which is observed under longwave UV. The fluorescence is attributed to the formation of the respective quinazolinones of these compounds. Medazepam, N-desmethyl diazepam, and oxazepam all show strong fluorescence whereas diazepam and 3-hydroxy diazepam fluoresce weakly, while N-desmethyl medazepam does not fluoresce at all. These compounds can be further characterized by acid hydrolysis in situ in a 105 “C oven when the N-methyl and Ndesmethyl 1,4-benzodiazepin-2-0nesare converted into their respective yellow colored benzophenones MACB and ACB. Those compounds which hydrolyze to the ACB derivative give a pink to purple colored spot of the diazo-chromophore when reacted with the Bratton-Marshall reagents (17), characteristic of the primary aromatic amine group.
The results of these experiments using the pharmaceutical trade formulation of medazepam (Nobrium) in man are in agreement with the findings of Schwartz and Carbone (5)who used ‘Gmedazepam administered to man for their metabolic studies.
(17) A. C . Bratton and E. K. Marshall, J. Biol. Chern., 128, 537 (1939).
RECEIVED for review June 1, 1970. Accepted September 8, 1970.
ACKNOWLEDGMENT
The authors wish to thank Dr. A. S. Leon and his staff for conducting the clinical studies and for the supervision of the human subjects at the Beth Israel Hospital, Newark, N. J. We also thank Messrs. T. Danielian and R. Mc Glynn for the drawings of the figures presented.
Quantitative Analysis of Triglyceride Mixtures by Mass Spectrometry Ronald A. Hitesl Northern Regional Research Laboratory, Peoria, Ill. 61604 A rapid and sensitive method has been developed for determining the molecular weight distribution of triglyceride mixtures that occur naturally as fats and oils. The mass spectrum of the fat is measured by placing the sample directly into the ion source, and the consequent fractionation of the sample, caused by molecular distillation, is corrected. Triglyceride compositions have been measured for kokum and cocoa butters, olive, peanut, cottonseed, corn, soybean, sunflower, safflower, and linseed oils. When observed values were compared to theoretical values, they had a high overall correlation. Several potential applications of this mass spectral technique to problems of lipid research have been tested. A NATURAL FAT may consist of several dozen different triglycerides. For example, Table I lists the 18 possible triglycerides formed from three fatty acids-palmitic, stearic, and oleic. Although the amount of each triglyceride occurring in a fat has considerable academic interest and practical significance, there are few methods for the quantitative analysis of triglyceride mixtures. Such procedures as fractional crystallization, ester distillation, countercurrent distribution, thin-layer chromatography, and gas chromatography either are time consuming, lack resolution, or require large samples (I). To overcome some of these problems and to provide an analytical technique suitable for research and industrial applications, a semiautomated mass spectrometric method was developed for measuring the triglyceride composition of fats. The method is both Present address, Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Mass. 02139 (1) M. H. Coleman, Aduan. Lipid Res., 1, 1 (1963); H. J. Dutton, “Progress in the Chemistry of Fats and Other Lipids,” Vol. VI, R.T. Holman, W. 0. Lundberg, and T. Malkin, Ed., Macmillan, New York, 1963,p 313. 1736
rapid (ca. ‘12 hour per sample) and sensitive (less than 100 pg of fat is needed) and involves no pretreatment of the sample. Good mass spectra of triglycerides can be obtained by introducing the sample directly into the ion source (2). Molecular ions, M+, and ions due to the loss of 18 amu from M+, (M - 18)+, are observed. Other features of these spectra have been reported elsewhere (2). Since there are no intense peaks within at least 200 amu of the molecular ion to act as interferences, it was predicted that the molecular weight distribution of a triglyceride mixture could be simply measured from its mass spectrum. This prediction has now been verified experimentally. Because molecular weight gives only the number of carbon atoms and double bonds in the triglyceride, it is impossible to distinguish between positional isomers (PSO us. POS us. OPS ; see Table I for explanation of abbreviations) or between isologs (dioleylstearin us. distearyllinolein). This limitation reduces the number of components that can be differentiated. In Table I, for example, the 18 possible triglycerides are grouped into 10 discrete molecular weights. EXPERIMENTAL
