Determination of Vitamin D in Multivitamin Mixtures after Separation by Partition Chromatography JAMES G. THEIVAGT and DONALD J. CAMPBELL Analytical Research Deportment, Abbott laboratories, North Chicago, 111.
b Determination of vitamin D in multivitamin mixtures by a chemical method is possible, if vitamin D can be separated from vitamin A. Partition chromatography offers a rapid, reproducible method that, for analytical purposes, is superior to adsorption chromatography. The separating ability of various partition columns can be quickly evaluated from approximate partition coefficients and the equations developed by Martin and Synge. After separation, vitamin D is estimated colorimetrically. Mixtures with A/D ratios as high as 50 to 1 have been separated and analytical results for a wide variety of samples have checked closely with the official U.S.P. biological assay.
T
successful application of chemical methods to the determination of vitamin D in multivitamin mixtures requires the quantitative separation of vitamin D from vitamin A. Prior to 1958, the most successful procedures for this purpose were those of Ewing, Schlaback, and Powell ( 2 ) and Tschapke and Plessing (8). These methods lack general applicatiou and difficulties are encountered in obtaining column materials with the required adsorption characteristics. The recent method of Wilkie, Jones, and Kline (9) is applicable to a nidi. variety of samples, but requires soniewhat elaborate preparation of the column materials and considerable care to run. -411 of the published methods have employed adsorption chromatography as the means of separating vitamin D. It was decided to investigate liquidliquid partition chromatography, b e cause inherently it is a better method of separation which depends, for the most part, only upon differences in the partition coefficient. This method minimizes destruction or alteration of materials often encountered in using strongly adsorbing column materials. Once prepared, a partition column can be used for many samples. This method separates vitamin A and vitamin D on a partition column using Celite 545 as the inert support, polyethylene glycol 600 (PEG 600) as the immobile phase, and iso-octane as the mobile phase. HE
I n partition chromatography, the search for a suitable immiscible pair can be hastened as follows. First, possible pairs can be devised from a list of solvents such as that prepared b y Craig ( I ) , which arranges solvents in order of increasing dielectric constants. The best pairs for separation are usually made up from solvents differing widely in dielectric value. Next, approximate partition coefficients, cy, are determined for each vitamin between equal volumes of the immiscible pairs chosen. The amount of solute in each layer is determined b y ultraviolet analysis. Typical results are shown in Table I. Partition chromatography columns operate under the same principles as countercurrent extractions using the Craig apparatus-that is, separations are based on differences in partition coefficients. Thus as cy approaches zero, the solute is moving as fast as the mobile phase through the column. I n the Craig apparatus the best separations are achieved when CY is unity. This affords good opportunity for minor components to be partitioned away. However, as pointed out by Trenner (Y), there is about five times as much mobile phase as immobile phase at each plate in the partition column. The Craig system employs equal volumes of solvent. Thus the partition coefficient of the major component should be about 5 to approach a “working” partition coefficient of unity. From Table I, i t can be seen t h a t vitamin A alcohol approximates this value. With a n (Y value of 10, the working partition coefficient becomes 2. This permits retardation of vitamin A
Table
I.
