(6) P. T. Kissinger. L. J. Felice, R. M. Riggin, L. A. Pachla, and D. C. Wenke, Clin. Chem. ( Winston-Salem, N.C.).20, 992 (1974). (7) W. D. Slaunwhite, L. A. Pachla, D. C. Wenke. and P. T. Kissinger. Clin. Cbem. ( Wlnston-Salem, N.C.),21, 1427 (1975). (8) R. M. Riggin, A. L. Schmidt, and P. T. Kissinger, J. Pharm. Sci., 64, 680 (1975). (9) L. A. Pachla and P. T. Kissinger, Anal. Cbem., 48, 364 (1976). (10) P. T. Kissinger. R. M. Riggin, R. L. Alcorn. and L.-D. Rau, Biochem. Med., 13, 299 (1975). (11) B. L. Oser, Ed., “Hawk’s Physiological Chemistry”, 14th ed, McGrawHill, New York, N.Y., 1965, p 1040.
(12) G. Ebinger and K. Adriaenssens. Clin. Chim. Acta, 48, 427 (1973). (13) I. Sankoff and T. L. Sourkes. Can. J. Biochem. Physiol., 41, 1381 (1963).
RECEIVEDfor review December 15, 1975. Accepted February 9, 1976. Financial support from the National Science Foundation, The National Institute of General Medical Sciences, and the Showalter Trust Fund is gratefully acknowledged.
High-pressure Liquid Chromatographic Determination of Ascorbic Acid in Selected Foods and Multivitamin Products S. P. Sood,’ L. E. Sartori,* D. P. Wittmer,* and W. G. Haney’ University of Missouri-Kansas City, School of Pharmacy, 5 100 Rockhill Road, Kansas City, Ma. 64 1 10
High-pressure liquid chromatography in the reversedphase, ion-pairing mode has been used to determine ascorbic acid in foods and multivitamin products. While several counterions were investigated for utility, tridecylammonium formate was selected. Workup procedures are minimal, requiring only dissolution of the analyte in water. With detection at 254 nm, solutions of ascorbic acid of 0.5 mg/100 ml can be determined.
The determination of ascorbic acid has been of considerable interest to analysts and was the subject of a recent review ( I ). Classically, 2,6-dichlorophenolindophenolvisual titration ( 2 ) , microfluorometry ( 3 ) ,or colorimetry of the 2,4-dinitrophenylhydrazone of dehydroascorbic acid ( 4 ) have been employed for this determination. However, these methods are often limited by the number of potential interfering substances found in the matrix containing the vitamin (j), and sample workup procedures are therefore complex or results subject to error. In addition, end points are ill defined, and problems with color development and fading are common. Polarographic (6),chromatographic (7), and turbidimetric (8) techniques have also been recently investigated for this determination, but appear also to suffer from the same or related limitations. As a result, a procedure utilizing high-performance liquid chromatography (HPLC) in the reversed-phase mode has been developed and evaluated.
EXPERIMENTAL Apparatus and Operating Conditions. A Model ALC 202 Liquid Chromatograph equipped with a Model 6000 pump and U6K injector (Waters Associates, Milford, Mass.) was used in the study, and column effluents were monitored with the 254-nm detector. Peak areas were determined using an electronic digital integrator (Varian Model 505). The flow rate was 3.0 ml/min. Column. A 30 cm X 4 mm i.d. pBondapak CIS column (Waters Associates) was used. pBondapak Cle has a monomolecular layer of octadecyltrichlorosilane chemically bonded to pPorasil beads having an average particle size of 10 Mm. The number of theoretical plates, based on ascorbic acid at 3.0 ml/min, was 2300, and k o was 1.9 ml. Reagents and Materials. Ascorbic Acid, Reference Standard, was obtained from the U.S.P. Reference Standards Laboratory. Present address, Marion Laboratories, 10236 Bunker Ridge Road, Kansas City, MO 64110. Present address, Waters Associates, Maple Street, Milford, Mass. 01757.
