Stability of Vitamin A Acetate under Laboratory Conditions

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<ul><li><p>960 </p><p>immiscible petroleum: the pulp is not dispersed and suspended in the petroleum in the way in which dry grass meal is seen to be. Or it may be that a protein-carotene linkage similar to that in the carrot, described by Kreula (8), is not easily ruptured by hot petroleum. </p><p>Thus the low extraction of carotene from whole leaves by 80' to 100' petroleum is probably mainly due to types of mechanical hindrance which can be overcome by dehydration and dis- integration, but not by either alone. This conclusion is in keeping with the fact (which the author has confirmed) that carotene cannot be satisfactorily extracted from fresh green leaves within a few hours by grinding under cold light petroleum or by soaking, without grinding, under acetone (which dehydrates the leaves), but can be easily extracted in a few minutes by B combination of grinding and the use of acetone. </p><p>Enzymic Destruction before Extraction. The problem of adapting the 80" to 100" petroleum technique to fresh leaves is not solved merely by grinding prior t o extraction, for carotene disappears from pulped leaf tissue sufficiently rapidly to inter- fere with accurate recovery. Under some conditions 25% may be lost from leaf pulp in 6 minutes (Z), which is about the shortest practicable time for thorough grinding and transference to the extraction vessel. The destruction is associated with lipoxidase activity (1 , 10, 14, and others). In the experiments in which leaves were ground and then extracted with 80 O to 100' petroleum the total yield of carotene, including that recovered from the residue, was less than that found by the control method. The loss could be considerably reduced by excluding osygen with car- bon dioxide during grinding, and almost eliminated by adding about 0.25 gram of quinol to 1 gram of leaves before grinding. Two difficulties, however, remain. The quinol makes the grind- ing physically difficult, and quantitative transference of the grist </p><p>A N A L Y T I C A L C H E M I S T R Y </p><p>to the extraction flask is tedious. No doubt these difficulties and the incomplete extraction could be overcome by the de- velopment of appropriate techniques-for instance, by first disintegrating leaves in cold 80 O to 100 a petroleum and quinol in a Folley-Watson (5) homogenizer and then heating in the same glass vessel. But then the main elegance of the 80' to 100" petroleum technique-its direct simplicity-will be lost and no improvement is had over the method using cold petroleum- acetone-quinol for the extraction. </p><p>ACKNOWLEDGMENT </p><p>The author thanks L. J. Harris and T. Moore for their interest in this work. </p><p>LITERATURE CITED </p><p>Booher, L. E., Hewston,, E. M., and Marsh, R. L., Food Research, </p><p>Booth, V. H., J . SOC. Chem. Ind . , 64, 162T (1945). Ibid., 64, 194T (1945). Edisbury, J. R., private communication, 1946. . Folley, S. J., and Watson, S. C., Biochem. J . , 42, 204 (1948). Hoffman, E. J., Lum, F. G., and Pitman, 9. L., J. Agr. Research, </p><p>Kernohan. G.. Science. 90. 623 (1939). </p><p>6, 493 (1941). </p><p>71,361 (1945). </p><p>Kreula, M . , Suomen Kemisti lehk, 18B, 69 (1945), Mann, T. B., Analyst , 69, 34 (1944). Mitchell, H. L., and Hauge, S. M., J. Biol. Chem., 163, 7 (1946). Moore, L. A., and Ely, R., IND. ENG. CHEM., ANAL. ED., 13, 600 (1941). </p><p>Nelson, W. A. G., Analyst , 72,200 (1947). Peterson, W . J., Biol. Symp08&amp;2, 12, 43 (1947). Sumner, J. B., and Dounce, A. L., Enzymologia, 7, 130 (1939). Zscheile, F. P., and Whitmore, R. A., ANAL. CHEM., 19, 170 (1947). </p><p>RECEIVED September 22, 1948. </p><p>Stability of Vitamin A Acetate under laboratory Conditions </p><p>M. E. CHILCOTE, N. B. GUERRANT, AND H. A. ELLENBERGER The Pennsylvania State College, State College, Pa. </p><p>The stability of