THE STABILITY OP VITAMIN A AND ITS ESTERS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By ALBERT JOSEPH PORLANO, B.Sc., M.Sc The Ohio State University 1959 Approved by ^ Adviser Department of Pharmacy ACKNOWLEDGMENTS I wish to express my thanks and appreciation to my adviser and friend, Dr. Loyd Harris, without whose assistance this study would not have been possible. I would also like to thank the American Foundation for Pharmaceutical Education for their generous financial assistance in the form of a fellowship. To my wife, Marie, who made my graduate studies have meaning by virtue of her kindness, cooperation and inspiration. To Dr. L. M. Parks for his understanding guidance and council. To all those who are too numerous to mention but are still near and dear to me, I give my heartiest thanks. May, 1959 Albert ur. Forlano ii TABLE OP CONTENTS Page Introduction .......................................... 1 Early History of Vitamin A ........................... 2 Quantitative Determination of Vitamin A ............... 7 Introduction.......................... 7 Biological Method ................................... 7 Spectrophotometrie Methods ......................... 8 Chemical M e t h o d s ........ .. .................... 13 Fluorescence M e t h o d s ............................. lip Vitamin A Alcohol ................................. lip Isolation and Chemical Characteristics.......... lip Ultra Violet Spectrum ............................. 16 Infrared Spectrum ........................... 17 Oxidation of Vitamin A ............................... 17 Photochemical Oxidation ........................... 17 Isolation and Characterization of Oxidation Products ....................................... 19 Effect of Solvents on Oxidation of Vitamin A . 22 Effect of Peroxides and Pro-oxidant Metals .... 2i| Effect of Acidity and Moisture in Oils Upon Oxidation ....................................... 25> Mechanism of Oxidation and Action of Antioxidants . 27 Spectroscopic Changes Occurring During Oxidation . 30 Miscellaneous Information Regarding Oxidation . 30 Activity of Specific Antioxidants ................. 32 iii TABLE OP CONTENTS (Contd.) Page Compounds Related to Vitamin A .................... 32 Axeropthene ..................................... 32 Vitamin A A c i d ............................ 33 Rehydrovitamin A ................................. 33 Vitamin A Methyl E t h e r ........................... 35 Retinene ....................................... 35 Vitamin A2 35 Anhydrovitamin A ....................... 37 8, 9> Dehydrovit amin A ........................... ij-3 Vitamin A E s t e r s ................................. 1|1|. Derivatives Other than Esters .................. 1|5 Isomerization in Vitamin A and R e t i n e n e s .......... Lf.6 Numbering Systems ................ i+6 Theories of Isomerization ...................... Ip7 Neovitamin A ................................... 5l Chromatography ............................. 53' Biopotency of Isomers and Congeners .............. 58 Description and Purpose of Study .................. 59 Experimental Part ................................. 61 Preliminary Studies ............................. 61 Determination of the Stability of Vitamin A Palmitate in the Presence of an Homologous Series of Patty Acids ........................ 67 Reagents 67 Decomposition Rates in "99%" Isopropyl Alcohol . 68 Preparation of Solutions ..................... 69 iv TABLE OP CONTENTS (Contd.) Page Rates of Degradation of the Vitamin A Ester . 70 Alumina Chromatography of these Samples ...... 73 Effects of Addition of Water to n99$" Isopropyl Alcohol Samples ................................... 80 Rates of Degradation of the Vitamin A Ester .... 82 Alumina Chromatography of these Samples ............ 87 Paper Chromatography of these S a m p l e s ............ 90 Action of Isopropyl Alcohol, Water, Cyclohexane and Patty Acids in these Solvents on Anhydrovitamin A 98 Cyclohexane Systems ............................. 98 "99$ " Isopropyl Alcohol Sy s t e m s .............. 106 "95$" Isopropyl Alcohol Systems ..... ......... 108 1185$ 11 Isopropyl Alcohol Systems . ................. 109 Paper Chromatography of the Isopropyl Alcohol Samples ......................................... 112 Preparation of Some New Esters of Vitamin A ...........116 Introduction ..................................... 116 Synthesis ......................................... 120 Preliminary Data ................................. 120 General Method of Preparation of the Esters . 122 Materials ...................................... 12I4. Infrared Spectra .......................... 127 Ultra Violet Spectra ........................... 130 Determination of R^ Values ..................... 