Problems in Gas Chromatographic Determination of Methionine, Methionine Sulfoxide, and MethionineSulfone Steven R. Tannenbaum, William G . Thilly, and Phillip Issenberg Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Muss. 02139 METHIONINE SULFOXIDE and methionine sulfone are formed from methionine under various conditions including the acid hydrolysis of proteins and as a consequence of the autoxidation of unsaturated fatty acids. Amino acids, including methionine, may be determined by gas chromatography of their N-acyl esters, but the literature appears confused as to the fate of similar derivatives of the sulfoxide and the sulfone. Only one investigation has dealt with the sulfone ( I ) while a number have reported with respect to the sulfoxide (1-3). None of these investigations was able to report separation of the methionine and methionine sulfoxide derivatives although a variety of acyl (acetyl and trifluoroacetyl) and ester (methyl and butyl) substituents and a variety of column liquid phases were used. In fact, some authors have suggested that methionine is converted to its sulfoxide, or vice versa, during derivatization or chromatography. The structure of n-butylN-trifluoroacetylmethionine has been verified by elemental analysis ( 4 ) . Evidence presented below indicates that under a number of conditions methionine sulfoxide is converted to an N-acylmethionine methyl ester and is chromatographed as such. EXPERIMENTAL Methionine and its oxidation products were converted to N-trifluoroacetyl methyl esters (TFAM) for gas chromatography. In all cases the amino acids were trifluoroacetylated after ester formation. The amino acids have been successfully esterified under a variety of conditions including: methanolic HC1; methanol and thionyl chloride; methanolic HC1 and dimethylsulfite. The latter conditions gave the highest degree of reproducibility with all three amino acids. In a typical preparation 1.0 mg of methionine in 2 ml of anhydrous methanolic HCl (1.2N) and 0.5 ml of dimethylsulfite were refluxed for two hours. The solvent was removed under reduced pressure, the resulting clear oil taken up in 2.0 ml of trifluoroacetic anhydride, and reflux continued for 30 minutes. The solvent was again removed under vacuum to give a clear oil, which was taken up in a quantity of ethyl acetate suitable for the subsequent chromatographic analysis-e.g., 0.25 ml. All chemicals were reagent grade, and the amino acids were chromatographically pure (Calbiochem, Los Angeles, Calif.). Thin-layer chromatography was performed on 1-mm layers of silica gel G on glass plates with n-propanol-water (2:l) as eluant. Gas chromatography was performed on a number of different columns. A Carbowax 20M support-coated open-tubular column (15.2-m X 0.50-mm i.d. ; PerkinElmer Corp., Norwalk, Conn.) was operated at 180 "C in the mass spectrometer inlet chromatograph (model 204-C, Varian-Aerograph, Walnut Creek, Calif.). Column effluent passed through a stainless steel capillary restriction (1.9-m X 0.50-mm i.d.) and fritted glass enricher (5), both main(1) D. E. Johnson, S. J. Scott, and A. Meister, ANAL.CHEM., 33,
669 (1961). (2) W. M. Lamkin and C. W. Gehrke, ibid., 37, 383 (1965). ( 3 ) C. Zomzely, G. Marco, and E. Emery, ibid., 34, 1414 (1962). (4) J. Hine, "Physical Organic Chemistry," McGraw-Hill, New York, N. Y . , 1956, p 176. ( 5 ) J. T. Watson and K. Biemann, ANAL.CHEM.,37, 844 (1965).
Table I. Thin-Layer Chromatography of Methionine and Derivatives RlIethionine
Methionine Methionine sulfoxide Methionine sulfone
Free amino acid 1.o 0.26
TFAM derivative 1.14 1.14
0.33
1.30
tained at 200 "C, into the ion source of a double-focusing mass spectrometer (Hitachi-Perkin-Elmer, RMU-7). Chromatograms were recorded from the total ion-current monitor located between the electrostatic and magnetic analyzers. Helium carrier gas flow rate was 6 cc/minute. Injector temperature was 225 "C, and the chromatograph detector oven was maintained at 235 "C. Mass spectra were scanned magnetically over the range m/e 4-400 in six seconds. Perfluorokerosene was introduced through a conventional liquid inlet for use as a mass marker. Ion-source temperature was 250 "C. RESULTS AND DISCUSSION Methionine, methionine sulfoxide, and methionine sulfone readily separate under thin-layer chromatography. The TFAM-methionine sulfone can be separated from the methionine and methionine sulfoxide derivatives, but the sulfoxide cannot be separated from the methionine derivative. The relative data are given in Table I. The free amino acids were located with ninhydrin, and the derivatives, with chloroplatinic acid. The TFAM-methionine was readily determined by gas chromatography and gave a single peak with reproducible results over a wide range of concentration. The TFAMmethionine sulfoxide gave a single major peak and a minor peak which was not resolvable from the solvent peak. Under no circumstances could TFAM-methionine be separated from TFAM-methionine sulfoxide. No peak could be observed for TFAM-methionine sulfone. The mass spectra of TFAM-methionine and TFAM-methionine sulfoxide were essentially identical, and the fragmentation pattern is that which would be expected for TFAMmethionine (6). A peak at m/e 259 was present (parent mass ion for TFAM-methionine), but no peak could be observed at m/e 275 (parent mass ion for TFAM-methionine sulfoxide), Peaks could be observed at mje 227 (M-CH3OH) and m/e 200 (M-CH3C0J but no corresponding peaks 16 mass units higher. This was true for all sulfur-containing fragment ions, which could be explained on the basis of -Sand sulfur. Even assuming the parent mass ion of not -S-
J.