Materials. Tristearin, 1,3-distearylpalmitin, 1,3-dipalmitoylstearin, and tripalmitin were the gift of W. F. Geddes, University of Minnesota. Estimated purity is better than 98%. 1,2-Dipalmitoylolein was used as purchased from Supelco, Inc. (purity 99+%). Kokum butter was supplied by E. S . Lutton, Procter & Gamble. The other vegetable oils came from commercial sources. None of these oils were refined or treated in a way that would alter the triglyceride composition; all were stored at less than 5 OC. The modified tallow samples came from an industrial source. An artificial (2) M Barber, T.0. Merren, and W. Kelly, Tetrahedron Lett., 1063 (1964).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 14, DECEMBER 1970
cocoa butter was prepared by selective hydrogenation of cottonseed oil stearin (3). The soybean-olive oil mixtures and the fractionation standard mixture were prepared by weight from the materials listed. The following fatty acid compositions were measured by conventional techniques ( 4 ) : A. Kokum butter (Garcinia indica): palmitic, 1.4%; stearic, 57.9 %; oleic, 40.7 %. B. Cocoa butter (Theobroma cacao): palmitic, 25.7 %; stearic, 33.8 %; oleic, 35.9 %; linoleic, 3.4%; linolenic, 1.2%. C. Olive oil (Olea europaea cesae): palmitic, 16.6%; palmitoleic, 4.4%; stearic, 3.1 %; oleic, 57.1 %; linoleic, 18.8 %. D. Peanut oil (Arachis hypogaea): palmitic, 10.8%; stearic, 4.4%; oleic, 48.5 %; linoleic, 28.0%; linolenic, 1.8 %; arachidic, 1.4%; eicosenoic, 1.2 %; behenic, 2.6%; lignoceric, 1.3%. E. Cottonseed oil (Gossypium species): myristic, 1.1 %; palmitic, 21.3%; palmitoleic, 1.0%; stearic, 2.8; oleic, 18.9 %; linoleic, 54.9 %. F. Corn oil (Zea mays): palmitic, 11.2%; stearic, 3.1 %; oleic, 27.0%;; linoleic, 56.8%; linolenic, 1.9%. G. Soybean oil (Glycine max): palmitic, 10.3%; stearic, 3.6 %; oleic, 22.8 %; linoleic, 55.0%; linolenic, 8.3 %. H. Sunflower oil (Helianrhus annuus): palmitic, 6.6 %; stearic, 4.4%; oleic, 15.7%; linoleic, 73.3 %. I. Safflower oil (Carthamus tinctorius): palmitic, 7.3 %; stearic, 2.9%; oleic, 14.4%; linoleic, 75.4%. J. Linseed oil (Linum usitatissimum): palmitic, 6.0%; stearic, 4.0%;; oleic, 20.5 %; linoleic, 16.0 %; linolenic, 53.5 %. Interesterificationof Cocoa Butter. Cocoa butter was dried at 110 "C under vacuum with stirring for 1 hour. After 1 % NaOCW3 was added, the mixture was held at 130 'C for 1 hour. Acetic acid was added to destroy the'catalyst, and the fat was dissolved in ca. 3 volumes of petroleum ether. The mixture was washed with water until neutral and dried over Na2S04. Solvent was removed under vacuum at 70 " C ; a yield of 85 was obtained. Mass Spectrometry. The mass spectrometer was a Nuclide Corp. 12-90-DF, a double focusing instrument of NierJohnson geometry. It was operated at a resolution of approximately 1 :1000. The ion source temperature was controlled at 250 f 1 "C and the direct inlet probe tempera4 "C. The electron energy was 70 ture was set at 289 eV. Spectra were repeatedly scanned from about m/e 700 to 900 in 5.8 minutes per scan. The peak intensities and corresponding magnetic field readings were determined by an Infotronics CRS-160 digitizer; output was on punched paper tape. Method. From 100 to 500 pg of the fat of interest is placed directly into the ion source of the mass spectrometer and heated at 289 zt 4 "C until thermal equilibrium is established and the total ion current is constant. This takes from 5 to 10 minutes. At least three mass spectra are then recorded and digitized over the mass range of approximately mje 700 to 900. This mass range was selected because it is sufficient to include all triglycerides formed from C-16 and (2-18 fatty acids, the most common fatty acids in vegetable and animal fats. The resulting mass spectral data on punched paper tape are then read into a computer (IBM 1130). To establish a direct proportionality to mass, the magnetic field readings are squared and then converted to masses by an extrapolation algorithm, which simulates the normal manual process of
*
~
~~~
(3) E. N. Frankel and J. C. Cowan, Northern Regional Research
Laboratory, unpublished work, 1969. (4) E. C. Horning, A. Karmen, and G. C. Sweeley, "Progress in the Chemistry of Fats and Other Lipids," Vol. VII, R. T. Holman, Ed., Macmillan, New York, 1964, p 167.