*
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Solutions of the ammonium salts were prepared and used as previously described (9). In brief, a solution of the quaternary ammonium halide in anhydrous methanol was mixed with silver oxide. M) was added Mobile Phase. The ammonium salt (2.0 X to deionized water (300 ml), and the pH was adjusted to 5.0 with 1% formic acid or sodium hydroxide solution. This solution was mixed with an equal volume of methanol and deaerated by vacuum. Calibration Curves. Samples of ascorbic acid of reference grade in phosphate buffer pH 5.0 were prepared to contain 0.5, 1.0, 2.5, 5.0, 7.5, 10.1, and 15.0 mg of ascorbic acid/100 ml. Aliquots (20.0 pl) of these solutions were injected into the chromatographic system, and the resulting peak areas were plotted against concentration for the calibration curve. Sample Preparation. Pharmaceutical Samples. One dosage unit of the sample was homogenized and transferred to a 100 ml volumetric flask with the aid of phosphate buffer pH 5.0 (50 ml). The flask was shaken for 2 min and diluted to volume with the buffer solution. The resulting mixture was filtered, the first 10 ml of filtrate discarded, and an aliquot of the remainder diluted to a final concentration of 5.0 mg/100 ml based on labeled claim. A 20pl. portion of this solution was injected, the resulting peak area determined, and the quantity of ascorbic acid in the sample calculated by reference to the previously derived calibration curves. Foods. A 100-g sample was homogenized with an equal weight of 6% HP03. A portion (10-30 g, accurately weighed) of this slurry was transferred to a 100-ml volumetric flask and diluted to volume with HP03. When necessary, this solution was further diluted to a final concentration range of 0.5-7.5 mg/100 ml. Samples were then treated and ascorbic acid content determined as above.
RESULTS AND DISCUSSION Chmmatographic procedures are a logical choice for the analysis of water-soluble vitamins because of the complex matrices in which they usually occur. Of the quantitative chromatographic techniques, HPLC is more attractive than GLC since derivatives need not be formed prior to analysis, and sample preparation time may therefore be reduced. However, the type of stationary phase employed in HPLC is a crucial factor in maximizing this conceptual advantage. Ion-exchange procedures have been employed in the determination of ascorbic acid ( I O ) , but the ease with which the columns are irreversibly “poisoned” ( 11) necessitates an involved sample preparation scheme. Likewise, normal-phase HPLC (12) suffers from the fact that highly polar constituents of the vitamin sample may be avidly adsorbed to the polar stationary phase. Some of these compounds are eluted only with methanol or water, after which original conditions are difficult, sometimes impossible, to restore. Reverse-phase chromatographic techniques would appear to offer significant advantages in the analysis of
Table I. Effect of Selected Salts on the Retention Volume of Ascorbic Acid Mobile phase Salta Ascorbic acid RVb, ml 0 None 1.8 1 Tetramethylammonium 1.9 hydroxide 2 Tetraethylammonium 2.3 hydroxide 3 Tetrapropylammonium 4.6 hydroxide 4 Tetrabutylammonium 7.9 hydroxide 5 Tetrahexylammonium 10.0 hydroxide 6 Tridecylammonium 11.35 formate 1.0 X mol. Flow rate of 2.5 ml/min in a solvent of methanol (300 ml) and water (300 ml). (I
water-soluble vitamins such as ascorbic acid. Polar solutes in an aqueous dispersion of the vitamin media would not be attracted to the lipophilic stationary phase and would therefore elute with the solvent front. Conversely, nonpolar constituents (for example, the oil-soluble vitamins) would have limited solubility in the aqueous dispersion, thereby minimizing their introduction into the chromatographic system. In such cases, retention volumes have been found to be reproducible ( 1 3 ) ,column lifetimes long, and sample preparation time short (14). However, in such a reverse-phase chromatographic system, water-soluble vitamins such as ascorbic acid are so polar even in the nonionic form that they have little affinity for the lipophilic stationary phase and also elute with the solvent front. It has Dreviouslv been shown ( 9 ) that addition of the appropriate salt to the mobile phase may result in retention of ionic analytes in a reverse-phase system. Thus, anionic analytes are retained when lipophilic
L- i 2
4
6
TIME Imin)
Figure 1. Chromatographic trace of an extract of a multivitamin capsule found to contain 101.2 mg of ascorbic acid (A) per dosage unit. See text for chromatographic conditions
Table 11. Analysis of Ascorbic Acid in Commercial Multivitamin Preparations Ascorbic acid Founda Dosage form Labeled constituents Labeled/dosage unit Capsule 9 vitamins, 8 minerals and extractives of 50 mg yeast and Streptomyces fermentation Capsule 9 vitamins and 6 minerals 250 mg Tablet 10 vitamins 333 mg Chewable 10 vitamins, fluoride, and fruit flavor 50 mg Tablet Liquid 7 vitamins, 2 minerals, and desiccated liver 100 mg Drops 10 vitamins, flavoring and coloring agents, 50 mg and preservatives 0 Percent recovered i% standard deviation). N = 8. According to U.S.P. XIX.