vitamin A acetate w-as studied under conditions comparable to those usually involved in biological and chemicophysical studies. The results show that vitamin A acetate, in the crystalline state or when dissolved in oils of low peroxide value, may be stored without appreciable deterioration for con- siderable periods of time. Vitamin A acetate may be </p><p>Ii A previous report, certain properties of crystalline vitamin I A acetate were discussed in relation to its use as a standard in vitamin A assays (4). Included were data obtained through a preliminary investigation of the storage stability of vitamin A acetate under certain experimental conditions, inasmuch as stability under these conditions is one of the primary requisites of an acceptable vitamin A standard. This investigation, en- larged to include a study of the stability of vitamin A acetate in a number of organic solvents, has been completed and the results are presented here. </p><p>STABILITY OF VITAMIN A ACETATE </p><p>In Crystalline State and in Oil Solutions. The procedure employed in preparing the crystalline vitamin A acetate used in </p><p>dissolved in any one of several organic solvents and the solution stored with more than the required stability for its use as a vitamin A standard in spec- trophotometric and other chemicophysical studies. Batches of crystalline vitamin A acetate having essentially the same absorption properties may be prepared in the same or in different laboratories. </p><p>these studies has been described ( 4 ) , as has the preparation of the oil solutions of vitamin A acetate in a commercially refined and deodorized cottonseed oil (Wesson oil), corn oil (Mazola oil), and peanut oil for the stability studies. (The peroxide values of cottonseed oil, corn oil, and peanut oil were, respectively, 2.0, 1.5, and 11.7. In the previous publication it was reported that the peanut oil was purchased through a laboratory supply company; therefore it may not have been a fresh product or even representative of the peanut oils generally used as vitamin -4 carriers.) However, the following brief description of the essential procedures seems worthy of repetition. </p><p>The oil solutions were made to contain approximately 10,000 U.S.P. units of vitamin A per gram of solution. Portions of these oil solutions (250 mg.) and of the vitamin A acetate in the </p></li><li><p>V O L U M E 21, NO. 8, A U G U S T 1 9 4 9 </p><p>crystalline state (4 to 7 mg.) w r e sealed in clear Pyrex ampoules in vacuo and in an atmosphere of nitrogen, and stored in the ab- sence of light a t 5 " C. and at room temperature (25" to 30" C.). Samples from freshly sealed ampoules and samples from ampoules removed from storage at intervals were examined spectrophotc- metrically by means of a Beckman quartz spectrophotometer in order to determine the effect of the time and condition of storage on thr vitamin A content. </p><p>.A summary of the results of these studies is presented in Table 1. </p><p>Dissolved in Certain Organic Solvents. To study the stability of vitamin .A acetate in solution in some of the solvents commonly used in spectrophotometric and other ehemicophysical investiga- tions pertaining to this vitamin, the folloiving procedure was adopted. </p><p>Duplicate portions of the crystalline vitamin (4 to 7 mg.), which previously had been stored in evacuated glass ampoules a t 5' C., werp weighed on a microbalance and dissolved in the following solvents: absolute ethanol (100% U.S.P. from U. S. Industrial C'homical Co.), isopropyl alcohol (Baker's c.P.), cyclohexane (Castman No. 702), benzene (Baker's c.P.), and EI mixture of 10% cyclohexane and 90% absolute ethanol. From each of these solu- tions more dilute solutions having optical densities ranging between 0.5 and 0.8 were prepared in duplicate for spectrophoto- metric examination. In all instances the vitamin A solutions were prepared and maintained at room temperature in amber glass volumetric flasks (3). For each of the above-mentioned solutions, one of the duplicate portions was examined spectro- photometrically within 60 minutes after removing the crystalline vitamin from the glass ampoule. At varying intervals up to and including 24 hours following the preparation of the solutions, the E:?m.