50 to 60 “C. We have determined that Beer’s law is obeyed in the concentration range 0.88 through 5.3 ppm of palladium for solutions at pH 4.0 and at a wavelength of 500 mp (molar absorptivity for the complex is 1.14 X lo4); therefore, the spectrophotometric titration method should be applicable under these conditions. The results of the titration shown in Figure 3 were obtained using 3.52 X millimoles (3.75 ppm) of palladium. The effect of all the platinum metals and various other ions on this method has been determined. This method is not applicable in basic solution (pH 8) since the reaction is too slow at 60 “C.

Shamir and Schwartz ( 1 ) have reported that solutions of nitroso R are stable for several months. We have observed that these solutions are not stable on standing and should be prepared daily if they are to be used in spectrophotometric titrations or mole-ratio studies. The spectrophotometric titration of palladium with nitroso R affords an excellent method for standardizing solutions of this reagent.

In forming the Pd(I1) complex, the nitroso R is clearly functioning as a bidentate ligand. This is supported by the absence of H+ ions in the potentiometric titration of the Na salt, and also by the number of Na atoms found per formula weight in the preparation. All of this is in accord with a coordination number of four, which is expected for a d-8 ion such as Pd (11).

The complex does not seem to dissociate appreciably in so- lution since the intersecting lines in Figure 4 show no curvature before and beyond the point where they meet. In addition, the complex is so stable that it was not decomposed into palladous hydroxide on boiling for 30 minutes at a pH of 12.

RECEIVED for review October 29,1969. Accepted October 19, 1970. The authors are grateful to the Unites States Naval Academy Research Council for financial support for this work.

Structure Elucidation of Aliphatic Aldehydes by Mass Spectrometry of Alkenyl Ethers

P. E. Manni, William G. Andrus,’ and Jack N. Wells Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, Lafayette, Ind. 47907

THE MASS SPECTROMETRIC identification of aldehydes and ke- tones has depended largely upon the recognition of fragments derived from alpha cleavage and the McLafferty rearrange- ment. The usefulness of such correlations in deducing the mass of substituents bonded to the carbonyl group and the degree of substitution at the alpha position, is well-docu- mented ( I ) . Recent applications of artificial intelligence to the interpretation of the low-resolution mass spectra of ke- tones promise to simplify structure assignment further (2) . Substituents on carbon atoms beta or further removed from the carbonyl group can in principle be distinguished by ob- serving fragments that arise by cleavage at branch points. However, this process and alpha cleavage suffer from the common disadvantage that often they do not represent the dominant mode of electron-impact induced decomposition; hence, weak peaks are produced in a mass spectrum and struc- ture assignment is complicated by the fact that their source cannot be established unequivocally. The introduction of ethylene ketals and acetals as fragmentation-directing deriva- tives has circumvented the problem in the case of alpha cleavage (3). Derivatives that would amplify the importance of cleavage at branch points would also be helpful.

1 Present address, The Upjohn Company, Kalamazoo, Mich.

(1) H. Budzikiewicz, C. Djerassi, and D. H. Williams, “Mass Spectrometry of Organic Compounds,” Holden-Day, Inc., San Francisco, Calif., 1967, Chap. 3.

(2) A. M. Duffield, A. V. Robertson, C. Djerassi, B. G. Buchanan, G. L. Sutherland, E. A. Feigenbaum, and J. Lederberg, J. Amer. Clzem. Soc., 91, 2977 (1969).

(3) H. Budzikiewicz, C. Ilrjerassi, and D. H. Williams, “Mass Spectrometry of Organic Compounds,” Holden-Day, Inc., San Francisco, Calif., 1967, pp 258-268.

The conversion of aldehydes to alkenyl ethers (alkenyl ethers as used here refers to alk-1-enyl methyl ethers) is repre- sented by

C,H,CH(R)CH,CHO - C,H,CH(R)CH&H(OCH,), - C4HgfiCH=CH0CH3

b R

Because beta substituents are at an allylic position in the alkenyl ether, cleavage a or b would be a primary mode of fragmentation. This fact and the facile conversion of alde- hydes to alkenyl ethers in high yield (4) suggested that the ethers would be useful derivatives for investigating the struc- ture of beta substituted aldehydes. Application of this tech- nique is demonstrated using heptanal, 3-methylheptanal, 3- ethylheptanal, and 3-isopropylheptanal.

