Table VIII. R a t i o of E l u t i o n Volumes for Polystyrenes with Molecular Weights of 1 X lo6 and 2 X l o 3 to the E l u t i o n Volume at Molecular Weight of 1.8 X lo6 Ratio of elution volumes, Ve(2)/Ve(l) Calculated
a1
PS PS 1 x 105 2 x 103
1.13 al = ro al = 0 . 5 r o 1.38 al = 0 . 1 ~ 1 . 1 4 ai = O.O1ro 1.02
Observed
PS 1
x
ps2
10s
x
103
(1.02-1.09)
(1.095-1.14)
Av. 1.065
Av 1.111
1.46 1.60 1.19 1.02
For estimation purposes, we have assumed values of K = 1 X and 01 = 0.75, then (S2)'/'
= 4 . 6 5 x 10-2@*58d-1/3
(7)
An effective radius for the polymer molecule is expressed by Equation 8 obtained by combining Equations 3 and 7
Assuming the effective radius, al, of polystyrene having molecular weight 1.8 X 106, to be successively a1 = ro, a1 = 0.5 ro, a1 = 0.1 ro and a1 = 0.01 ro, we calculated the ratios of elution volumes of polystyrenes having molecular weight 1 X lo5 and 2 X lo3 to that of 1.8 X lo6 using Equations 4 and 8. The results are shown in Table VIII. End-to-end distances of polystyrenes calculated by D. F. Alliet and J. M. Pacco (7) are 1490 8, (PS 1.8 X 106 mol wt), 280 8, (1 X lo5 mol wt), and 35.2 8, (2 X lo3 mol wt). If half of these values are considered to be equal to the effective radius of the polymers, then the value of a1 falls between 0.106 ro and 0.166 ro. Comparison of the calculated and observed values a t a1 = 0.1 ro shows that the experimental results are in reasonable agreement with theory.
RECEIVEDfor review February 22, 1974. Accepted May 17, 1974. A portion of this work was supported through National Science Foundation Grant No. GP 28613. This paper is part XXXI of a series on Column Fractionation of Polymers. (7) D. F. Alliet and J. M. Pacco. 6th International Seminar, Oct. 1968, Florida, Preprint, p 274.
Determination of Vitamin D2 in Gelatin-Protected Vitamin A AcetateNitamin D2 Beadlets by High Pressure Liquid Chromatography David F. Tomkins' and Ronald J. Tscherne2 Quality Control Department, Hoffmann-La Roche Inc., Nutley, N.J. 07 1 10
Vitamin concentrates containing vitamin D2 (ergocalciferol) and vitamin A acetate (trans-retinyl acetate) usually are prepared by dispersing the vitamins in starch-coated, gelatin-protected beadlets, or by dissolving the vitamins in oil. The vitamins are stabilized with antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),or ethyoxyquin. Chemical methods for the determination of vitamin D2 in gelatin-protected beadlets involve laborious techniques for the removal of interfering substances. The method outlined in the United States Pharmacopeia requires exhaustive purification including saponification, extraction, and separation on several columns prior to reaction with antimony trichloride and colorimetric determination ( I ) . T o a large degree, the accuracy of the compendia1 determination is dependent on the manipulative skill of the analyst. Since no known chemical reaction is sufficiently specific to permit determination of vitamin D2 in the presence of vitamin A acetate, this interference remained the most important obstacle to the accurate determination of vitamin Dz. Although vitamin A acetate and vitamin D2 can be separated by thin-layer chromatography (TLC), the vitamins Present address, Corporate Research, Monsanto Chemical Company, St. Louis, Mo. 63166. A u t h o r t o whom a l l correspondence should be addressed. (1) "United States Pharmacopeia," 18th rev., Mack Publishing Co.. Easton, Pa., 1970, p 915.