and allows vitamin D and other components to be partitioned away. In the system chosen, LY*/CYD = 10/0.8 or approximately 10. However, the separation is not of t h a t order. Actually, in running a separation as described, the peak concentration of vitamin D appears at about 50 ml. while vitamin A peaks at about 160 ml. or a 3/1 ratio. This separation can be predicted by the ratio of R values as calculated using the equation developed b y Martin and Synge (4) where movement of maximum concentration of solute R = movement of solvent above bed Ai Am A, where
A,
+ + +
Ai
( d m )
cross-sectional area of immobile phase A , = cross-sectional area of mobile phase A , = cross-sectional area of support The calculation for Ai is as follows: volume of immobile phase A, = total volume of column X cross-sectional area of column .4% and A , are calculated in the same manner, using the volumes of the mobile and stationary phases, respectively. The column described has a height of 15 em. and a radius of 1.1 cm. and contains 25 grams of Celite 545, 10 ml. of PEG 600, and 40 ml. of iso-octane. The density of Celite is 3.3 grams per nil. Thus, -4, = 0.51 sq. cm.; A , = 2.66 sq. em.; ii, = 0.67 sq. cm. From Table I, LYA = 10 and CYD = 0.8. The corresponding R values calculated for vitamin A and D are 0.4 and 1.2, respectively. =
Partition Coefficients of Vitamins A and D in Several Systems
Solvent Pairs Immobile Mobile Acetonitrile Iso-octane Ethylene glycol Mineral oil Propylene glycol Chloroform Ethyl alcohol 90% Iso-octane HzO 10% PEG 200 Iso-octane PEG 600 Iso-octane Iso-octane Mg. of solute in immobile phase. mg. of solute in mobile phase
Approximate Partition Coefficient, aa Vitamin -4palmitate Vitamin D 0.02 0.36 0.00 0.42
0.32 0.00 0.00 0.66
0.01 0.04
0.37 0.80 0.80
10 (vitamin A alcohol)
VOL. 31, NO. 8, AUGUST 1959
1375
This means the vitamin D nil1 come off the column three times as fast as vitaniin A. As stated earlier, the peak concentration of vitamin D appears a t 50 ml. Thus from the R values, vitamin A should reach a peak a t 1.2/0.4 X 50, or 150 ml. This is very close to the experimental value of 160 nil. I n this manner, using a value approsimations and Martin and Synge's calculations, a reasonably methodical approach can be made to tlic choice of partition systems for difficult separations. PROCEDURE
Reagents. Iso-octane (2,2,4-trimethylpentane), 99% pure from Phillips Petroleum Co., Bartlesville, Okla., is shaken with P E G 600 and used only after i t has cleared on standing. Polyethylene glycol 600 (PEG 600), romniercial grade from Union Carbide Chemicals Co. Sitanlin Dz standard solution. Prepare an ethylene dichloride solution to contain 10 y (400 U.S.P. units) of crystalline vitamin D1 (Calciferol) per nil. Store under refrigeration. h i t i mony trichloride solution. Prepare as described by Wilkie, Jones, and Kline
column \\ ith three bninll portions of iso-octane. Add larger amounts of equilibrated iso-octane and maintain a flow rate of 2 to 3 ml. per minute. Collect the fraction which has previously been found to contain vitamin D, and dilute to 25 nil. with iso-octane. Place 10 ml. of this solution in a centrifuge tube with 10 ml. of water and shake. Centrifuge and decant the clear iso-octane into a flask containing 1 gram of anhydrous sodium sulfate. This is the sample solution. This water wash removes traces of P E G 600 (which form a haze when the antimony trichloride reagent is added). Determine the absorbance at 500 mp of the following three solutions exactly 1 minute after adding the antimony trichloride reagent, using a blank of ethylene dichloride (9). Any colorimeter which will provide linearity a t 500 nip is suitable. A I 1 ml. of sample solution, 1 ml. of ethylene dichloride, and 10 ml. of antimony trichloride reagent. -42. 1 ml. of sample solution, 1 ml. of a 1 : 1 mixture of acetic anhydride and ethylene dichloride, and 10 ml. of antimony irichloride reagent. .id.1 nil. of sample solution, 1 ml. of vitamin U standard solution, and 10 ml. ut antimony trichloride rpagent.
ralculation.
(9).