EXPERIMENTAL

Mass spectrometric analysis of gas chromatographiceffluents was performed with the LKB 9000 instrument fitted with an 8-ft x 0.125411. glass column containing 3 SE-30 on 100-120 mesh Chromosorb G (acid washed and silanized). Alkenyl ether spectra were measured with the Hitachi RMU-6A spectrometer. Normally an electron energy of 70 eV was employed. Except for molecular ions and several peaks mentioned in the Discussion, mass spectra include only peaks of at least 10% relative intensity.

Preparative gas chromatography was conducted in a Varian A90-S chromatograph equipped with a thermal conductivity detector. A 5-ft X 0.25 in. stainless steel column packed

(4) C. V. Viswanathan, F. Phillips, and V. Mahadevan, J. Chro- matogr., 30, 405 (1967).

ANALYTICAL CHEMISTRY, VOL. 43, NO. 2, FEBRUARY 1971 265

with 5% QF-1 on acid-washed and silanized Chromosorb W (60-80 mesh) was used. Reaction gas chromatography was performed with the same instrument using a 6-ft x 0.125-in. aluminum column filled with 20 % ethyleneglycol succinate and 2% phosphoric acid; the first 6 inches of packing were removed from the column and replaced with anhydrous p-toluenesulfonic acid. Analysis of effluents from the reaction column was accomplished on a Beckman GC-5 chromato- graph equipped with a hydrogen flame ionization detector and fitted with a 6-ft X 0.025-in. stainless steel column containing 3 % SE-30 on Chromosorb G (42-60 mesh). Helium was the carrier gas in all cases.

Reagents. A 10% w/v solution of BF3 in methanol was obtained from Eastman Organic Chemicals. Pentane was purified by passage over silicic acid. Heptanal was purchased from Matheson, Coleman and Bell; other aldehydes were synthesized.

Synthesis. Aldehydes were prepared from acids of general formula RCH=CHCO?H. The alpha, beta unsaturated acids were either purchased (crotonic acid from Matheson, Coleman and Bell) or synthesized (2-pentenoic acid and 4- methyl-2-pentenoic acid) (5) ; synthetic products showed physical and spectral properties in agreement with published data. The synthesis of 3-methylheptanal is presented as an example of the reaction sequence used. New compounds were characterized by spectral data and elemental analyses ; these data are given only for the aldehydes. The yields of aldehydes from 3-substituted acids were 15-22z. No attempt was made to improve yields because sufficient sample was available for this study.

3-Methylheptanal. 3-Methylheptanoic acid was prepared from sec-butyl crotonate and n-butylmagnesium bromide (6). Treatment with thionyl chloride gave the acid chloride which was allowed to react with ethylenimine in the presence of triethylamine to form an N-acylaziridine. Reduction of the N-acylaziridine with lithium aluminum hydride (7) furnished 3-methylheptanal, bp 49-50 "(75.9 mm; IR 2700 (aldehydic CH), and 1730 cm-l (C=O): NMR 6 0.95 (m, 6, CH3), 1.25 (m, 6, CH?), 1.85 (m, 1, CHCH2CHO), 2.20 (m, 2, CH2- CHO), and 10.40 ppm (t, 1, J = 1 Hz, CHO); mass spectrum m/e (re1 intensity) 128 (0.3), 113 (l), 85 (14), 84 (loo), 71 (50), 69 (50), 57 (42), 56 (85), 55 (77), 44 (27), 43 (100).

A 2,4-dinitrophenylhydrazone was formed and recrystal- lized from ethanol-water; mp 79-79.5 "C.

Anal. Calcd. for C14H20N404: C, 54.54; H, 6.54. Found: C, 54.93; H, 6.72.