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are relatively unstable on dry chromatographic adsorbents (2). Furthermore, TLC estimations are only semiquantitative and require lengthy sample extractions from vitamin formulations. Gas-liquid chromatography (GLC) has also been applied to vitamin Dz determinations with varying degrees of success. A review article by Sheppard and coworkers ( 3 )outlines recent advances in GLC determination of fat-soluble vitamins. These methods require preliminary sample purification. In addition, GLC determinations of the thermally unstable vitamin D2 result in the formation of cyclization products ( 4 ) . Alternate techniques including mass spectrometry ( 5 ) and gel filtration (6) also require tedious extraction procedures. Recent advances in commercially available instrumentation and column packings have enabled High Pressure Liquid Chromatography (HPLC) to be used for the separation and determination of fat-soluble vitamins (7-12). Williams (2) G. W. Johnson and C. Vickers, Analyst(London), 89, 257 (1972). (3) A. J. Sheppard, A. R. Prosser, and W. D. Hubbard, J. Amer. OilChem. Soc., 49, 619 (1972). (4) P. P. Nair and S. deleon, Progr. Biochem. Pharmacob, 3, 498 (1967). (5) K . Tsukida and K. Saiki, lnt. J. Vitamin Nutr. Res., 42, 242 (1972). (6) F. Ueda, T. Makino, A. Kazama, and K. Watanalee, J. Vifaminol. (Kyoto), 17, 142(1971). (7) Chromatronix. Berkeley, Calif., "Liquid Chromatography Application," Number 1, (1972). (8) Waters Associates, Framingham, Mass., "Applications Bulletin," AN71-111 (1971). (9) Reeve Angel, Clifton, N.J., "Applications Bulletin," 2.5 M 7/72 (1972). (10) F. G. Theivagt and D. F. Cambell, Anal. Chern., 31, 1375 (1959).
ANALYTICAL CHEMISTRY, VOL. 46, NO. 11, SEPTEMBER 1974
(13) has described the use of a small diameter silica column for the separation of fat-soluble vitamins. However, this method requires a sample extraction procedure which only partially separates fat-soluble vitamins from multivitamin preparations. This extraction procedure was ineffective for the separation of vitamins from gelatin-protected beadlets. This paper describes a HPLC method for the determination of vitamin D2 in gelatin-protected beadlets utilizing a column of small diameter silica. The procedure eliminates lengthy extractions and results in rapid, reliable determinations of vitamin D2. A unique extraction procedure is described which provides complete separation of vitamin D2 from a gelatin matrix.
EXPERIMENTAL Apparatus. A DuPont Model 820 Liquid Chromatograph equipped with an ultraviolet detector system (254 nm) was used for analytical separations. A DuPont Zorbax Si1 chromatographic column (25 cm X 2.1 mm i.d.) was used for all determinations. In addition, a “guard” column (5 cm X 2.1 mm i.d.) packed with Corasil I1 (Waters Associates) was connected between the injector assembly and the chromatographic column, using low “dead-volume” connectors. Sample injections were made with a 5 - 4 Hamilton High Pressure syringe (No. H P 305 N). A Honeywell Model 194 dual channel dual pen recorder was used for monitoring. - the detector output. R e a -g e n t s a n d Solutions. Certified ACS ethvl acetate and soectral quality heptane were used in the mobile phase solutions. Reagent grade p-nitrophenylacetonitrile,and spectral quality dimethyl sulfoxide (DMSO) were purchased from Matheson, Coleman & Bell. Samples of starch-sugar-gelatin matrix vitamin A acetatehitamin Dz beadlets were manufactured by Hoffmann-La Roche. A s h c k internal standard solution was prepared by diluting 100 mg of p-nitrophenylacetonitrile to 1 liter with dichloromethm e . A standard vitamin Da solution was prepared by dissolving 12 mg of reference standard vitamin D2 (The United States Pharmacopeia] Convention, Inc.) and diluting to 100 ml with DMSO. All solutions were stored in low actinic glassware. Column Preparations. The “guard” column was dry-packed with Corasil I1 adsorbent and connected between the injector and the Zorbax Si1 column. The “guard” column and the chromatographic column were allowed t o equilibrate with the mobile phase until a stable base line was attained. Mobile Phase P r e p a r a t i o n . Spectral quality heptane was water saturated by overnight equilibration with water. T h e watersaturated heptane was passed through a column of 23% (w/w) water saturated silica gel, prepared by adding 30 ml of water t o 100 g of silica gel (100-200 mesh). T h e saturated heptane was diluted with an equal volume of spectral quality heptane to give a 50% water-saturated heptane solut,ion. The mobile phase was prepared by diluting 40 ml of ethyl acetate and 120 ml of dichloromethane to 1 liter with 50% water-saturated heptane. S a m p l e P r e p a r a t i o n . A l.Gg sample of vitamin A acetate/vitamin 1~~beadlets (containing 500,000 I.U./g of vitamin A acetate and 50,000 I.U./g of vitamin Dz) was weighed into a low actinic .iO-ml centrifuge tube and 15.0 ml of DMSO were added. The tube was shaken vigorously and placed in a water bath a t approximately 60 “C for 20 minutes. To ensure complete dispersion of the gelatin, the solution was shaken a t frequent intervals for the first few minutes of heating. After dispersion was complete, the samples were allowed to cool to room temperature. The gelatin was then precipitated by the addition of 25.0 ml of the internal standard solution and the tube was centrifuged a t 3000 rpm for 10 minutes. The supernatant liquid was injected directly onto the column. S t a n d a r d P r e p a r a t i o n . Fifteen milliliters of the standard vitamin D L solution were pipetted into a low actinic centrifuge tube and 25.0 ml of the internal standard solution was added. T h e standard was run concomitantly with the sample. Procedure. The column pressure was adjusted to 1300 psi (0.6 ml/min) a t ambient temperature. Injection volumes of 4 p1 of standard and sample solutions were used for determinations. Peak (11) R. C. Williams, F. A. Schmit, and R . A. Glenny, J. Chromatogr. Sci., 10, 494 (19?2). (12) Nester/Faust, Newark, Del., “Liquid Chromatographic Application Bulletin,” No. 10-71 (1971). (13) R. C. Williams, “DuPont L.C. Application Lab Report,” No. 72-09 (1972).
