Table I. Experimental Data for Solutions Containing M Ti4+and Fe3+ 4x
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
... ... ... ... ...
... ... ... ... ...
0.096 0.096 0.0108 0.0144 0.018 0.0216 0.144 0.192 0.192 0.223 0.384 0.480 0.432 0.576
7.69 3.85 0.29 0.39 0.48 0.58 0.58 0.77 0.77 0.58 0.77 0.96 0.58 0.58
0.752 0.760 0.752 0.757 0.760 0.762 0.757 0.736 0.746 0.757 0.753 0.760 0.752 0.759 0.760 0.754 0.759 0.758 0.760
0.507 0.507 0.506 0.507 0.508 0.508 0.507 0.420 0.500 0.507 0.507 0.507 0.506 0.508 0.507 0.508 0.508 0.507 0.510
;i-2 30 12
[Bez'l/ IF 1
Figure 1. Absorbance vs. [Be2+]/[F-] ratio for solutions of 0.5 M
F-I The fluoride ion concentration was varied from 0.0125 to
0.0125 0.025 0.0375 0.0375 0.0375 0.0375 0.25 0.25 0.25 0.3875 0.5 0.5 0.75 1.0
1.0 M and, a t all concentrations, the interference could be
removed by addition of beryllium. The upper limit of 1.0 M fluoride concentration was set only by the use of a 50-ml volumetric flask, higher concentrations of fluoride not being possible within a total volume of 50 ml, because of the need to include beryllium solution, buffer solution, Tiron solution, etc. However in practice when analyzing a solution, obtained for example by dissolving a mineral or ore containing iron andlor titanium in a mixture of hydrofluoric and sulfuric acids, the fluoride ion concentration would rarely approach 1.0 M . Whenever the beryllium concentration of the solutions exceeded approximately 0.15 M , a transient, colorless, gelatinous precipitate was formed during the final stages of neutralization with concentrated ammonium hydroxide. This precipitate, presumably of beryllium hydroxide, completely redissolved on standing for 5 to 10 minutes. Stirring the solution during the addition of the ammonium hydroxide kept the quantity of precipitate to a minimum.
The violet iron-Tiron and yellow titanium-Tiron complexes were stable for long periods as reported by Yoe and Jones ( I ) and Yoe and Armstrong (2). The presence of the beryllium-fluoride complex appeared to have no affect upon this property. Solutions containing excess beryllium ( [Be2+]/[F-] 2 0.6) gave the same absorbance values for a t least 24 hours. This work confirms that the determination of titanium and iron using Tiron can be carried out, in the presence of up to 1 M fluoride, simply and quickly by the addition of beryllium.
LITERATURE CITED (1) J. H. Yoe and A. L. Jones, lnd. Eng. Chem.. Anal. Ed., 18, 111 (1944). (2) J. H. Yoe and A. R. Armstrong, Anal. Chem., 19, 100 (1947). (3) M. Fujimoto, Bull. Chem. SOC.Jpn, 29, 783 (1956). (4) G. V. Potter and C. E. Armstrong, Anal. Chem., 20, 1208 (1948).
RECEIVEDfor review July 17, 1975. Accepted October 6, 1975.
Determination of Ascorbic Acid in Foodstuffs, Pharmaceuticals, and Body Fluids by Liquid Chromatography with Electrochemical Detection Lawrence A. Pachla and Peter 1.Kissinger' Department of Chemistry, Purdue University, West Lafayette, Ind. 47907
A chromatographic method for the analysis of ascorbic acid utilizing a thin layer amperometric detector with a carbon paste electrode is described. Application of the new method is evaluated for multivitamin preparations, fruits, juice concentrates, milk products, urine, and serum. When possible, standard redox tltration procedures were used to corrobo384
rate the chromatographic method. When compared to ciassicai titrations or colorimetric redox procedures, the new assay features straightforward sample preparation and improved sensitivity and seiectivlty. A sample rate as high as 20/hr is possible, depending on the particular sample under study.
ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976
Because of increasingly strict government regulation of vitamins and other food additives, there is great interest in improved assay procedures for these substances in a variety of sample matrices. In particular, speculations on the prophylactic and therapeutic value of Vitamin C have led to intensified research on human ascorbic acid metabolism and increased interest in the shelf life of fortified food products and multivitamin preparations. These efforts have stimulated research on new analytical methods for Vitamin C ( 1 - 4 ) . Recently we have been developing the technique of high performance liquid chromatography with electrochemical detection (LCEC) for trace organic analysis. This approach has been demonstrated for the routine analysis of picomole amounts of phenolic compounds in tissue, body fluids, and pharmaceutical preparations (5-7). While electrochemical methods for ascorbic acid have been available for many years (&IO), they lack sufficient selectivity or sensitivity to be used for trace levels of the vitamin in complex biological media. Two brief examples of the application of LCEC to ascorbic acid determination in urine (11) and brain tissue (12) have appeared. In the present paper, detailed procedures are given for urine, blood, plant matter, fortified and unfortified food products, tablets, and multivitamin preparations.