3-Ethylheptanal. The compound was prepared from 2- pentenoic acid; bp 56-58 "C/7.0 mm; IR 2725 (aldehydic CH), and 1728 cm-1 (C=O); NMR 6 0.90 (m, 6, CHJ, 1.30 (m, 8, CH?), 1.80 (m, 1, CHCHCHO), 2.30 (m, 2, CH2CHO), and 9.66 ppm (t, 1, J = 1 Hz, CHO); mass spectrum m/e (re1 intensity) 142 (0.3), 113 (3), 98 (87), 85 (lo), 83 (lo), 70 (40), 69 (53), 67 (lo), 57 (52), 56 (loo), 55 (48), 44 (5), 43 (44).

The semicarbazide was formed and recrystallized from ethanol-water ; mp 11 1.5 "C.

Anal. Calcd. for C10H21N3: C, 60.27; H, 10.62. Found: C, 60.23; H, 10.59.

3-Isopropylheptanal. The compound was prepared from 4-methyl-2-pentenoic acid; bp 86-88 "C/8 rnm; IR 2700 (aldehydic CH), and 1730 cm-' (C=O); NMR 6 0.90 (m, 9, CH3), 1.25 (m, 6, CH2), 1.80 (m, 2, CH, CHCHCHO), 2.20 (m, 2, CH2CHO), and 10.10 ppm (t, 1, J = 1 Hz, CHO); mass spectrum mje (re1 intensity) 156 (0.3), 113 (19), 112

( 5 ) A. I. Vogel, "Practical Organic Chemistry," 3rd ed., John Wiley and Sons, Inc., New York, N. Y., 1956, pp 465-466.

(6) J. Munch-Petersen in "Organic Syntheses," Vol. 41, J. D. Roberts, Ed., John Wiley and Sons, Inc., New York, N. Y., pp 60-64.

(7) H. C . Brown and A. Tsukamoto, J. Amer. Chem. Soc., 83,2016 (1961).

(55), 99 (2), 84 (19), 83 (13), 81 (l l) , 71 (13), 70 (33), 69 (loo), 68 ( l l ) , 57 (78), 56 (73), 55 (50), 44 (7), 43 (55).

The 2,4-dinitrophenylhydrazone was prepared and re- crystallized from ethanol-water ; mp 104 "C.

Anal. Calcd for C16H24N402: C, 57.13; H, 7.19 Found: C, 56.78; H, 7.38.

Dimethyl Acetals. Each aldehyde was treated with BF3- methanol essentially as described by Morrison and Smith (8), and the mixture of aldehyde and acidic methanol heated until the infrared spectrum of an aliquot showed constant carbonyl absorption. Complete disappearance of the car- bonyl band was not achieved because of methyl ester forma- tion (see below). Base was added, the dimethyl acetals were extracted from aqueous methanol into pentane. Because of the negative temperature coefficient observed for the forma- tion of cyclohexanone dimethyl ketal (9) more recent experi- ments have been performed by allowing the aldehyde to react with acidic methanol at 0 O C for 1 hour; quantitative conversion has been obtained.

Alkenyl Ethers. Initial attempts to effect alkenyl ether formation using a column of ethylene glycol succinate and phosphoric acid at 175 "C (IO) failed because the acetals rapidly hydrolyzed. Replacing the fore-portion of the column with p-toluenesulfonic acid gave high yields of alkenyl ethers which were stable even on chromatographic columns that readily hydrolyzed dimethyl acetals.

Typically, dimethyl acetals in pentane were concentrated in a nitrogen steam and the concentrate was injected onto the reaction column at 175 OC with a helium flow of 60 ml/min. The total effluents were collected in tubes immersed in dry ice-acetone. Infrared spectra of the effluents showed two bands near 1600 cm-1 as expected for a mixture of cis- and trans-alkenyl ethers (11). Chromatographic analysis indi- cated a slight excess (ca. 55:45) of the cis-isomer. The configuration of alkenyl ethers was deduced from retention data (12) and NMR analysis. The effluents contain two other compounds. One is a methyl ester formed by air oxidation of the aldehyde during or preceding dimethyl acetal forma- tion; ester formation is minimized by using freshly distilled aldehydes and short reaction times (0.25-1 hr) at low tem- perature (0 "C). The other component is unreacted dimethyl acetal.