Column Preparation. Mix 25 grams of Celite 545 in 150 ml. of iso-octane. Add slowly with vigorous stirring 10 nil. of P E G 600 and continue mixing until the slurry is uniform. Add ali(pot5 of the slurry to a 28-cm. chromatography tube 22 mm. in inside diameter, and pack to a height of 15 cm., using the technique of Martin (3, 4 ) . Determine the volume in nhich vitamin D is recovered as follows: ildd 2 ml. of iso-octane containing 0.6 mg. of vitamin D to the column and rinse it into the column with three small portions of iso-octane. Maintain a flow rate of 2 to 3 ml. per minute by regulating the height of iso-octane above the column bed. Follow the recovery of vitamin D by measuring the absorbance of small fractions of the eluate a t 263 mp, 'S'itaniin D is recovered in a volume of less than 25 ml., usually between the 35th and 75th ml. of eluate. This volume and the interval of recovery remain constant for each column and are used for subsequent analysis. Basic Method. Place a n accurately measured amount of sample expected t o contain 10,000 units of vitamin D in a n Erlenmeyer flask. -4dd 2.5 ml. of 50% potassium hydroxide solution for each gram of sample, and 50 ml. of ethyl alcohol. Reflux on IL steam bath for one-half hour. Cool, and extract the nonsaponifiable material by the method of Wilkie, Jones, and Kline (9). Evaporate the extracts to drvness under a stream of nitrogen, and immediately add 4 ml. of iso-octane to dissolve the residue. Allow the iso-octane above the chromatographic column to flon into the paper disk. Add 2 ml. of the estract solution and rinse it into the 1376
ANALYTICAL CHEMISTRY
fl, - A , _ _ X units of vitamin 13 per ml. of -4 - A , 1
standard solution units of vitamin D per ml. of sample solution units vitamin D Der of sample soILition then -d. weight of sample ( g . ) per ml. of sample solution units per g. of sample =
T o re-use the column, it is necessary to remove the vitamin A that remains. This is done by allowing 200 or 300 ml. of equilibrated iso-octane to pnss through the column. T o test for complete removal of vitamin A, add 10 ml. of the antimony trichloride reagent to 1 ml. of eluate-the solution will be colorless if sufficient niobile phase has been used.
Modification of Basic Method. I n some cases, interfering vitamin A degradation products may not be completely separated by t h e partition column alone. Several materials show promise in removing all or a large part of these interfering substances. Among the materials tested were alumina, silicic acid, and Florex XXS. Of these, Florex XYS,which has been used fsr the separation of vitamins A and D by Schmall (6), proved to be a convenient material to use. If the value A2 is more than 25% of AI, the use of Florex XXS is required as follows. Add 10 ml. of the vitamin D fraction from the partition column to a 6-mm. tube tapered a t one end, plugged with glass wool, and containing 3 grams of Florex XXS (Floridin Co.,
Tallahassee, Fla.) tamped in pl'iw. Apply vacuum or air pressure in order to get a flow rate of 3 ml. per minutr. Rinse with 10 ml. of iso-octane and &;card the eluate. Elute the vitamin D with 50 nil. of reagent grade benzene, and evaporate to dryncsq on a steam bath using nitrogen protection. I n all evaporations, do not apply heat in excess of 40" C. to the solutions n-hen the volume is less than 10 mi. Add 10 ml. of ethylene dichloride to the residue and use the colorimetric annlJ-sis as above, DISCUSSION AND RESULTS
Tlie analysis has been applied successfully to a number of pharmaceutical products containing vitamins A and D, including multivitamin tablets with and without minerals, multivitamin liquids, and fish liver oils. Sonic. low potency multi>itaniin s i r u p wquire an extraction of the vitamin D prior to the saponification. Petroltuni ether is a suitable solvent to w e : but to ensure complete extraction of the Iitaniin D and to avoid emulsions, it is desirable to maintain a 1 : 1 : 1 ratio of water (and sample), ethyl alcohol, and petroleum ether. The petrolrum ether extract is washed with n atrr and evaporated to dryness. The residue is then treated in the manner described for other samples. The P E G 600-iso-octane column is stably and inert. Columns have been used for 3 months on a wide variety of sumplo3 and the vitamin D has been quantitatively recovered in the fraction initially determined, using the standard solution of vitamin D. I n some instances, material present in the particular saniple may alter the partition coefficient sufficiently to affect the collection volume. Although this has not been observed, i t could be corrected by collecting a larger volume of eluate. T o ensure that vitamin A is not present in this large volume, the movement of the vitamin A zone down the column can be readily followed with a weak ultraviolet source (9). Sample size may be varied over a wide range, because the limiting factor is only that the final assay solution should contain at least 100 IT.S.P. units of litamin D per ml. The amount of sample solution applied to the partition column can be varied between 0.1 and 5 ml. without altering the recovery volume of vitamin D. Samples in vhich the ratio of vitamin A to vitamin D is 50 to 1 have been analyzed satisfactorily. However, the major consideration is not the ratio of vitamin A to vitamin D, but rather the amount of vitamin A degradation products which might be encountered. -4s much as 500,000 units of vitamin A liavc, been intioduced into a column and
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