Table I. Possible Triglycerides Formed from Stearic, Palmitic, and Oleic Acidsa Triglyceride Abbreviation Mol Wt Tripalmitin PPP 806 1,3-Dipalmitoylolein POP 832 1,2-Dipalmitoylolein PPO 832 1,3-Dipalmitoylstearin PSP 834 1,2-Dipalmitoylstearin PPS 834 1,3-Dioleylpalmitin OPO 858 1,2-Dioleylpalmitin 858 OOP 1-Palmitoyl-3-oleplstearin PSO 860 1-Palmitoyl-3-stearylolein POS 860 1-Oleyl-3-stearylpalmitin OPS 860 1,3-Distearylpalmitin SPS 862 1,ZDistearylpalmitin SSP 862 Triolein 000 884 1,3-Dioleylstearin os0 886 1,2-Dioleylstearin 00s 886 l13-Distearylolein sos 888 1,ZDistearylolein SSQ 888 Tristearin sss 890 a Steroisomers are excluded.
counting; i.e., starting from one known mass all other peaks are assigned masses on the basis of their appearance at regular intervals. The intensities are then normalized to 9999 units for the largest and corrected for contributions of 2H, 13C, 1 7 0 , and ISO. These are large corrections at the high masses involved here; tristearin, for example, has a molecular 1/M composition of Cj7Hi100Sfor which the calculated M ratio is 0.636 and the M 2/M ratio is 0.211. Next, to measure as much of the total ion current for each component as possible, the M+ and (M - 18)f intensities are summed, This step presents problems only with a triglyceride containing nine double bonds because its molecular ion would fall at the same mass as the (M - 18)+peak of the corresponding saturated triglyceride. The only instance of such interference that can occur in fats from natural sources is trilinolenin us. tristearin. Fortunately, both of these triglycerides do not occur together in high concentrations in naturally occurring fats. The several replicates are averaged before correction factors are applied (see below). The relative standard deviation of these averages is less than 5%. The average values are now renormalized and the resulting molecular weightintensity table is printed. . Some comments on the method are in order. Although the amount of sample recommended (100 to 500 pg) is large relative to direct introduction probe requirements, this much sample is needed to ensure codistillation of all triglycerides of interest for the duration of the measurement (15 to 30 minutes). It is expected that a faster mass spectrometer scan rate would allow a considerable reduction in sample size requirement. Unlike most quantitative mass spectrometric techniques, this method does not use molar sensitivity correction factors. These factors relate peak intensity at a given mass to the concentration of the compound represented by that mass. These factors are not applied in this case because various structurally different triglycerides are represented by the same mass; thus there can be no unique Correction factor for each mass.