Mass spectra of alkenyl ethers discussed below were ob- tained from samples of the trans-ether isolated by preparative gas chromatography. Each isolate was examined for traces of aldehyde which might have been produced on the reaction column by partial dimethyl acetal hydrolysis (SE-30 was unable to separate aldehydes completely from the correspond- ing alkenyl ethers); neither NMR nor mass spectrometry in- dicated aldehyde contamination.

RESULTS AND DISCUSSION

Peaks arising from the McLafferty rearrangement are prom- inent in the mass spectra of beta substituted aldehydes. The M-44 peak is very intense (50% or greater) in all spectra studied, but the intensity of mje 44 diminishes to ca. 5 when R is ethyl or i-propyl. The intensity of ions formally derived by cleavage at a branch point (M-57, M-R) is also sensitive to structural variation. Whereas M-57 shows an intensity of 50% when R is methyl, intensities of 10% and 2 % are noted for 3-ethylheptanal and 3-i-propylheptanal, respectively.

(8) W. R. Morrison and L. M. Smith, J. Lipid Res., 5, 600 (1964). (9) D. G. Kubler and L. E. Sweeney, J . Org. Chem., 25, 1437

(10) V. Mahadevan, C. V. Viswanathan, and F. Phillips, J . Lipid (1960).

des . , 8 , 2 (1967).

60 (1963). (11) H. R. Warner and W. E. M. Lands, J. Amer. Chem. SOC., 85,

(12) R. A. Stein and V. Slawson, J . Chromatogr., 25, 204 (1966).

266 ANALYTICAL CHEMISTRY, VOL. 43, NO. 2, FEBRUARY 1971

The M-R peak is of appreciable intensity (19x) only in the case of 3-i-propylheptanal. These results show that cleavage at branch points is not a predominant mode of fragmentation for beta substituted aldehydes.

Deuterium-labeling studies (13,14) have revealed rearrange- ments in the fragmentation of hexanal and heptanal that re- sult in the loss of C-2 and C-3, and C-5 and C-6 as ethylene. Scheme I outlines two plausible mechanisms (13) for the re- arrangements; only migration of butyl is shown in Scheme I although the migration of R is also possible. If both se- quences are operative in beta substituted aldehydes, peaks should appear a t M - R C H S H 2 , M--CeHI2 and/or M- C3H6. In no case d o these ions amount to more than 5 Z of the base peak indicating that these pathways are of minor im- portance for the compounds studied.

H I

H

/ 6 5 3 2

CH3CH2CHzCH2CHCHzC=O? I I R H

\

H 0 +. Scheme I

The mass spectra of cis- and trans-alkenyl ethers are sub- stantially the same at 70 eV. The spectra, summarized in Table I, are in vivid contrast to the aldehyde spectra with re- spect to the importance of M-57 and M-R. One of these ions is among the most intense peaks in each alkenyl ether spectrum. This preference toward allylic cleavage facilitates the identification of beta substituents. Both the mass of beta substituents and the degree of branching affect the intensities of M-57 and M-R. When R is hydrogen, only M-57 is noted. However, as R approaches butyl in mass, M-R increases rela- tive to M-57. In the case of i-propyl M-R is more intense. The resulting series of leaving ability, i-CaH, > n-C4H9 > C2HS > CHs > H parallels the data of Marshalland Williams (15) in their study of ethylene ketals and acetals. Thus, the fact that M-43 is more intense than M-57 in the spectrum of 3-i-propyl-1-methoxy-1-heptene signifies the presence of i- propyl rather than n-propyl.

A potential problem to interpretation arises when two sub- stituents have a large difference in mass. Loss of the larger group could be favored to such an extent that only a minute peak would indicate the presence of the smaller substituent. However, when the original compound is a beta monosub- stituted aldehyde the mass of the smaller substituent can be established readily. Examination of the mass spectrum of the

(13) R. J. Liedtke and C. Djerassi, J. Amer. Chem. Soc., 91, 6814

(14) C. Fenselau, J. L. Young, S . Meyerson, W. R. Landis, E.

(15) J. T. B. Marshall and D. H. Williams, Tetrahedron, 23, 321

(1969).