EXPERIMENTAL Apparatus. Waterjacketed glass columns, 50 cm X 2 mm i d . , (Altex Scientific Co., 1450 Sixth Street, Berkeley, Calif. 94710, No. 251-02) were dry packed with the following pellicular high performance strong anion exchange packings: Vydac SAX (The Separations Group, 8738 Oakwood Avenue, Hesperia, Calif. 92345, No. 301), Zipax SAX (E. I. du Pont de Nemours and Co., Inc., Instrument Products Division, Wilmington, Del. 19898, No. 820960005) and Bondapak AX/Corasil (Water Associates Inc., P.O. Box 246, Milford, Mass. 01757, No. 27440). All liquid chromatographic data were obtained from commercially available modular components, including an amperometric electrochemical detector (Bioanalytical Systems Inc., P.O. Box 2206, W. Lafayette, Ind. 47906, model LC2). A short precolumn was used in series with the injector to prevent clogging the narrow analytical column with protein or particulate matter ( 2 3 ) . The instrument was interfaced to a Digital Equipment Corporation 8/E computer for peak integration. Data were also evaluated by manual measurement of peak heights. The program, written in SABRE and FORTRAN 2, is available upon request. Reagents. Distilled water, deionized and finally distilled from alkaline permanganate, was used for all solutions. Anhydrous L ascorbic acid was used as obtained from Sigma Chemical Co. All other chemicals were reagent grade. T h e “extracting solution” consisted of 3% metaphosphoric acid-8% acetic acid. T h e multivitamin dosage forms were commercially available products from T h e Upjohn Co. (Kalamazoo, Mich.). Food products were obtained commercially, while the biological samples were collected from apparently healthy male subjects. A stock 1 mg/ml solution in the extracting solution was used to prepare calibration standards containing 1-100 pg/ml by diluting the stock solution with 0.05 M HCLO4. P r e p a r a t i o n of Assay Solutions. Tablets. Five tablets were accurately weighed and then pulverized to a fine powder. The assay solution was then prepared by one of the following methods: i) A known amount of the total mass corresponding to approximately 5 mg of AA was transferred to a clean dry 100-ml volumetric flask and diluted to volume with cold 0.05 M HC104 just prior to chromatographic analysis. ii) A portion of the pulverized powder corresponding to ca. 50 mg of AA was transferred to a clean dry 100-ml volumetric flask and diluted to volume with cold extracting solution. One milliliter of this solution was pipetted into a second 100-ml volumetric flask and brought up to volume with cold 0.05 M HC104 yielding a nominal 5 pg/ml solution. Liquids. Liquid formulations were quantitatively diluted with cold 0.05 M HClO4 to obtain a final assay solution of about 5 pg/ml for the chromatographic assay. Capsules. Five capsules were mechanically ruptured in the presence of the extracting solution and quantitatively transferred into
a 100-ml volumetric flask and diluted to volume. This solution was then quantitatively diluted to yield a nominal 5 /rg/ml solution of AA. Milk Products. Twenty-five ml aliquots of fresh homogenized milk were pipetted into a 100-ml volumetric flask and the net weight was noted before diluting to volume with cold 0.05 M HC101. The solution was then shaken and ca. 8 ml was transferred into a 12-ml glass centrifuge tube and centrifuged a t 1500 x g for 5-8 minutes. Four microliters of the resulting deproteinized supernatant was then used for the chromatographic assay. Powdered milk samples were weighed and diluted with cold water to yield a nominal 20 /rg/ml solution which was then worked up further in the same manner as the fresh milk samples. Baby Foods and Frozen Juice Concentrates. Thirty-gram portions of the thawed juice concentrates and 85-100 gram portions of the baby food products were accurately weighed into 250-ml polypropylene centrifuge bottles and centrifuged at 27000 X g for 15-20 minutes a t 5 OCC. The supernatant was transferred into a 200-ml volumetric flask. The remaining residue was extracted with a 20-ml aliquot of the extracting solution, mechanically shaken for 5 minutes, and centrifuged. After transferring the supernatant to the volumetric flask, the residue was again extracted with a 50-ml aliquot and the isolation procedure was repeated. The combined supernatants were diluted to volume and used for subsequent analysis. Whole Fruits a n d Fortified Cereals. Fifty- to sixty-gram samples of whole fruits and 15-gram samples of the fortified cereals were weighed and then homogenized in a Waring Blendor with 50 ml of the extracting solution. The