132 Stability Testing of Esters ........................ 136 Stability Against Base ...................... 137 Saponification Equivalents of the Esters .... 138 v TABLE OP CONTENTS (Contd.) Page Stability of the Esters in 0.0113 N HCl - Anhydrous Ethanol .......................... 139 Stability of the Esters in 0.0113 N HCl - Ethanol TJSP ................................. lij.0 Stability of Esters in u99%" and "95%" Isopropyl Alcohol with and withoutPatty Acid ......... 1I4.I Vitamin A A c r y l a t e ....... .................. 1 )|? Vitamin A Chloroacetate .................... II4J4. Vitamin A Sorbate .......................... 1I4.5 Vitamin A o( Chloropropionate................ 11^.7 Vitamin A Crotonate ............... 1)4.8 Paper Chromatography of the Reaction Mixtures . II4.9 Discussion ....................................... 1J?2 Effect of Patty Acids .......................... 152 Mechanism of Action of Patty Ac i d s ........... 153 Reaction Products .............................. 155 Action of Water ................................ 156 Mechanisms of Decomposition of Vitamin A Esters and Alcohol ................................... 157 Summary and Conclusions .......................... 162 Bibliography .............. 165 Autobiography ..................................... I7I4. vi LIST OP TABLES Table Page 1 . Conversion Factors for Vitamin A and Its Acetate • . ........................ 13 2. Extinction Values for Vitamin A Alcohol and Acetate ..... 16 3. Percent Vitamin A Destroyed by Aeration in One Hour ................................... 22 ’I4.. Stability of Vitamin A in Various Concentrations of Patty Acid . , ........................... 25 5. Time Required for 20 Percent Loss of Vitamin A . 26 6. Extinction of Esters Before and After Saponification...................... Ijl(. 7. Summary of Alumina Chromatography Systems . 56 8. Bioactivity of the Various Congeners ........ 56 9. Assay Values for Vitamin A in Various Ointment Bases ..................................... 63 10. Assay Values for Vitamin A in n-Hexane and "99%" Isopropyl A l c o h o l .......... 6l| 11. Rate Constants for Degradation of Vitamin A Palmitate in "99%" Isopropyl Alcohol Solutions 70 12. U.V. and I.R. Spectra of Chromatographed Fractions from "99%" Isopropyl Alcohol Systems 75 1 3 . Rate Constants for the Degradation of Vitamin A Acetate in "95%" Isopropyl Alcohol ........ 82 ll(.. Initial Rates of Formation of Anhydrovitamin A in Units of Vitamin A Lost in "95%" Isopropyl Alcohol ................................... 83 15. Comparison of Total Vitamin A Loss to Conversion into Anhydrovitamin A in "95%" Isopropyl Alcohol ................................... 81| vii LIST OP TABLES (Contd.) Table Page 16. IJ.V. and I.E. Spectra of "9$%" Isopropyl Alcohol-Vitamin A Acetate Control I4.5 0 . 88 N/30 Palmitic Acid h£>0 ............... 89 N/30 Caproic Acid 1|5° 89 17. R f Values of Chromatographed "95%" Isopropyl Alcohol Control Samples .............. 91 18. Rf Values of Chromatographed "95$" Isopropyl Alcohol, N/30 Caproic Acid I4.5 0 Samples . 92 19. Rf Values of Chromatographed "95$" Isopropyl Alcohol, N/3 0 Palmitic Acid i|5° Samples . 93 20. Rates of Formation of Vitamin A Substance in Acetic and Caproic Acids with Anhydrovitamin A In Cyclohexane ......................... 102 21. Rates of Transformation of Anhydrovitamin A into a Vitamin A Substance in "99%" Isopropyl Alcohol ......................... 108 22. Rates of Transformation of Anhydrovitamin A into a Vitamin A Substance in "95%" Isopropyl Alcohol .............. Ill 23. Rates of Transformation of Anhydrovitamin A into a Vitamin A Substance in "85%" Isopropyl Alcohol ......................... 112 21).. R f Values of Substances Pound in "99%" Isopropyl Alcohol Systems ................ 111). 25. Rf Values of Substances Pound in "85%" Isopropyl Alcohol Systems ................ 115 26. K„CL of Acids Related to this Study ........... 119 27. I.R. Carbonyl Peaks of New Esters (/i) .... 130 28. IJ.V. Extinction Coefficients of New Esters . 131 29. Rf Values of Vitamin A Esters and Related Compounds ............................... 136 30. Time Required to Consume 1 HI. 0.01N NaOH . 137 31. Saponification Equivalents of Esters ......... 138 viii LIST OP TABLES (Contd.) Table Page 32. Rates of Elimination of Vitamin A Esters and Related Compounds in 0.113N HCl - Anhydrous Ethanol ..................................... IlpO 33* Rates of Elimination of Vitamin A Esters and Related Compounds in 0.0113N HCl-Ethanol TJSP . lljl 31}.. Rates of Decomposition of Vitamin A Acrylate in Isopropyl A l cohol........................... ll+lj. 35. Rates of Decomposition of Vitamin A Chloro- acetate in Isopropyl A l cohol...................ll\$