Selke, and L. C . Leitch, ibid., p 6848.

(1967).

Table I. Partial Mass Spectra (70 eV) of trans-3- Alkyl-1-Methoxy-1-Heptenes

mle 170 156 142 127 113 99 95 85 81 71 69 67 55 41

a Parent peak.

Intensities 3-Methyl 3-Ethyl

19a 15" 3 43

100 12

100

12 60 10

3-&Propyl 56

65 9

19

14 100

21 19 10 12 18 16

aldehyde leads to its molecular weight and the degree of branching at the alpha carbon while data from the alkenyl ether spectrum reveals the mass of the heavier beta substituent. The mass of the lighter fragment is then obtained by subtrac- tion of known masses from the molecular weight of the al- dehyde or alkenyl ether. A more serious situation arises when the aldehyde is beta disubstituted and the appreciable loss of only one of the three allylic groups occurs. Unless the mass of a second substituent is known, the calculation de- scribed above yields only the sum of the masses of the re- maining groups. In some cases the weak peaks produced by cleavage of small allylic groups can be used to identify beta branches, but these peaks must be interpreted cautiously be- cause the effect of other moieties upon fragmentation in the region of the alkenyl ether group remains to be established.

Another interesting aspect of alkenyl ether spectra is the m/e 71 peak, the intensity of which is related to the nature of R. As R becomes a better leaving group with respect to butyl, the intensity of m/e 71 increases. When R is lost to a greater extent (R = i-propyl) m/e 71 is the base peak in the spectrum. These data infer that m/e 71 arises from M-R. The conclusion is supported by the appearance in each spec- trum of a metastable ion at m/e 39.7. A mechanism for the genesis of m/e 71 similar to that invoked to explain the forma- tion of m/e 99 peaks in the mass spectra of beta substituted ethylene ketals (16) is outlined in Scheme 11. Deuterium labeling experiments have

- R. +* C,HgCHCH=CHOCHa -

I k

\ + CH3CH2CHZCHz-CH- CH-CHxOCH, - H- 1 +

CHZ=CHCH=OCH, + C4Hs m/e 71

Scheme I1

shown that the hydrogen transferred in cholestan-7-one eth- ylene ketal is derived about 65 from C-1 (16). This speci-

(16) Z. Pelah, D. H. Williams, H. Budzikiewicz, and C. Djerassi, J. Amer. Chem. Soc., 86, 3722 (1964).

ANALYTICAL CHEMISTRY, VOL. 43, NO. 2, FEBRUARY 1971 267

ficity would not be expected to prevail for alkenyl ethers de- scribed above. Rather, hydrogen transfer reactions closely akin to aliphatic ethers (1 7) and thioethers (18) would be an- ticipated.

To obtain the most information from an alkenyl ether mass spectrum, it should be interpreted in conjunction with the mass spectrum of the aldehyde precursor. In most cases only the mass of beta substituent(s) will be discernible, though at times a difference in leaving aptitude may indicate the structure of the fragment. However, even in the former situation, data provided by alkenyl ether spectra can reduce the number of structures that must be considered for a particular aldehyde.

(17) C. Djerassi and C. Fenselau, J. Amer. Chem. SOC., 87, 5141

(18) S. D. Sample and C. Djerassi, ibid., 88, 1937 (1966). (1965).

ACKNOWLEDGMENT

The authors thank Dr. Fred Regnier, Department of Bio- chemistry, Purdue University, for his assistance in obtaining mass spectral data, and Dr. J. E. Sinsheimer, College of Pharmacy, University of Michigan, for measuring the NMR spectra of alkenylethers.

RECEIVED for review November 7, 1969. Accepted October 16, 1970. This work represents a portion of the Dissertation of William G. Andrus, Jr., Purdue University, 1969. A brief account of these data was presented at the 158th National Meeting of the American Chemical Society, New York City, September 8, 1969. This work was supported, in part, by a National Institute of General Medical Sciences Fellowship No. 1-Fl, GM-34, 131-01, and by the American Foundation for Pharmaceutical Education.

