no vitamin A was found in the fraction which would contain vitamin D. If desired, the iso-octane used during the analysis can be recovered b y passing it through a silica gel column. Vitaniins A and D are removed, and iso-octane spectrophotonietrically equal to the initial solvent can be obtained. I n Table I1 are typical results on d i f h e n t types of multivitamin products. As can be seen, good checks TI itli the U.S.P. biological assay are obtained. This method is also suitable for stability studies, because lox biological assay values were corroboratcd by this method. The method appears to l i a ~ e good reproducibility. Triplicate analyqes were performed on niultivitaniin tablets 1%ith minerals and values of 2150, 2090. and 2110 units of vitamin D \yew found. The U.S.P.biological method gave 2000, 2100, and 2200 units. Duplicate analyses can be conipleted in 6 hours and, with a little experience, srx era1 columns may be used simiiltancously b y one analyst. Because the U.S.P.assay requires a special rat colony and takes about 2 mebs to coinp k t e , the advantages of this method are evident. During this work, it was observed that the partition chromatography systrm used also makes possible the
Table
II.
Comparisons of Chemical and Bioassay Results
Sample Multivitamin tablet Multivitamin tablet vith minerals Sirup Vitamin D in corn oil Fish liver oil Stability Samples Multivitamin drops, 12 months Multivitamin tablet, 36 months
Chemical Analysis, U.S.P. Units 1,328 2,150 983 1 ,039,000 52,000 Theory 1250 875
separation and rr’covcry of other nonsapoiiifiablc substances froni multivitamin preparations. These include vitamin E (a-tocopherol), vitainin -4alcohol, and several vitamin A4 dtgradation products such as anh~-drovitamin A. Vitamin E c:tn be srparated from vitamin A and quantitatiwly determined b y ultraviolet means in some instances. Vitamin A alcohol can be srparated from ni:tterials which prevent its determination by ultraviolet spectrophotonwtry despite the use of a Morton-Stubbs (6) type of correction. Subsequent papers will explore these other possible uses.
Bioassay, U.S.P. Units 1,235 2,200 1,000 1,000,000 50,000
TS2 540
752 604
Part I, “Separation and Purification,” p. 149, Interscience, S e n York, 1956. ( 2 ) Ewing, D. T., Schlaback, T I]., Powell, I f . J., SAL. CHEM.26, 1406 (1954). (3) Martin, -4. J. P., Biochem. Soc. Symposiu S o . 3, 11 (1949). (4) hlartin, A. J. P., Synge, R. 1,. M., Biochem. J . 35, 1358 (1941). (5) lIort>on,I