-maxima of each type of solution was determined a t specific wave lengths, as was also the optical density of the solution. These measurements were also made by means of the Beckman quartz spectrophotometer while using the hydrogen discharge tube as the source of illumination. </p><p>When no significant differences could be detected in the extinc- tion ratios or the Ei7m.-rnaxima of any of the solutions within the first 24-hour period, the spectrophotometric absorption examina- tion was continued a t specific intervals during the next 130 hours, the solutions remaining a t room temperature (approximately 25" C.) in the interim. In the meantime, solutions of vitamin A acetate in absolute ethanol, isopropyl alcohol, and cyclohexane, having optical densities ranging between 0.6 and 0.7, were pre- pared by diluting portions (120 to 136 mg.) of a Wesson oil solu- tion of the vitamin with the appropriate solvent. The maxima of these latter solutions were determined immediately and then a t specific intervals during the next 165 hours. These solutions were likewise stored a t room temperature. The sta- bility of vitamin A acetate and of Tesson oil solution of vitamin A acetatc. in the above organic solvents is shown in Figures 1 and 2 . </p><p>In addition, optical densities a t specific points over the spectral range of 220 to 400 ms were determined for the freshly prepared </p><p>cc IN CYCLOHEXANE OR IN BENZENE - IN ISOPROPANOL - IN W%ETHANOL-IO%CYCLOHEXANE IN ETHANOL </p><p>I I I I I I '*o 24 48 72 96 120 144 </p><p>STORAGE PERIOD IN HOURS </p><p>Figure 1. Relative Stability of Crystaliine Vitamin A Acetate at Room Temperature </p><p>Dissolved in Different Solvents </p><p>961 ___-- </p><p>Table I. Stability of Vitamin -4 Acetate and Its Oil Solutions </p><p>[ a s indicated by change in extinction coefficient (B:Fm.-326 mp)] </p><p>Time in Storage </p><p>Days </p><p>0 30 60 </p><p>120 151 187 244 307 </p><p>0 3 1 61 </p><p>117 152 184 217 301 </p><p>0 30 60 </p><p>101 120 156 189 257 290 </p><p>0 30 73 90 </p><p>124 189 249 </p><p>Retention of Vitamin A Stored at Room Temperature </p><p>Retention of Vitamin A Stored a t 5' C. </p><p>Under I n Under I n nitrogen vacuum nitrogen vacuum </p><p>, I 9% 7% % I n crystalline state </p><p>1P J 100 100 1IJO </p><p>83', '97 a </p><p>83 </p><p>89 80, 97a </p><p>. . . </p><p>. . </p><p>101 100 100 99 </p><p>100 99 99 </p><p>100 101 </p><p>96, 98" 96 </p><p>93 91,99" </p><p>.,. </p><p>9 $4 </p><p>100 Y9 </p><p>100 99 99 </p><p>. . . </p><p>Dissolved in Wesson oil 100 100 100 100 98 100 100 101 90 99 98 . . . 89 98 98 96 84 97 97 98 . . . 97 96 99 88 95 94 100 87 94 93 98 </p><p>Dissolved in Wesson oil 100 100 100 100 98 100 100 101 90 RQ RX _ _ _ - . . . .. 89 98 98 96 84 97 97 98 . . . 97 96 99 88 95 94 100 87 94 93 98 </p><p>100 100 97 83 88 83 83 82 80 </p><p>Dissolved in corn oil 100 99 98 96 97 96 97 96 . . . </p><p>Dissolved in peanut oil </p><p>100 98 </p><p>98 97 99 99 </p><p>100 98 </p><p>. . . 100 98 </p><p>97 98 97 99 97 </p><p>... . . . </p><p>100 100 100 100 </p><p>79 . . . a Ampoules filled with nitrogen by means of manifold system; other </p><p>ampoules charged with nitrogen by vacuum desiccator. </p><p>? w w 0 4 </p><p>3Hc I N CYCLOHEXANE I N lSOPROPANOL </p><p>w I N E T H A N O L </p><p>8 ,I I I I 1 1 1 0 24 48 72 96 120 1 4 4 168 </p><p>STORAGE P E R I O D I N H O U R S </p><p>Figure 2. Relative Stability of Vitamin A Content of Wesson Oil Solution of Vitamin A Acetate Dissolved in </p><p>Different Solvents </p><p>solutions of crystalline vitamin A acetate in the above-mentioned