+
+
RESULTS AND DISCUSSION
Fractionation Correction. Because a direct introduction inlet system is used, a correction must be made for the consequent molecular distillation. This correction, called the fractionation correction, is obviously a function of the rate of
ANALYTICAL CHEMISTRY, VOL. 42, NO. 14, DECEMBER 1970
e
1737
0 PPO
Carbon Number Figure 1. Fractionation correction factor (Fa)us. triglyceride carbon number (see Table I for explanation of point labels) molecular distillation of a given compound in the mixture. This rate for component i, W,, is given by W,
=
kP,T1/2M,-I12
(1)
where k is a constant, Pc is the vapor pressure of i, T is the absolute temperature, and M i is the molecular weight of i (5). A correction factor for i, F,, relative to a standard component (tristearin for example) is defined:
(”)
-1,Z F. - W, - P, 2 1 WSTD PSTDM S T D
(2)
where Tis constant. Because measurements are limited to those triglycerides with 48 to 54 carbon atoms in the alkyl chains, the molecular weights vary between 806 and 890 amu. Thus the factor ( M ~ / M s T D )varies - ~ / ~ between 1.00 and 1.05, assuming that the standard is tristearin. This factor is neglected. For triglycerides it has been shown that log Pi
=
(3)
-kCc
where C, is the number of carbon atoms in triglyceride i (6). (It is assumed that unsaturation has no effect on vapor pressure.) Hence log Fz = -k(Ci - CBTD) = -kCc
+ k’
(4)
Thus, a plot of log Fius. Cashould be linear. To test this hypothesis, a mixture of PPP, PSP, PPO, SPS, and SSS of known composition was analyzed according to the technique described above. The ratios of observed to ( 5 ) E. W. Berg, “Physical and Chemical Methods of Separation,”
McGraw-Hill, New York, 1963, p 40. (6) E. S. Perry, W. H. Weber, and B. F. Daubert, J. Amer. Clzem. Soc., 71, 3720 (1949). 1738
0
expected results were calculated and normalized so that the factor for SSS was 1.00. The results from two runs averaged together are shown in Figure 1. If the PPO datum is omitted, the carbon number and the fractionation correction are highly correlated. This experiment confirms the feasibility of correcting for the fractionation of homologs from a direct introduction inlet. In practice, the intensities for those triglycerides having a molecular weight between mje 798 and 806 are divided by the correction factor for 48 carbons (4.57 from Figure 1); between mje 807 and 834, by 2.77; and between mje 835 and 862, by 1.68. Of course, all experiments and calibrations are run at a constant direct inlet probe and source temperature. The datum point for PPO lies far from the line in Figure 1; in fact, it deviates by a factor of 2.18. This deviation is attributed to different molar intensities for the molecular ion peaks of PSP and PPO rather than to differences in vapor pressure as a function of unsaturation. This deviation is not unexpected since one would predict that the introduction of a double bond into the molecule would have a significant effect on the stability of the molecular ion and thus would affect the observed relative intensities. To correct for this effect, all fully saturated triglyceride intensities (molecular weights 806, 834, 862, and 890) are multiplied by 2.18. In making this correction, it has been assumed that the fractionation correction factors for all unsaturated triglycerides fall on a line parallel to that for saturated triglycerides regardless of degree of unsaturation. Vegetable Oils. To determine the accuracy of the method, the oils listed in Table I1 were analyzed. Observed results were then compared to those values predicted by the 1,3random (saturates) 2-random (unsaturates) theory of triglyceride distribution (1). In simplified terms this theory assumes that unsaturated fatty acids are preferentially esterified at the 2-position of the triglyceride. For example, instead of all 18 triglycerides mentioned in Table I being present in an oil, only those with oleic acid in the 2-position would occur. These would be POP, OOP, POS, 000, OOS, and SOS. The amounts of these various triglycerides can be predicted from the known fatty acid composition in the following manner : Let A( be the overall percentage of fatty acid i as measured by gas chromatography, Bd the percentage of i in the 2-position relative to the 2-position only, and C, the percentage of i in the 1- or 3-position. It follows that
CA, = CB, = 2.
z
Z C , = 100%
(5)
1.