Table I. Recovery of Vitamin Dz from Gelatin-Matrix
Sample No. Amount added, m g
1
0 .ooo
2 3 4 5 6 7
0.364 1,096 1.824 2.300 3.536 5.480
Experimental amount found, mg
Recovery,
0.000 0.393 1.096 1.768 2.384 3.560 5.400 Av recovery, % =
Yo
100 108 100 97 103 101 99 101
areas were determined by multiplying peak height by peak width a t half-height. Calculation of sample vitamin D2 content was identical t o internal standard calculations described in current literature and the factor, l mg = 40,000 I.U., was used to convert milligrams of vitamin D2 to International Units.
RESULTS AND DISCUSSION This vitamin extraction from a gelatin matrix was developed after an evaluation of a variety of vitamin extraction procedures. Dispersion of the sample in warm DMSO was found to be a rapid method for the removal of vitamins from a gelatin matrix. However, the direct injection of this solution resulted in peak distortion after several determinations. The addition of dichloromethane (which is miscible with DMSO) to the sample solution eliminated peak interferences associated with dissolved gelatin. Although the dichloromethane precipitated nearly all the gelatin from sample solution, a “guard” column protected the chromatographic column from the small amount of gelatin remaining in the supernatant liquid. The “guard” column was replaced when peak broadening and variation of standard response ratio were observed. Recovery studies indicated the complete extraction of vitamin Dz from placebos-ie., material having the same matrix as the sample with the exception of vitamin Dz-at levels ranging from 9 to 35 pg/ml (See Table I). No variation of response ratio was found for standard solutions containing various amounts of placebo over the range of 0.2 to 2.0 g. Heating of vitamin D2 in DMSO at approximately 60 “C for up to 45 minutes caused no apparent change in detector response; however, one hour of heating resulted in an 8% decrease. To minimize decomposition, it is recommended that low actinic glassware be used and temperatures be kept below 70 OC. The sample solutions were stable for approximately one day when protected from light and heat. Vitamin A acetate instability was not a critical factor in vitamin D2 determinations. An adsorption column was chosen because large concentrations of vitamin A acetate interfered with vitamin D2 determinations when reverse phase separations were attempted. Under reverse phase conditions, the vitamin D2 was retained only slightly longer than vitamin A acetate and, as a result, was incorporated into the tail of the very large vitamin A acetate peak. The small diameter silica adsorbent had both a high sample capacity and provided the required efficiency for the separation. Since the retention time was dependent upon the surface activity of the silica packing, it was imperative that the water content of the mobile phase be maintained constant for reproducible separations. High operating pressure of the colrequired the use of an internal standard metod to eliminate injection variation. The operating conditions described represent the most favorable compromise between speed of determination and maximum separation of the vitamins present in the sam-
ANALYTICAL CHEMISTRY, VOL. 46, NO. 11, SEPTEMBER 1974
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d I IA E
e 4
; W v)
0
$5 I I :
A'
2
5W.L 10 IS TIME ( m m )
20
Figure 1. Sample chromatogram of vitamin D2 beadlets containing vitamin A acetate