solvents. Similar ultraviolet light absorption measurements were made on absolute ethanol solutions of a sample of the crystal- line vitamin A acetate supplied for a U.S.P. collaborative study (18, 14). The concentrations of the solutions used in these latter studies m r e adjusted so that the optical densities fell within the limits of 0.5 to 1.9, as suggested by Vandenbelt et al. (IS). The optical density data expressed as extinction ratios (9) are presented in Table 11. For comparison, previously reported data for crystalline vitamin -4 acetate when dissolved in absolute ethanol and in cyclohnane (7,8,1Z) N I c also prrsented in Table 11. </p><p>MANIPULATION OF BECKMAN QUARTZ SPECTROPHOTOMETER </p><p>In carrying out the above measurements, the sensitivity knob of the Beckman spectrophotometer was set a t three turns from </p></li><li><p>962 </p><p>the extreine clockwise position. The slit width was then varied to adjust the galvanometer needle to 1007, transmission when the solvent occupied the absorption cell. In following this proce- dure of instrumentation, the slit width, with rare exception, never varied more than 0.04 mm. from a setting of 0.40 mm. when transmission measurements were made at 325 mp. This practice was believed justifiable, inasmuch as varying the slit width in steps from 0.2 to 0.55 mm. and compensating by sensitivity ad- justments produced no appreciable differences in the optical den- sities of a series of solutions of varying concentrations of a dis- tilled vitamin A ester concentrate (obtained from Distillation Products, Inc.) in isopropyl alcohol when the measurements were made a t 325 mp. </p><p>The performance of the Beckman spectrophotometer \vas checked routinely by means of an isopropyl alcohol solution 2- phenylazo-p-cresol dye (obtained from Shohan Laboratories) as suggested by Taylor (11) and Kreider (5). For this purpose, a 15 mg. 7 0 solution of the dye in isopropyl alcohol was kept in the rc- frigerator. The solution under this condition of storage remaincvl practically stable for 3 months. </p><p>A N A L Y T I C A L C H E M I S T R Y </p><p>tioiis through an extended capillary into small glass ampoules. The oil-filled ampoules were then charged with carbon dioxide in a vacuum desiccator, removed, and immediately sealed. In con- trast to the previously mentioned nitrogen-flushed ampoules, only a very small gas space remained in these latter ampoules after seal- ing. These ampoules were stored in the absence of light at 5' C. and a t room temperature (25" to 30" C.) as were the nitrogen- charged ampoules. Subsequent spectrophotometric examination of the contents of the ampoules revealed that much higher sta- bility of vitamin A had been attained. After 138 days of storage at 5" C. and at room temperature, the average E:Fm.-325 mp values obtained on the contents of triplicate ampoules indicat,ed that the oil retained 100 and 98qC, respectively, of its original vitamin content. </p><p>The above observatioris indicate that vitamin A acetate in the , cry stall in^ state and vhen dissolved in a refined cott,onseed oil is </p><p>sufficiently stable to Lvarrant its use as a vitamin Stability of Vitamin A Acetate Dissolved in Organic Solvents. </p><p>The data presented in Figures 1 and 2 show that, vitamin A ace- tate, in the crystalline state or when dissolved in Wesson oil, reniainrd stable for 24 hours under all the conditions investigated except when the oil solution of the vitamin was dissolved in ab- solute ethanol. Even the extinction coefficient of this solution was sufficiently stable to permit the use of the solution as a spectrophotometric st,andard over the time interval usually required. The extended stability of crystalline vitamin acetate and of Kesson oil solutions of the vitamin, when dissolved in cyclohwane or in isopropyl alcohol, is a further a...</p></li></ul>

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