and
+
A , = (22% &)/3 (6) The theory assumes B, = 0 if fatty acid i is saturated and Br = nAe if fatty acid i is unsaturated (n is the normalization factor for unsaturates only). Ce for all i follows from Equation 6. The percentage of triglyceride XYZis
XYZ = inC,BuCzI1O4 (7) where m = 2 if XYZ is unsymmetrical about the 2-position and m = 1 otherwise. A fat containing less than 33 % ufisaturated fatty acids is excluded in this treatment. As an example, the following compilation can be given for kokum butter. Fatty acid Palmitic Stearic Oleic
ANALYTICAL CHEMISTRY, VOL. 42, NO. 14, DECEMBER 1970
A, 1.4 57.9
40.7
B,
Ci
0.0 0.0 100.0
86.85 11.05
2.10
Table 11. Observed and Theoretical Triglyceride Compositions for Vegetable Oils Kokum Cocoa Olive Peanut CottonCorn Soybean Sunflower Samower Linseed oil oil butter butter oil oil seed oil oil oil oil Average number double bonds ~
Carbon Double Mol Wt No. bonds 48 48 48 50 50 50 50 50 52 52 52 52 52 52 52 54 54 54 54 54 54 54 54 54 54
2 1 0 4 3 2 1 0 6 5 4 3 2 1 0 9 8 7 6 5 4 3 2 1 0
802 804 806 826 828 830 832 834 850 852 854 856 858 860 862 872 874 876 878 880 882 884 886 888 890
Correlation coefficient a OB = observed, TH
0.41 OBa TH
0.46 0.99 1.11 1.30 1.46 1.58 1.62 1.65 2.13 OB TN OB TH OB TH OB TH OB TH OB TH OB TH OB TH OB TH 0 . 7 0.1 0.8 0 . 8 0.8 0 . 3 0.3 0.4 0.4 0.8 0 . 4 1.2 2.3 1.2 2.3 13.413.2 2.5
0.1 1.2 0.3 0.1 4.2 0 . 6 0 . 9 4.5 0 . 4 1.6
1.3 0.4 1.1 0.5 8.5 4.6 3.0 2.414.716.0 1.4 0 . 5 1 0 . 1 9 . 8 1 4 . 3 1 8 . 8 5.4 3 . 6 3 0 . 7 3 4 . 6 0 . 7 1 . 7
0.7 1.5 5.1 1 . 2 4.9 18.9 19.2 11.2 69.2 75.4 20.4 0.991=
0.6 5.4 7.5 4.3 0.3
1.4 0.8 1.9 0.5
0.7 0.2 8.5 5.3 0 . 4 1 . 7 0.8 1 . 4 0 . 8 4 . 6 3 . 0 3 . 4 3 1 . 5 2 2 . 0 1 7 . 7 1 2 . 0 1 3 . 5 1 1 . 2 1 2 . 9 1 1 . 2 1 3 . 5 1 3 . 1 4.8 9.7 8 . 0 1 4 . 9 6 . 0 1 1 . 1 4.2 8.4 2.2 4.8 2.0 5.0 1 . 1 9.1 1 . 3 4 . 5 1.1 3.7 1 . 0 2.8 0.4 1 . 6 0.2 1.3 0.7 1 . 3 0.4 0.7 0 . 5 0.5 0.5 0.4 0.6 0.2 0.6 0 . 2 0.5
0.3 0.4 2 . 0 0 . 6 4 . 1 3 . 5 26.9 0 . 1 9 . 7 5 . 8 21.1 12.2 16.4 0.918.618.022.420.3 6.1 3 . 2 19.5 20.0 14.2 15.1 1 . 3 10.7 2 . 2 3.4 1 . 1 4 . 2 0 . 7 22.8 0 . 4 0 . 2 0 . 3 0 . 7
0.99+
0.96
1.1 1.5 0.4 0.1 0.6 0 . 2 0.6 0.5 8 . 0 1 . 3 1.9 0 . 7 1 . 5 0 . 3 0 . 8 0 . 2 1 . 0 0 . 3 0 . 1 2 . 7 0 . 3 0..9 0 . 2 0 . 6 0 . 1 0 . 2 0 . 1 0 . 2 0 . 3 0 . 2
0.92
15.3 15.8 8.3 2.6 0.5
0.90
0 . 0 0.1 0.0 0 . 1 1.4 1 . 1 1.5 1 . 8 9 . 0 7 . 9 31.5 19.7 30.1 22.5 26.4 26.2 21.1 22.6 9.515.5 9.212.3 2 . 1 5.0 2.5 4.1 0.4 0.9 0.6 0.8 0.2 0.1 0.2 0.1 0.92
0.96
46.4 39.0 25.9 25.0 9.412.8 1.0 3.6 0.4 0.7 0.2 0.1 0.99
56.8 19.6 5.7 0.9 0.3
42.5 24.3 9.8 2.3 0.3
14.2 11.9 20.0 14.9 10.6 3.7 1.6 0.6 0.4
0.97
5.4 3.2 4.6 1.9 1.0 0.2 15.2 13.6 21.5 14.4 10.4 5.1 1.9 0.6 0.1
0.98
theoretical,
The percentages of triglycerides in kokum butter are: POP = 1 x 2.10 x 100.0 x 2.10/104 = 0.044 OOP = 2 x 11.05 x 100.0 x 2.10/104 = 0.46 POS = 2 X 2.10 X 100.0 X 86.85/104 = 3.6 000 = 1 X 11.05 X 100.0 X 11.05/104 = 1.2 0 0 s = 2 X 11.05 X 100.0 X 86.85/104 = 19.2 SOS = 1 X 86.85 X 100.0 X 86.85/104 = 75.4 In this example, all the possible triglycerides have different molecular weights. If this condition were not so, the calculations would have included a final step in which all percentages referring to the same molecular weight are summed. In this way, a theoretical molecular weight-intensity table is generated. These operations were programmed for a computer using the gas-chromatographic fatty-acid data given under Experimental. The calculations for each oil appear in the columns headed “TT”’ (for “theoretical(’) in Table 11. The corresponding experimental values appear in the columns headed “OB” (for “observed”). If the theoretical value for a component at a given molecular weight was less than 0.050 %, neither a theoretical nor an observed value is recorded. The observed values were normalized so that their sum was equal to the sum of the theoretical values. The oils are arranged in Table I1 so that average unsaturation increases from left to right. Correlation coefficients between observed and theoretical values for each oil are also reported. Statistical analysis of the 140 datum pairs shows that the overall correlation coefficient is 0.96. Kokum and cocoa butters and olive and linseed oils show particularly good agreement. The other oils had larger differences, reaching more than 10% absolute for mje 878 in cottonseed, corn, and safflower oils. The precision of the observed values is good: two different runs of safflower oil, for example, show no differences larger than 0 . 6 z (56.8 us. 57.473. Examination of the values in Table I1 reveals greater dis-
agreement between observed and theoretical values as the average unsaturation of the oil increases. This effect indicates that molar sensitivities vary as a function of the number of double bonds in the triglyceride. Thus, the assumption that the fractionation correction factors for all unsaturated triglycerides fall on a line parallel to that in Figure 1 and passing through the PPO point is probably not valid. Nevertheless, thc agreement between observed and theoretical values throughout Table 11is gratifying, considering that the theoretical assumptions are not exact, that no molar sensitivity corrections were used, and that the measurements cover a wide dynamic range. The errors here are on the order of those for other techniques for the analysis of triglycerides. The possible use of this technique in research and industrial environments can be illustrated by various examples. Interesterification. Because a single fat does not have the correct properties for many applications, new fats can be made by chemically scrambling the fatty acid moieties among all glycerol positions. Since this process produces a random distribution of triglycerides, it is called randomization or interesterification. As an example of the mass spectral technique described here, natural and randomized cocoa butter and natural and randomized tallow (beef fat) were analyzed (Figure 2). Note the great change in the cocoa butter composition upon randomization; there is a change in physical properties as well. This change in properties results from the nonrandom structure of the natural material; namely, the preferential esterification of unsaturated fatty acids at the beta-position. Also shown in Figure 2 are the corresponding data for a partially hydrogenated tallow having about 30 % palmitic acid, 5 5 % stearic acid, and 15% oleic acid. Unlike cocoa butter, the difference between the natural and randomized
ANALYTICAL CHEMISTRY, VOL. 42, NO. 14, DECEMBER 1970
e
1739
Natural
j
Modified Tallow
Figure 2. Partial triglyceride composition of natural and randomized cocoa butter and tallow. Only selected triglycerides are shown. The order of the letters is not significant (e.g.,PSO = POS = OPS) Table III. Triglyceride Analysis for Artificial utter Prepared from Cottonseed Oil Stearin Artificial cocoa Natural cocoa No. double bonds butter, % butter, % 0
2.8
1
37.4 25.6 21.9 9.0 2.4
2 3 4 5
6 Average no. double bonds per triglyceride
1.0
64.1 23.6 8.7
2.6
0.9
... ~ . .