Utes, a long retention time for vitamin D2 (ca. 14 minutes) was chosen because vitamin A alcohol (retinol) and degradation products from vitamin A acetate elute shortly after the vitamin D2 peak. Peak area measurements using peak height multiplied by the width a t half-height proved to be very reliable when the photometer attenuation was reduced to 0.04 absorbance unit full scale. This attenuation gave sample vitamin Dz peaks of nearly full scale deflection and could be easily and accurately measured. The vitamin Dz content of several lots of vitamin A acetatehitamin Dz preparations, ca. 500,000 I.U. vitamin A acetate and 50,000 I.U. vitamin Dz, were determined. Results of triplicate chromatographic determinations for ten lots of gelatin-protected beadlets as well as results for the compendial method are compared in Table 11. A statistical evaluation of both methods reveals the compatibilitjr of results. The average standard deviation for the chromatographic method was 0.22 whereas the cornpendial method was 0.3. These results demonstrated that the chromatographic method is as precise as the wet chemical method; however, the determination time using the chromatographic method was substantially decreased. The chromatographic shown in Table 11 were obtained in two days, while the U.S.P. results required nearly ten days of
(A) Vitamin A acetate: ( B ) vitamin DP: (0 p4trophenylacetonitrile
Table 11. Vitamin D2 Determination in Gelatin-Protected Beadlets USP results X 10-4 fI.U./g)
Chromatographic results X 10 -4 (I.U./g) Individual preparations -____ ____.
Sample No.
1 2 3 4 5 6 7 8 9 10
5.61 5.46 5.46 5.50 4.31 5.38 5.66 5.81 5.30 5 .00
5.63 6.04 5.46 5.50 4.25 5.48 5.11 5.51 5.30 4.88
5.31 5.56 5.62 5.60 4.17 5.43 5.78 5.22 5.13 5.27
Avg.
std dev
5.51 5.84 5.51 5.53 4.24 5.43 5.52 5.51 5.24 5.05
0.18 0.24 0.09 0.06 0.08 0.06 0.40 0.35 0.10 0.23 0.22
Av std dev Table 111. Precision of Vitamin DPDeterminations Vitamin D? content X 10-4 (I.U./g) Run No.
1 2 3 4 5 6 7 8 9 10
Av Std dev Re1 std dev
Replicate sample preparations
5.29 5.30 5.13 5.21 5.60 5.32 5.31 5.10 5.31 5.15 5.27 0.14 2.7%
Replicate injections of one preparation
5.20 5.38 5.22 5.08 5.32 4.93 5.29 5.23 5.23 5.23 5.21 0.13 2.5%
ples. The chromatogram shown in Figure 1 exemplifies the type of separation achieved. Internal standard retention time was more dependent upon dichloromethane concentration than was the retention time of vitamin D,; consequently, the relative retention times could be varied. Although no variation in response ratio was observed for vitamin Dz retention times from approximately 7 to 14 min-
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Individual preparations
5.6 5.1 6.1 5.4 4.7 5.6 6.2 6.1 5.7 5.9
5.5 5.7 5.8 5.6 4.2 5.3 5.9 5.7 5.5 5.4
Av
std dev
5.6 5.4 6 .O 5.5 4.5 5.4 6.1 5.9 5.6 5.6
0.1 0.5 0.3 0.2 0.5 0.3 0.3 0.4 0.2 0.5 0.3
testing. Precision of replicate vitamin Dz analyses was determined for individually prepared samples and also for ten injections of one sample. The results and standard deviation of these determinations are summarized in Table 111. Peak areas of the vitamin Dz and the internal standard solutions were linear with respect to concentration from 8 to 120 pg/ml. The limit of detection, i.e., the minimum amount of vitamin D2 that gave a response equal to twice the noise of the detector, was approximately 24 ng. This new approach to the separation and determination of vitamin Dz has wide application to a variety of vitamin preparations. The suggested extraction procedure gives rapid and reliable removal of vitamin D2 from a gelatin matrix, reducing the time required for determination from nearly one-half day, using the USP XVIII methotto less than one hour. The method works equally well for preparations of vitamin D3 (cholecalciferol); however, vitamin Dz will not separate from vitamin D3 in the system described. Preliminary work indicated that vitamin A acetate determinations are possible if special care is taken to reduce decomposition.
RECEIVEDfor review February 14, 1974. Accepted May 16, 1974.
ANALYTICAL CHEMISTRY, VOL. 46, N O . 11, SEPTEMBER 1974
