Substitutive Solvents for Chloroform in the Identification Test For

190 J. Food Hyg. Soc. Japan Vol. 38, No. 3 Report Substitutive Solvents for Chloroform in the Identification Test for Ergocalciferol and Cholecalciferol in the Japanese Standards for Food Additives (ReceivedDecember 13, 1996) Hajimu ISHIWATA*1, Chlakl KAT0*1,*2, Takiko SUGITA*1, Yoko KAWASAKI*1 and Takashi YAMADA*1 (*1National Institute of Health Sciences: 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158, Japan; *2Faculty of Environmental Health Sciences, Azabu University: 1-17-71, Fuchinobe, Sagamihara-shi, Kanagawa 229, Japan) Key words: Japanese Standards for Food Additives, identification test for vitamin D, ergocalciferol, cholecalciferol, chloroform, toluene 2. Analytical methods Introduction Ergocalciferol and cholecalciferol were iden- Clean analysis is now being proposed as a tified by the Japanese official method described means of protecting the earth's environment in "Identification (1)" under "Ergocalciferol" in and the health of analysts1),2). Nevertheless, the JSFA4),5) and a modified method, namely: 0.3 toxic reagents such as chloroform, benzene, mer- mL of acetic anhydride and 0.1 mL of sulfuric cury compounds, etc., are still used in many acid were added to 0. 5 mg of ergocalcif erol or tests in the Japanese Standards for Food Addi- cholecalciferol dissolved in 5 mL of chloroform, tives (JSFA), sixth edition3). While the methods diethyl ether, ethyl acetate, n-hexane, toluene, or or reagents for these tests must be changed, xylene. The mixture was shaken, then the color modification of the official methods may affect of the reaction mixture was observed by the the standards and evaluation of whether a prod- naked eye, and the absorption spectrum and uct would pass or fail the test. Equivalent re- optical density were determined. sults must be obtainable with a modified Results and Discussion method. We have studied organic solvent substitutes for the chloroform used to identify er- 1. Color development in the identification test gocalciferol and cholecalciferol in the JSFA, The same identification method for ergocal- aiming to obtain similar results with minimum ciferol and cholecalciferol in the JSFA is also changes of the official method, and found that used in the Pharmacopoeia of Japan6),7) and the chloroform can be replaced with toluene. United States Pharmacopeia8),9). Both compounds in chloroform are identified by the Experimental Procedure change in the color of the reaction mixture. The 1. Reagents and equipment description of the change in color is as follows: a Ergocalciferol and cholecalciferol were of red color develops and immediately changes to guaranteed reagent grade (Tokyo Chemical In- green through purple and blue. Chloroform is dustry Co., Ltd., Tokyo). Other reagents were of used in the test only as a solvent to dissolve JIS special grade (Wako Pure Chemical Indus- ergocalciferol and cholecalciferol in all the stan- tries, Ltd., Osaka). A Shimadzu UV-240 spectro- dards described above, so it should be possible photometer was used. to replace it with another solvent. Diethyl ether, ethyl acetate, n-hexane, toluene, and xylene June 1997 Modification of Identification Method for Vitamin D's as Food Additives 191 Table 1. Color Development of Ergocalciferol and Cholecalciferol by the Modified Official Methoda) Using Various Solvents a) To 5 mL of each solvent containing 0. 5 mg of ergocalciferol or cholecalciferol, 1.0 mL of acetic anhydride and 0. 1 mL of sulfuric acid were added. b) Color developed in the emulsion. Table 2. Wavelengths of Amax of the Absorption Spectra and Optical Densities of the Reaction Mixtures Obtained by the Modified Official Methoda) a) Experimental conditions were the same as described in Table 1. Determinations were carried out after allowing the reaction mixtures to stand for 20 min. The results were the average of duplicate examinations. were assessed as possible replacements for chlo- (a mixture of ortho-, meta-, and para-) was pale roform. red and changed to pale yellowish green. Trans- The color of ergocalciferol and cholecalciferol parent reaction mixtures and the same color as in the reaction mixture with diethyl ether and in the description in JSFA4) were obtained when ethyl acetate solutions was between lemon o- and m-xylene were used, but the reaction yellow and pale bluish-green. The reaction mix- mixture with p-xylene was turbid and the color tures with n-hexane, toluene, and xylene as sol- of the ergocalciferol reaction mixture was pale vents formed emulsions when shaken, and the green. The reaction mixture with n-hexane was sulfuric acid layer (green) adhered to the surface turbid, and the color of the emulsion changed of the test tube because of insufficient solubility from brown to green without ever appearing of sulfuric acid. Thus, these methods were not red. The reaction mixture with n-hexane sepa- suitable for the identification test, though the rated into two layers after standing for 1 min. color development of the emulsion was the same The sulfuric acid layer (lower layer, about 1 mL) as in the description of the official method. was green, and the n-hexane layer was colorless. The volume of acetic anhydride was, there- Toluene was found to be the most suitable of the fore, increased to 1.0 mL to dissolve sulfuric solvents tested as a replacement for chloroform acid. The results of observations of color devel- in the identification test for both compounds. opment with the improved methods are summa- 2. Absorption spectra of the reaction mixtures rized in Table 1. Transparent reaction mixtures The wavelengths of the )max and the optical were obtained when toluene and xylene were densities in the absorption spectra of the reac- used, and the changes in color, except for ergo- tion mixture after standing for 20 min are calcif erol in xylene, were the same as in the shown in Table 2. The spectra of the reaction description in the official method. The color of mixtures in chloroform and toluene when 1.0 the reaction mixture of ergocalciferol in xylene mL of acetic anhydride and 0. 1 mL of sulfuric 192 J. Food Hyg. Soc. Japan Vol. 38, No. 3 color development for the identification of ergocalciferol and cholecalciferol in JSFA4),5). D2 in chloroform D2 in toluene Conclusion D3 in chloroform D3 in toluene Several organic solvents were assessed for clean analysis as potential substitutes for the chloroform used to identify ergocalciferol and cholecalciferol in the JSFA. Diethyl ether, ethyl acetate, n-hexane, toluene, and xylene were ex- amined, and toluene was found to yield results similar to those obtained with chloroform. References 1) Anonymous: Japanese Pharmacopoeial Forum, 5, 126 (1996). 2) Horiuchi, Y.: Pharmaceutical Industry, 299, 544-545 (1995). Fig. 1. Absorption spectra of the reaction 3) Ministry of Health and Welfare, Japan: The Jap- mixtures of ergocalciferol or cholecalcif- anese Standards for Food Additives, Sixth Ed., erol in chloroform or toluene (1992). D2: ergocalciferol, D3: cholecalciferol 4) ibid.: p. 181. a) Experimental conditions were the 5) ibid.: p. 257. same as described in Table 1. Determina- 6) Ministry of Health and Welfare, Japan.: The tions were carried out after allowing the Pharmacopeia of Japan, Thirteenth Ed., p. 347- reaction mixtures to stand for 20 min. 348 (1996). 7) ibid.: p. 577-578. acid were added to the ergocalciferol and chole- 8) Authority of the United States Pharmacopeial calciferol solutions are shown in Fig. 1. The Convention Inc.: The United States Pharmaco- toluene solution yielded the same ;imax as the peia, The National Formulary, p. 365-366 chloroform solution and the highest optical den- (1995). sity of the solvents substituted. These results 9) ibid.: p. 599-600. indicated the equivalency of toluene to chloroform as a solvent in terms of the description of.

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