2.08
1.48
distributions is small; thus, there is less specificity for unsaturated acids in the beta-position of tallow than in coca butter. Other data from lipase hydrolysis confirm that animal fats have less beta-unsaturated specificity than do vegetable fats ( I ) . This comparison of molecular weight distributions before and after randomization allows one to obtain some qualitative information on intramolecular triglyceride structure. Furthermore, triglyceride analysis is often the only means of determining whether a randomization reaction has been effective. Artificial Cocoa Butter. A possible domestic source of a cocoa butter substitute is selectively hydrogenated saturated fractions of vegetable oils. To test the similarity of such experimental reaction products to natural cocoa butter, they can be analyzed by the technique described here. Table I11 lists results for a product obtained from cottonseed oil stearin. Because palmitic and stearic acids behave similarly with regard to physical properties of a fat, the data are compared to natural cocoa butter on the basis of the number of double bonds in the triglycerides. The values given in Table 111 are derived from a molecular weight us. intensity table (such as Table 11) by summing all values representing the same degree of unsaturation regardless of carbon number. The following conclusion can be drawn from Table 111: Although the synthetic and natural fats are somewhat similar (note 0 and 2 double bond values), the synthetic sample has, on the average, considerably more double bonds per triglyceride than does the natural sample (2.08 us. 1.48). Therefore, in future experiments a more saturated oil fraction should be used or the extent of hydrogenation should be increased somewhat. By comparing the triglycerides in this manner, rather than the fatty acids, one takes into account the eFect of the nonrandom structure of the natural fat. 1740
e
I 0.2
I
I
0.4 0.6 Soybean oil/olive oil
I 0.8
I 1
1.0
Figure 3. Plot of intensity ratio of peaks at m/e = 878 and 884 as a function of soybean to olive oil ratio for several mixis a hypothetical unknown (see text) tures. The point “XYy Adulteration. Frequently it is necessary to determine if an oil has been diluted with another, less expensive oil. The mass spectral technique described could be a simple and rapid means of making such a determination. As an example of this forensic application, the problem of determining the amount of soybean oil present in olive oil has been studied. Because olive oil contains from 35 to 60% triolein (mol wt 884) and soybean oil contains from 15 to 25% trilinolein (mol wt 878), the ratio of the intensities of these two molecular ion peaks should be directly proportional to the ratio of soybean to olive oil: -Z878 = k soybean oil Zsaa olive oil Unfortunately, the proportionality constant, k , is expected to differ from oil to oil because of the natural variability of composition of oils from the same plant species. This variation is demonstrated by Figure 3 which shows a plot of Iga4us. the soybean-olive oil ratio for several mixtures. Those data lying on the line represent mixtures all prepared from the same oils, but the point labeled “ X ’ represents a mixture prepared from different soybean and olive oils. The deviation of “X” from the calibration line is obvious and gives a 15% error in the calculated olive oil percentage (calculated, 89%; real, 77%). However, this error is often acceptable. Therefore, this technique, which needs no sample preparation, could find application as a means of rapidly estimating the presence of large amounts of a diluent in an unknown oil. Alternately, however, the determination of the concentration of mixtures of known oils could be carried out with high accuracy. ACKNOWLEDGMENT
The assistance and encouragement of W. K. Rohwedder, H. J. Dutton, and J. C. Cowan are gratefully acknowledged. RECEIVED for review May 7, 1970. Accepted September 17, 1970. Presented in part at the 17th Annual Conference on Mass Spectrometry and Allied Topics, Dallas, Texas, May 18-23, 1969. The author thanks the National Research Council for a Postdoctoral Research Associateship during 1968-69. The Northern Regional Research Laboratory is headquarters for the Northern Utilization Research and Development Division, Agricultural Research Service, U. s. Department of Agriculture. Mention of trade or company names is for identification only and does not imply endorsement by the Department.
ANALYTICAL CHEMISTRY, VOL. 42, NO. 14, DECEMBER 1970