homogenates were quantitatively transferred to 250-ml polypropylene centrifuge bottles and centrifuged at 27 000 X g for ca. 20 minutes at 5 “C. The supernatant was transferred into a 200-ml volumetric flask. The remaining residue was then mixed with ca. 25-ml portions of the extracting solution and centrifuged. All the supernatants were combined and diluted to volume. Powdered Water-Soluble Products. Appropriate amounts of these products were weighed and transferred into dry 100-ml volumetric flasks. Just prior to assay, each sample was diluted to volume with the extracting solution to yield a nominal 100-300 pg/ml solution. Biological Samples. Aliquots (0.5 ml) of the urine samples were diluted between 1 : l O and 1:lOO with cold water depending on the ascorbic acid level and used immediately for the chromatographic assay. Much smaller aliquots of urine could be used for studies with laboratory animals. Blood serum was diluted 1:lO and injected directly. For these samples, the precolumn becomes an essential component of the chromatograph. Replacement of the precolumn is generally needed after between 100 and 1000 samples have been processed. Titrimetric Assay. All pharmaceutical dosage forms and multivitamin preparations were analyzed by the standard U.S.P. iodimetric procedure ( 1 4 ) . The food products were assayed by titration with N-bromosuccinimide ( 1 5 ) .The following aliquots of the food products assay solutions were pipetted into 50-ml Erlenmeyer flasks containing 3 ml of 8% KI and 3 drops of soluble starch solution: 10 ml of the juice aliquots and 25 ml of baby foods, whole fruits, and powdered water-soluble solids. N-Bromosuccinimide (0.06 M ) was then added drop-wise to the mechanically stirred solution until the blue starch complex end point just appeared and persisted. Chromatographic Assay. The chromatographic columns were waterjacketed to maintain a constant temperature 01 25 O C . Acetate buffer (0.07 M , p H 4.75) was employed in the assays using the Zipax SAX columns whereas acetate buffer (0.07 M , pH 5.25) was used for columns packed with Vydac SAX. In each case, the flow rate was 300 pl/min. The detector potential was set at 800 mV vs. the Ag/AgCl reference. Four-microliter injections were used for those solutions having a 4-6 pg/ml concentration of AA and 2-microliter injections were used for the solutions having a concentration of ca. 50 pg/ml of AA. The concentration of the samples was determined by comparing the area or peak height to that of an aqueous calibration standard or by using the standard addition method. T h e Zipax SAX column was used in all of the assays while the Vydac SAX column was employed only for the pharmaceutical dosage forms. All solutions prepared from food products as indicated above were used directly for the titrimetric assay; however, these solutions were subsequently diluted to yield ca. 4-6 /rg/ml of AA with cold 0.05 M HC104 for the chromatographic assay. Because of its high ionic strength and strong acidity, it is not desirable to inject
ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976
Figure 1. Chromatogram for ascorbic acid (AA) in homogenized milk diluted 1:4 ( A ) Sample containing 13 ng of AA. ( B ) Sample following standard addition of 20 ng of AA. (C) 20-ng standard. Zipax SAX, 2.1 mm X 50 cm glass column. Mobile phase: 0.07 Macetate buffer, pH 4.75 at 0.30 ml/min. Applied potential: 0.700 V vs. Ag/AgCI
Table I. Ascorbic Acid in Multivitamin Products Iodimetric
Adeflor M Adeflor M Adeflor M Adeflor M Unicap Unicap Unicap M Unicap M Unicap M Unicap M Unicap M Unicap Senior Unicap Therapeutic Unicap Therapeutic Zymacap Ferritrinsic Ferritrinsic Ferritrinsic Adeflor B Drops
100 mg/tab 100 100 100 50 50 50 50 50 50 50 90 300 300 100 50 50 50 83.3 mg/ml
RESULTS Table I summarizes the data obtained for various commercial multivitamin formulations. Retention times were such that a sampling rate of 12-20/hr could be used depending on the chromatographic packing material chosen. Multivitamin formulations in general d o not present any particular problem for either iodimetry or LCEC except in those preparations which contain ferrous sulfate. For these examples, the chromatographic approach is superior. T h e sensitivity and selectivity of the chromatographic method was given a more severe test on various food products. T h e precision of the chromatographic method for the food products was ca. +2% for all products at a maximum sampling rate of 15/hr. In several of the samples listed in Table 11, the titrimetric method was unable t o provide a clear distinct end