rod was not cooled by water, the ionization source was kept at 200 O C and the sample was introduced by means of a glass cell. The mass spectra were recorded, as the ionization current indicated on the recorder was watched. The mass spectrometric conditions for EI-MS were as follows. The ion source temperature was held at 210 "C during the EI-MS runs.
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, MARCH 1975
573
I
ca
% I5O0 1
C I I CHI) c120
%I M O 1
a./
OMt-HzO
200
,
250
I
'0°1
OMS 2 8 5 ( C i a )
OM-HzO
Figure 2. CI-MS spectra of fatty acids in coconut oil. The top
1
Figure 1. Mass spectra of myristic acid (top), palmitic acid (middle), and stearic acid (bottom), obtained by CI-MS employing isobutane as reagent gas
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 p A of emission current. The scan speed was 6. The pressure in ionization source was 0.5-1 Torr.
RESULTS AND DISCUSSION Mass Spectra of Fatty Acids by CI-MS. Figure 1 shows the mass spectra of myristic acid (C14:o), palmitic acid (CIS:()), stearic acid (C18:o), obtained by CI-MS employing isobutane as reagent gas. The patterns are quite simple. The ion-molecule reaction of fatty acids in the CI source is caused by the proton transferred, and the quasimolecular ion is recorded as (M l)+. The quasi-molecular ion, (M l ) + , of C12:o is recorded a t rnle 201 with an intensity of 90-93% of the total ion quantity. As for the fragment ions, apparent (M - 17)+ which is actually the dehydrated proton-transferred ion, (M 1) HzO, is recorded with an intensity of below 10%. The quasimolecular ion, (M l)+,of C14:o is recorded a t rnle 229 with an intensity of 85-91%. The dehydrated ion peak, (M - 17)+, is recorded a t rnle 211 with an intensity of about 10%. The quasi-molecular ion (M + l)+,of C16:o and C1s:o are mle 257 and mle 285 with an intensity of 86-90% and 80-89%, respectively. And the dehydrated ion peaks, (M 17)+, are recorded a t mle 239 with an intensity of 5-10% and a t mle 267 with an int,ensity of 10-13%. No high-intensity ions, other than quasi-molecular ions (M 1)+and (M - 17)+,are recorded. As is clear from these data, the ion peaks of CI-MS directly indicate the quasi-molecular ions and, hence, CI-MS permits direct qualitative determination of fatty acid mixture samples. I t follows that GC, which has hitherto been thought essential to analysis of fatty acids, can be dispensed with. The whole analytical procedure can be simplified. Methane and isobutane were used as reagent gas. Isobutane was more suitable because it produces fewer dehydrated fragment ions and fewer fragment ions of alkanes. That molecular ions are recorded provides the advantage that information on the degree of saturation is easily ob-
+ +
+
+
+
574
was
obtained with methane, and the middle with isobutane, as reagent gas. The bottom spectrum was obtained using an El source
tained. C1s:o, Cls:~,and CIS:^, for example, can be easily differentiated from the difference in mass number of 2 amu. As for the relative intensities of QM+, QM+ 1, and QM+ 2 of fatty acid isotopic peaks of fatty acids (QM+ = loo%), QM+ 1 and QM+ 2 of C1~:owere 20.9 and 3.3%, respectively, and those of Cl6:0, 18.3 and 2.8%, and those of c14:o,17.9 and 2.1%. I t must be remembered that the peaks of the QM+ 2 ion of unsaturated fatty acids may overlap the QM+ of saturated fatty acids of the same number of carbon atoms. But, since the intensity of this interfering peak is very small-about 3.3% for Cls:o-and the direct inlet method is used, from which a high quantitativeness cannot be expected, this overlapping may not cause serious errors. Analyses of Various Fatty Acids by CI-MS. Figure 2 shows the CI-MS spectra of fatty acids extracted from coconut oil by hydrolysis. The top spectrum was obtained with methane, and the middle with isobutane, as reagent gas. The bottom spectrum was obtained using E1 source. In the CI-MS spectra, whether the reagent gas be methane or isobutane, caproic acid (c6)gives its quasi-molecular ion a t rnle 117, caprylic acid (Cs) a t rnle 145, capric acid (Clo) a t mle 173, lauric acid ((212) a t m/e 201, myristic acid (C1.J a t mle 229, palmitic acid (c16)a t mle 257, stearic acid (C18:o) a t rnle 285, oleic acid (C18:1)a t mle 283, and linoic acid (Cle:~)a t mle 281. The peak a t mle 183 is the only dehydrated peak derived from Clz. There is no other dehydrated peak recorded. As for the fragment ions recorded along with these quasimolecular ions, in the case of isobutane as reagent gas, there are recorded only a few small peaks-a dehydrated C12 and peak (largest one) a t rnle 183 with an intensity of 11%and others a t mle 103 with 2% intensity, a t rnle 110 with 3% intensity, a t mle 127 with 2% intensity, and a t rnle 211 with 2% intensity. In the case of methane as reagent gas, there are more fragment ions recorded compared with isobutane gas-at mle 183 with 18% intensity, a t rnle 103 with 11%intensity, a t mle 137 with 11%intensity, a t rnle 131 with 7% intensity, a t rnle 155 with 7% intensity, and a t mle 211 with 4% intensity. In the case of the E1 source, fragment ions of fatty acids are recorded a t rnle 115,157,101,201,171,and 143 with an intensity of 62,59,54,32, 31, and 29%, respectively, relative to the peak a t rnle 131 ( = 100% intensity). The quasi-mo-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, MARCH 1975
+
+
+
+
+
C rJ S
Figure 3. Gas chromatogram of the fatty acids methylesters extracted from coconut oil by hydrolysis detected by an FID Conditions: 2-m X 3-mm i.d. glass column packed with 15% diethylene glycol succinate on Shimaiite 80-100 mesh; 190 OC isothermal
Figure 4. Spectra of the fatty acids extracted from rapeseed oil by hydrolysis, measured by CI-MS and ELMS
lecular ions, used in qualitative determination, have low in, for Clz, 9% tensities--'?% for cg, 4% for CS, 7% for C ~ O28% for C14, 6% for CIS, 3% for C l s : ~0.7% , for C l s : ~and , 0.6% for cl8:S.
