Thin Layer Chromatographic and Colorimetric Methods for Monitoring the Purity of S-2-(3-aminopropylamino)ethylphosphorothioic Acid (WR-272 1) L. C. Washburn and R. L. Hayes Medical Division, Oak Ridge Associated Universities, Oak Ridge, Tenn. 37830
The radioprotective agent, S -2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721), has evoked much interest in recent years because of reports ( 1 - 3 ) that the drug protects normal tissues against radiation damage to a much greater extent than it does tumor tissue. The radiation resistance of the normal tissues in drug-treated animals is approximately doubled, while that of malignant tissues remains essentially unchanged. This allows the use of larger radiation doses to tumors without increasing normal tissue injury. Another advantage of the drug is that most of the radioprotection which the drug can afford is obtained a t doses far below the toxic level ( 4 ) . WR-2721 thus holds promise as an adjunctive aid in the treatment of cancer by radiotherapy. Because of a proposed clinical investigation of WR-2721 in the radiotherapy of solid tumors, we sought methods for quality control to allow the detection of impurities in WR2721 preparations. The major impurities commonly found are the analogous mercaptan and symmetrical disulfide, which can be formed by hydrolysis of WR-2721 and a resultant oxidation of the mercaptan so formed (Scheme I). The mercaptan has an LDjo value of approximately one-third that of WR-2721 itself, and disulfides generally are even more toxic than mercaptans ( 5 ) .Although WR-2721 is unusually stable compared to most radioprotective agents, the increased toxicity of the decomposition products relative to WR-2721 made it desirable to explore methods for monitoring the purity of WR-2721 preparations.
oxidation
RSH
mercaptan
RSSR
symmetrical disulfide
EXPERIMENTAL A procedure involving thin layer chromatography (TLC) was developed that provides good separation of WR-2721 from the mercaptan and symmetrical disulfide. Eastman Chromagram Sheet 6061 silica gel without fluorescent indicator was used, along with a sandwich-type developing chamber which holds an entire silica gel sheet. The optimum solvent system for development of the chromatogram was found to be methanol-chloroform-concentrated ammonium hydroxide (2:l:l by volume). After the approximately 4.5 hours required for development, the spots were visualized with ninhydrin spray reagent (Sigma Chemical Co.). Using this system, WR-2721 has an R f value of 0.20, compared t o 0.42 for the analogous mercaptan and symmetrical disulfide. Although the mercap-
(1) J. M. Yuhas and J. B. Storer, J. Nat. Cancerlnst., 42, 331 (1969). (2) J. M. Yuhas. J. Nat. Cancerhst., 48, 1255 (1972). (3) J. M. Yuhas, J. Mat. CancerInst., 50, 69 (1973). (4) J. M. Yuhas. Radiat. Res., 44, 621 (1970). (5) T. R. Sweeney, in "Biological Aspects of Radiation Protection," T. hara and 0. Hug, Ed., lgaku Shoin Ltd., Tokyo, 1969, pp 164-168.
tan and disulfide are not separated using this TLC method, it is possible to easily determine each in the presence of the other using the colorimetzic method described below. Because the TLC development time was lengthy, we sought a simple colorimetric method to rapidly detect significant quantities of mercaptan or symmetrical disulfide contaminant in samples of WR-2721. The method developed is a modification of the nitroprusside test of Hammett and Chapman (6). T o detect the disulfide colorimetrically, it must first be reduced to the mercaptan. T h e best agent found for reducing the disulfide was aqueous sodium sulfite, which, unlike sodium or potassium cyanide ( 6 ) , does not catalyze the hydrolysis of WR-2721 to the mercaptan. A sufficient quantity of the drug sample t o be tested is dissolved in aqueous sodium sulfite solution (12.6 mg/ml). After 5 minutes, 1 ml of the solution is saturated with solid ammonium sulfate, made basic with 2 or 3 drops of concentrated ammonium hydroxide, and treated with 3 drops of 1% sodium nitroferricyanide. In the presence of mercaptan or disulfide impurities, a bright pink color is formed which generally fades within 5 minutes. Sodium sulfite itself gives a pale yellow to yellow-orange color with the nitroferricyanide reagent, but this does not appreciably interfere with the pink color. However, a blank (no WR-2721 added) should be run alongside and the colors compared. A detectable pink color (compared to the blank) was observed a t a mercaptan or disulfide concentration as low as 0.02 mg/ml of test solution. For distinguishing between mercaptan and disulfide impurities, one need only omit the sodium sulfite reducing agent in the above procedure. Mercaptans give the pink color in the absence of sodium sulfite; disulfides do not. A blank is also desirable in this test as a means of color comparison.
DISCUSSION We have made no attempt to spectrophotometrically quantitate the color formation in this test. This could conceivably be done although the fleeting nature of the pink color could lead to an appreciable error in quantitation. Even the purest WR-2721 preparations which we have synthesized and used in animals have contained a certain amount of impurity, chiefly the mercaptan. However, a preparation is generally considered unsuitable for use only if it contains appreciable amounts of impurity, say greater than 1%.Using the colorimetric procedure outlined above, one can easily and quickly monitor a WR-2721 preparation for impurities of that magnitude a t intervals during its storage and use. The TLC method, although requiring a longer time for the analysis, is a valuable supplementary technique. ACKNOWLEDGMENT The authors are indebted to M. H. Heiffer, Walter Reed Army Institute of Research, for providing samples of the mercaptan and symmetrical disulfide, which were used as standards in this study. RECEIVEDfor review May 13, 1974. Accepted August 19, 1974. This work was supported by the United States Atomic Energy Commission.
Suga(6) F. S.Hammett and
S. S.Chapman, J. Lab. C/in. Med., 24, 293 (1938)
ANALYTICAL CHEMISTRY, VOL. 46, NO. 14, DECEMBER 1974
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