Chromatographic 102.3 (2.16)
Visual titrationb 101.74 (2.44)
108.4 (2.49) 103.7 (1.78) 98.4 (2.00)
110.7 (2.76) 104.6 (2.21) 99.2 (2.64)
97.6 (2.13) 102.4 (2.76)
99.4 (7.46) 104.4 (6.42)
Table 111. Comparative Analysis of Ascorbic Acid in Selected Food Products _
Sample
_
_
~
Ascorbic acid contentn ~
Composition Chromatographic Tomato juice 14.4 (3.22) 2 Lemon 34.0 (2.50) 3 Asparagus 19.4 (2.40) 4 Greenpeas 6.3 (3.10) 5 Fruit drink 24.9 (2.76) 6 Red beets 8.3 (3.02) 'i Pineapple 16.4 (3.47) 8 Grapefruit juice 15.2 (3.44) 9 Infant formula 12.3 (3.76) Expressed as mg of ascorbic acid/100 g (% standard deviation), N = 8. 1
a
_
Titrimetric 19.2 (1.64) 34.5 (1.24) 20.2 (1.55) 9.2 (1.42) 27.0 (1.22) 21.4 (9.40) 18.1 (2.44) 31.0 (1.94) 34.3 (2.00)
Turbidimetric 14.2 (5.21) 34.6 (4.04) 19.0 (4.33) 6.9 (4.86) 26.4 (4.11) 9.20 (4.74) 17.4 (5.21) 20.8 (7.20) 19.7 (6.46)
ANALYTICAL CHEMISTRY, VOL. 48, NO. 6, MAY 1976
797
100
--
75
al
m
-Pa r c
6
-k
A
v) W
z
g
5c
W v)
a K W 0 0
Y
2!