Aflatoxin Detection by Thin=Layer Chromatography-Mass Spectrometry

W. F. Haddon, Mabry Wiley, and A. C. Waiss, Jr. Western Regional Research Laboratory, Agricultural Research Service, U. S. Department of Agriculture, Albany, Calq. 94710

NUMEROUS RECENT PUBLICATIONS describe the isolation and identification of aflatoxins at low levels (1-4). These papers reflect concern over the possible presence of these carcinogenic compounds in agricultural commodities. It is now common practice to use thin-layer chromatography (TLC) to identify aflatoxins on the basis of R , value and fluorescence at levels which can be as low as several parts per billion (1,3).

A common complaint against all TLC methods used for aflatoxin detection is their vulnerability to interference from nonaflatoxin compounds (3). Nonfluorescing materials of coincident R , value can mask the presence of fluorescent spots from aflatoxins below threshold values which can be surprisingly high (5). Fluorescing artifacts can, on the other hand, falsely indicate the presence of aflatoxins. These prob- lems have led to chemical confirmatory tests for aflatoxin BI, which is the most toxic known aflatoxin. A confirmatory method proposed originally by Andrellos and Reid (6) and based on the reaction of aflatoxin B1 with volatile acids has been dramatically successful in this regard, but often requires more than 1 pg of aflatoxin and cannot be used to confirm aflatoxins B2, GP, and MI.

We have found that low and nigh resolution mass spectral techniques can provide unambiguous qualitative identification of aflatoxins isolated from individual TLC spots with sensi- tivity approaching that of the TLC-fluorescence method. The mass spectral method is not limited to particular aflatoxins and appears to have approximately equal sensitivity for BI, B2, GI, G2, and MI. The mass spectrometer is now used in

(1) W. A. Pons, Jr., and L. A. Goldblatt, J . Amer. Oil Chem. SOC.,

(2) M. Wiley, J. Ass. ODc. Anal. Chem., 49, 1223 (1966). (3) L. Stoloff, ibid., 50, 354 (1967). (4) I . F. H. Purchase and M. Steyn, ibid., p 363. (5) A. D. Campbell, ibid., p 343. (6) P. J. Andrellos and G. R. Reid, ibid., 47,801 (1964).

42,471 (1965).

this laboratory to confirm the presence or absence of aflatoxins in a variety of agricultural commodities in amounts typically as low as 20 ppb. Positive identification can be obtained in many cases with 50 ng or less of aflatoxin. The use of mass spectrometry extends the lower limit of reliable detection two orders of magnitude or more below existing chemical con- firmatory tests for these compounds.

EXPERIMENTAL

TLC Analysis. TLC analysis reported here utilizes the procedure for aflatoxin isolation described by Pons and Goldblatt for cottonseed products (1). The samples used in this study were experimental aflatoxin-contaminated cotton- seed meal. TLC plates are prepared from pure silica gel (G-HR, Brinkmann Instrument Company). The TLC plates are developed using 9 parts of CHCI, and 1 part acetone. The R , value for aflatoxin B1 under these analytical conditions is 0.56.

Mass Spectral Analysis. Mass spectra were obtained on a CEC Model 21-110 mass spectrometer with electron multi- plier detection. Samples were introduced directly into the ion source using a heated direct introduction probe. The aflatoxins volatilize between 185 and 205 "C in the mass spectrometer. Aflatoxins in pure form used to establish mass spectral fragmentation patterns were obtained by column chromatography on silica gel. The purity of BI, B1, GI, and G2 was greater than 98 by mass spectral analysis. Aflatoxin MI contained about 10% aspertoxin. Because aspertoxin is more volatile than MI, it could be distilled from MI within the mass spectrometer, thus yielding a spectrum of pure MI.

TLC-Mass Spectrometry. Aflatoxins cannot be run directly from TLC plates because such compounds adsorb strongly on silica gel and cannot be volatilized in the mass spectrometer without thermal decomposition. Developed chromatograms are first spotted with 1 p1 of distilled water to dislodge the aflatoxins from the most active sites of the silica gel. The gel is then loaded into a microcolumn formed by

268 ANALYTICAL CHEMISTRY, VOL. 43, NO. 2, FEBRUARY 1971