Detection of methanesulfonic acid in ethyl methanesulfonate

ANALYTICAL

BIOCHEMISTRY

%i,

Detection

307-m

(1973)

of Methanesulfonic

in Ethyl

Acid

Methanesulfonate

The mutagenic effect of alkylating agents has been demonstrated in a wide variety of organisms (1,2). Ethyl methanesulfonate (EMS) has been used extensively as a model system for studying the mutagenic action of monofunctional alkylating agents in organisms ranging through phage (3,4), plants (5), insects (6,7), and mammals (8,9). A major objective of research in our laboratory has been to measure quantitatively the dose of EMS in terms of alkylation of DNA in the reproductive cells (10) and in parallel experiments to measure the genetic consequence of a given dose to the reproductive cells. This dosimetry will permit comparisons among different species in which differences in physiology are separated from any differences in the genetic effect of alkylation. Ethyl methanesulfonate is commonly administered in aqueous solution. Although acid-catalyzed (11) hydrolysis of EMS is relatively slow in aqueous media, initial traces of methanesulfonic acid (MSA) in the feeding solutions would result in sporadic mutagen dosages,caused by accelerated EMS hydrolysis. Thus, establishment of the EMS purity is of paramount importance in order to administer specific dosages. This communication describes a convenient method for detecting traces of methanesulfonic acid in ethyl methanesulfonate. We utilize the welldocumented reaction of sulfonic acids with diazoalkanes (12) and subsequent separation via gas chromatography. The necessity of making a derivative of the MSA is due to the low vapor pressure of MSA. Quantitative analyses were obtained by employment of an aromatic hydrocarbon (e.g., mesitylene) as the internal standard. Analytical procedure. A standard solution of ethyl methanesulfonate was prepared by carefully weighing ca. 10 mg of EMS into a lo-ml volumetric flask and then filling with a solution containing mesitylene (1 mg/ml) in anhydrous ether. To 1 ml of this solution was added 1 ml of freshly prepared diazomethane solution, prepared by standard procedures (13). (Caution: Diazomethane is extremely hazardous and should be used only with adequate ventilat’ion.) The solution was covered lightly and allowed characteristic

to stand at ambient temperature yellow coloration had dissipated. 307 Chpyrighht @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.

for several

hours

until

the

308

SHORT

COMMUNICATIONS

TABLE Retention

1 Data

RP

~~ Ethyl methanasulfonate Methyl methanesulfonate Methanesulfonic acid Mesitylene Ether

FA*

2.75 1.50 c

0.31 0.28

4.25 0.25

1.00 -

0 Retention time. * Response factors; relative to the standard mesitylene. c The elution of methanesulfonic acid required 20-30 min

at 300°C.

The separation of the esters was achieved using a Perkin-Elmer Model 990 Gas Chromatograph equipped with flame ionization detector (Column: 10% OV-101 on Chromasorb Q, 100/120 mesh, 6 ft X l/s in.; column temperature: 100°C (isothermal) ; injector and manifold temperature: 250°C; carrier gas: nitrogen (25 ml/min) ; 3-,ul sample sizes). The retention data are tabulated in Table 1. Substitution of diazoethane (14) in the above procedure permits the facile detection of MSA in MMS. Use of this method is readily adapted to radiolabeled EMS; however, to determine radiochemical purity, a column splitter and adequate traps are required. Extension of this procedure to use biological fluids is currently under consideration in these laboratories. ACKNOWLEDGMENTS This investigation was supported 0032095 from the National Institute liam R. Lee, and by NIH Research of Neurological Diseases and Stroke

in part by NIH Research Grant Number ESof Environmental Health Sciences to Dr. WilGrant Number NS 09930-0181 from the Institute to Dr. George R. Newkome. REFERENCES

1. LOVELESS, A. (1966) Genetic and Allied Effects of Alkylat,ing Agents, Pennsylvania State University Press, University Park. 2. FISHBEIN, L., FLAMM, W. G., AND FALK, H. L. (1970) Chemical Mutagens, Aeademic Press, New York. 3. GREEN, D., AND KRIEG, D. R. (1961) Proc. Nut. Acad. Sci. USA 47, 64. 4. BROOKES. P., AND LAWLEY, P. D. (1963) Biochem. J. 89, 138. 5. VELEMINSKY, J., ZADRAZIL, S., AND GICHNER, T. (1972) Mutation Res. 14, 259. 6. JENKINS, J. B. (1967) Genetics 57, 78S793. 7. LEE, W. R., SEGA, G. A., AND BISHOP, J. B. (1970) Mutation Res. 9, 323. 8. VOGEL, F., AND F~QHRBORN, G. (1970) Ch emical Mutagenesis in Mammals and Man, Springer-Verlag, New York. 9. GENEROSO, W. M. (1972) Newsletter Environ. Mutagen Xoc. 6, 14.

SHORT 10. 11. 12. 13. 14.

309

COMMUNICATIONS

SEGA, G. A., GEE, P. A., AND LEE, W. R. (1972)

Mutation Res. 16, 203. OLAH, G. A., Ku, A. T., AND OLAH, J. A. (1970) J. Org. Chem. 35, 3908. ARNDT, F., AND MARITUS, C. (1932) Ann. 499,264. DE BOER, TH. J., AND BACKER, H. J. (1954) Rec. Trav. Chim. 73, 229. MARSHALL, J. A., AND PARTRIDGE, J. J. (1968) J. Org. Chem. 33, 4090. CHARLES GEORGE PATRICIA WILLIAM

Department of Zoology and Physiology, Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana ?‘0805 Received January 22, 19‘78; accepted

and

1 Reprint requests Physiology.

should

be made

S. AARON R. NEWKOME M. SEAMSTER R. LEEI

and

March

to Dr.

15, 1978

William

R. Lee,

Department

of Zoology