0
4
2
6
TIME (min)
Figure 2. Chromatographic trace of an extract of tomato juice found to contain 17.7 mg of ascorbic acid (A)/100 g. See text for chromatographic conditions
quaternary ammonium compounds are added to the mobile phase. Conversedly, cationic analytes in a mobile phase containing lipophilic sulfonate salts exhibit dramatic retention as compared to that in the absence of the salt. Since the retention volumes of analytes appear to be generally explicable in terms of the relative lipophilicity of the ion pair formed within the system, the technique is referred to as the reverse-phase, ion-pairing approach to HPLC. In using this solvent selection approach, lipophilic tertiary and quaternary ammonium compounds were dissolved in the aqueous component of the mobile phase, and the p H was adjusted to 5.0. At this pH, ascorbic acid injected into the system and the ammonium compounds in the mobile phase are ionic, and the expected effects are observed (Table I). On the basis of these data, mobile phases 5 and 6 were selected for examination of potential interferences in foods. Mobile phase 5 was rejected after several food extracts (e.g., baby cereal and tomato juice) exhibited peaks corresponding to the retention volume of ascorbic acid but were not reduced by prior treatment with ascorbic acid oxidase ( 4 ) . No such interferences were encountered with mobile phase 6. Using this chromatographic system, calibration curves were prepared daily for 10 days. The average correlation coefficient was 0.998, the percent standard deviation of slope was 1.36, and the average y intercept was 4.3 integrator units. These data indicate that the procedure is amenable to use of a single-point standard. 798
ANALYTICAL CHEMISTRY, VOL. 48, NO. 6, MAY 1976
A chromatogram of an extract of a multivitamin capsule is presented in Figure 1, and data regarding accuracy and precision of the procedure in analysis of representative multivitamin preparations are presented in Table 11. Results generally are comparable with those of the titrimetric procedure. It is noteworthy, however, that the precision of the visual titration procedure is considerably reduced in determination of both liquid products. This is apparently due to the interference of coloring agents in these products with end-point determination. Results of the determination of ascorbic acid in food extracts using the chromatographic procedure were compared (Table 111) with results of the 2,6-dichlorophenolindopheno1 titration and a turbidimetric procedure, and a representative chromatogram is presented in Figure 2. Results of the titration procedure were taken for comparison because of the wide use of the method. However, the turbidimetric procedure has recently been shown to be considerably more selective, with stannous ion being the only demonstrated interference. The fact that both the chromatographic and turbidimetric procedures give comparable results that are generally lower than that of the titration procedure is indicative of this increased specificity. Those cases where the chromatographic procedure gives lower results than the turbidimetric procedure can be attributed to interference of stannous ion with the latter procedure. The increased precision of the chromatographic procedure over the turbidimetric procedure is a reflection of the less complex sample workup of the former and the variables in turbidity development in the latter. An initial subject of concern was the stability of the chromatographic system under these conditions of minimum sample preparation. T o date over 300 food and multivitamin extracts have been examined with no discernible change in the chromatographic characteristics. This may be attributed in part to the responsiveness of the detector to ascorbic acid (0.5 mg/100 ml is readily determined) and the consequent injection of small quantities of the initial sample. In addition, the chromatographic system was flushed nightly with methanol as recommended by the supplier, ensuring removal of lipophilic substances from the column. In conclusion, it appears that application of the reversephase, ion-pairing approach to HPLC to the analysis of ascorbic acid results in an accurate and precise procedure. This general approach should be applicable to the determination of other water-soluble vitamins, and studies regarding these applications are currently in progress.
LITERATURE CITED (1) B. R. Hajratwala, Aust. J. Pharm. Sci., 5 , 33 (1974). (2) "The United States Pharmacopeia", 19th Revise, Mack Publishing Co., Easton, Pa., 1975, p 120. (3) M. J. Deutsch and C. E. Wecks, J. Assoc. Off. Anal. Chem., 48, 1248 (1965). (4) J. H. Roe, M. J. Oesteriing, and C. M. Damron, J. Biol. Chem., 174, 201 ( 1948). (5) M. Zobel. Ernahrungsforschung, 16, 257 (1971). (6) J. Lindquist and S.M. Farroha, Analyst(London). 100, 377 (1975). (7) J. E. Schlack, J. Assoc. Off. Anal. Chem., 57, 1346 (1974). (8) J. W. Ralis. J. Agric. Food Chem., 23, 609 (1975). (9) D. P. Wittmer, N. 0.Nuessie, and W. G. Haney, Anal. Chem.. 47, 1422 (1975). (10) R. C. Williams, D. R. Baker, and J. A. Schrnit, J. Chromatogr. Sci., 11, 618 (1973). (11) M. Singh, J. Assoc. Off. And. Chem., 57, 358 (1974). (12) D. P. Wittmer and W. G. Haney, J. Pharm. Sci., 63, 588 (1974). (13) R. E. Huettemann and A. P. Shroff, J. Chromatogr. Sci., 13, 357 (1975). (14) L. B. Bighley and P. M. McDonald, J. Pharm. Sci., 64, 549 (1975).
RECEIVEDfor review October 3, 1975. Accepted January 26, 1976.