For the purpose of comparison, the fatty acids extracted from coconut oil by hydrolysis were methyl esterified and gas chromatographed with a polyester column. Figure 3 shows the chromatogram, which is quite similar to the pattern obtained by CI-MS. The quantitative values of the fatty acids relative to Clz (= loo), measured on the basis of peak areas in GC and on
the basis of ion intensity in CI-MS, are shown in Table I. There is rather good agreement between the data of GC and that of CI-MS, except for C6 which was counted as 1 (GC) and 18 (CI-MS) and C8 which was counted as 24 (GC) and 12 (CI-MS). These data show that CI-MS will become a useful tool not only for separation and identification but also for quantitative determination. When samples are introduced, they are heated either by the ion source temperature or by heating the probe through which samples are introduced. The intensities of the quasiANALYTICAL CHEMISTRY, VOL. 47, NO. 3, MARCH 1975
*
575
lox+
2x
pl
3
c
0
N N
rc)
----c
Figure 5. G a s chromatogram of the methyl esters of fatty acids of rapeseed oil, by a capillary column ( 4 ) Conditions: 1504 X 0.01-inch capillary column coated with 1.4% butane diol succinate; 170 O C isothermal
Table I. Quantitative Values of the Fatty Acids Extracted from Coconut Oil by Hydrolysis Relative to Clz ( = loo), Measured on t h e Basis of P e a k Areas i n GC a n d on the Basis of Ion Intensity i n CI-MS GC
C1 (CH4)
Table 11. Quantitative Values of Fatty Acid Components in P e a n u t Oil, Corn Oil, Cottonseed Oil, a n d Linseed Oil by Hydrolysis Relative to Base P e a k ( = 100) Measured on the Basis of Ion Intensity in CI-MS Using Isobutane a s Reagent Gas
C1 (iso-C4HI0)
Corn
Cottonseed
Linseed
4 12 13
5
...
...
6
c14
9
2
1
3
ClO
18 41 12
c 12
24 14
6
c12
100
100
100
c16:1
37 15 4 10 2
31 23 3
28 18
c18:0
6 3 27
5
c18:2
5
18 8
c18:3
7 8 6 27 100 12
1
c6
C8
c14 c16
c18:0
c18:1 c18:2
8
c16:0
c1*:1
C20:O C20:l
c20:z
molecular ions thus obtained vary to some degree, because of the variation in the temperature a t sample introduction. This naturally causes errors in quantitative determination. Our present study, therefore, was limited to separation and identification. (We believe a high reliability can be expected if temperature-controlled sample injection ports are used.) Figure 4 shows the spectra of the fatty acids extracted from rapeseed oil by hydrolysis, measured by CI-MS and EI-MS, and Figure 5 shows the gas chromatogram of the methyl esters of fatty acids of rapeseed oil, by a capillary column. From the CI-MS spectrum (isobutane as reagent gas), c12:0, c 1 4 : 0 , cl5:0, c16:0, cl6:l, c16:2, c16:3, c 1 7 : O ~cl8:0,ClS:l, c18:2, c18:3,c19:0,c19:1,c20:0, c20:1,c20:2, c 2 0 : 3 , c22:0, c22:1, C22:2, C 2 4 : 0 , C24:1, and c 2 6 : o were identified. The degree of unsaturation can be easily determined. This is one of the greatest advantages of mass spectrometry. No dehydrated ion peaks are recorded. In the CI-MS with methane as reagent gas, the dehydrated ions of the CIS group are recorded a t mle 263 and 265, those of the C21 group a t mle 291 and 293, and those of the C22 group at rnle 319 and 321. Besides, fragment ion peaks are recorded between mle 100 and 250 with intensities between 5 and 20%. 576
Peanut
c20:3 c20:5
100 56 13 5 52 24
5
...
6 52 100 13 5
c22 :0
12
c 2 2 :1
10
5 12 10 5
c22:2 c22:3 c22 :5 c22 :6
c24:5 c24:6 c24:7 c 2 6 :0
... ... ... ... ...
... ...
...
...
... ...
10
... ...
c21:4
2
...
... ...
c21:3
1
10
4 4 6 4
...
...
... ...
... ...
26 6 4
... ...
... 5
11 24
.
...
5
... 15 60 84 100 4 16 14 22
... ...
...
5 6
3 4
... ... ...
...
... ...
Comparison with gas chromatograms ( 4 ) by a capillary column shows that there is good qualitative agreement between the two data. I t must be noted that C26:" which cannot be detected by GC can be well identified by CI-MS. The EI-MS spectrum is shown a t the bottom of Figure 5. Table I1 shows the hydrolyzed fatty acid components of various vegetable oils determined by CI-MS. The QM+ intensities given are relative to C18:1 (100) for peanut oil, C18:2 for corn oil, and C 1 8 : 2 for cottonseed oil, and C18:3 for linseed oil.
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, MARCH 1975
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 palmi-
tate (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.
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