0 TFAM-methionine sulfoxide to be unstable, no peaks corresponding to hypothetical fragment ions could be observede.g., M-CH3S0. ~
~~
(6) K. Heyns and H. F. Grutzmacher, Fortschr. Chem. Forsch., 6, 536 (1966). VOL. 40, NO. 1 1 , SEPTEMBER 1968
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Thin-layer and gas chromatographic and mass spectral evidence lead to the conclusion that methionine sulfoxide is converted to TFAM-methionine in the course of derivative formation. It is of interest that the yield of TFAM-methionine from methionine sulfoxide is calculated to be 5 2 z of theoretical (based upon gas Chromatographic peak areas) over a wide range of concentration of starting material. Allowing for the error in the method, this could imply that methionine sulfoxide is converted to methionine via a disproportionation reaction similar to that observed for cystine monoxide (7) and for a number of sulfoxides (8). Direct deoxygenation (9), however, cannot be overruled on the basis (7) W. E. Savige and J. A. Maclaren, “The Chemistry of Organic Sulfur Compounds,” Vol. 2, Pergamon Press, New York, N. Y., 1966, p 367. (8) H. H. Szmant, “The Chemistry of Organic Sulfur Compounds,” Vol. 1, Pergamon Press, New York, N. Y., 1961, p 154. (9) D. N. Jones, M. J. Green, and M. A. Saeed, Chem. Commun., 674 (1967).
of our results. At the same time the failure to find a gas chromatographic peak for the N-ac yl ester of methionine sulfone may be because sulfones are prone to elimination reactions at high temperatures and produce the corresponding vinyl compounds (4). In the analysis of amino acids by ion exchange chromatography, inadvertent oxidation of methionine can be recognized by the appearance of separate peaks for methionine sulfoxide and sulfone (IO). This would not appear to be the case for gas chromatography of N-acyl esters, and care should be taken in recognition of this fact, both with sample preparation and treatment prior to analysis. RECEIVED for review May 8, 1968. Accepted June 6, 1968. Work supported in part by grant NsG-496 to Massachusetts Institute of Technology from the National Aeronautics and Space Administration. (10) P. B. Hamilton, ANAL.CHEM.,35, 2055 (1963).
Spectrophotometric Method for Estimating Alkyl Ester HydroIys is R. S. Roy Science College, Mosul Unioersity, Mosul, Iraq ESTERS UNDERGO hydrolysis ( I ) . Both alkyl esters and alkyl acids exhibit maximum absorbance at 204 mp, but the molar absorptivity of an ester is higher than that of the corresponding acid (2). This difference in molar absorptivities can be utilized in determining the concentrations required for estimating the velocity constant of an ester and its acid during hydrolysis at any interval. THEORY. The hydrolysis of an ester may be represented by the following equation: RCOOR’
+ H20 e RCOOH + R’OH
Table I. Absorbance and k Values for Methyl Acetate Hydrolysis at 22” C = 46.7, e, = 34.0, u = O.O3M, [HCI] = 0.15M Time (min) 10 40 90 Absorbance 1.385 1.360 1.290 k (min-1) x 10-3 3.9 3.0 3.6
and
(1)
where R and R’ are alkyl groups.
If
= the molar absorptivity of the ester at 204 mp ea = the molar absorptivity of the acid at 204 mp A = the absorbance of the ester solution (1-cm cell) at
time t a = the concentration of the ester at time 0 ce = the concentration of the ester at time i
Values of ca and ce can be calculated from Equations 4 and 5 , respectively. For calculating k , the velocity constant of hydrolysis, insertion of ce in the kinetic equation for a reaction of the first order (S), gives
ca = the concentration of the acid at time t it follows that the absorbance of the solution containing both ester and acid (during hydrolysis at time t ) is given by
A A because Ce = a
= (U
Cere
+
CaEa
- C J E ~ + CaEa
(2)
(3)
- ea.
On rearrangement, it can be shown that
The rate of hydrolysis of an alkyl ester can be followed by determining the absorbance of the ester solution at 204 mp from time to time. This general method can be applied to all alkyl esters in the absence of absorbance of other constituents interfering with ester absorption band. Results obtained for methyl acetate hydrolysis using a Unicam SP 800 spectrometer and presented in Table I have given a value for k of 0.0035 min-l at 22 “C which agrees with the values obtained by the usual titration method.
-~
RECEIVED for review October 5 , 1967. Accepted November 17, 1967.
(1) B. Capon and B. C. Ghosh, J . Chem. SOC.,1966,472. (2) A. E. Gillam and E. S. Stern, “An Introduction to Electronic Absorption Spectroscopy in Organic Chemistry,” Edward Arnold (Publishers) Ltd., London, 1962, pp 56 and 61.
(3) E. A. Moelwyn-Hughes, “Physical Chemistry,” Pergamon Press, 1964, p 1115.
(4) ~
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0
ANALYTICAL CHEMISTRY