point and therefore no comparison data could be obtained. Titrimetric methods based on oxidation of ascorbic acid give results with a strong positive bias for samples such as limes. In fact, many of the examples in Table I1 illustrate this problem to some extent in that the titrimetric results were higher than the corresponding LCEC value. This strongly supports our contention t h a t the new method has superior selectivity against other oxidizable species. T h e sensitivity of the LCEC approach is clearly demonstrated in Figure 1 which illustrates chromatograms obtained directly from homogenized milk diluted fourfold. In this case quantitation was achieved by standard addition. T h e present scheme was developed originally for biological fluids and Figure 2 depicts a chromatogram for a diluted urine sample. In samples following typical popular dosages of ascorbic acid (0.2-1.0 g/day), it is usually possible t o carry out the analysis directly on samples which have been diluted 100-fold with cold 0.05 M HC104. In a study of ascorbic acid excretion by men in their early 20’s following a 1.0-g morning dose, we find t h a t ca. 45% of the total is excreted unchanged in 12 hours. This is in excellent agreement with a n early report (16). 366
109 116 116 51.1 47.2 59.7 59.3 58.4 57.2 58.1 102 31 5 335 95.7 60 60
114 113 114 51.6 na 57.4 56.0 51.0 56.2 58.1 98.1 336 335 na na 57.3 na na
Table 11. Ascorbic Acid Content of Food Products Sample
the extracting solution prior to dilution. The other solutions (e.g., pharmaceuticals and milk products) were used directly as prepared for the chromatographic assay.
Baby food Peaches Applesauce Apricots Bananas Fruit juices Orange Tangerine Grapefruit Hawaiian Punch Lemonade Tomato Artificial fruit drinks Tang Tang w/Grapefruit Fortified cereals Cheerios Rice Krispies Whole fruits Orange Grapefruit Lime Milk products Infant Formula Instant Breakfast Whole Milk a1 Whole Milk =2 Whole Milk =3 Dry Milk Miscellaneous Vitamin Premix Real Lemon Concentrate a Label value.
0.250 0.397 0.247
0.250 0.386 0.153 0.246
100 103 105 100
1.5_6 0.742 1.47 2.84 0.136 0.785
1.47 0.726 1.38 2.72 0.131 0.614
106 102 107 104 104 128
0.741 0.424 0.291
0.538 0.893 0.012 0.0080 0.0038 0.0056
... ... ...
DISCUSSION Stability of Ascorbic Acid. Many so-called stabilizing solutions were examined during the course of this investigation; however, only two solutions were found t o be reasonably satisfactory. Cold dilute perchloric acid (0.05 M ) is a good medium to inhibit oxidation of ascorbic acid and both samples and standards were frequently diluted with this solution and stored refrigerated for several hours. T h e classical extracting solution, 3% metaphosphoric acid-8%
ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976
maximum sample rate of 20/hr can be achieved. A third stationary phase, Bondapak AAX behaves similarly to the Zipax SAX; however, on-column decomposition of the vitamin limited its use for trace analysis. For all of the stationary phases evaluated, the retention time for ascorbic acid is very sensitive to ionic strength and therefore the mobile phase should be prepared carefully in large quantities.
CONCLUSION We believe the LCEC approach affords convenience of sample preparation, sensitivity, and selectivity superior to any method for ascorbic acid published to date. Although the present method is not directly suitable for dehydroascorbic acid, assay of the latter can be achieved by reoxidation to ascorbic acid prior to analysis (19). T h e simple instrumentation required for ascorbic acid is also applicable to the assay of many other reducing agents and to a variety of phenolic natural products. A list of those compounds which we have worked with to date is available upon request.
* * *
Figure 2. Chromatogram for human urine diluted 1:lOO with major peaks for uric acid (UA) and ascorbic acid (AA) (19ng) Zipax SAX, 2.1 mm X 50 cm glass column. Mobile phase: 0.05 M acetate buffer, pH 4.75 at 0.33 ml/min. Applied potential: 0.700 V vs. Ag/AgCI
acetic acid, affords excellent stability for vitamin C; however, samples in this medium could not be chromatographed directly because of its high ionic strength. This resulted in a decrease in retention time on the anion exchange columns. In some samples (e.g., blood) the stability of ascorbic acid is particularly poor and sample handling prior to workup must be carried out in the shortest possible time. At the present time, our methodology is insufficient for accurate measurements a t normal serum levels (5-15 lg/ml) (17, 18). Another problem encountered was that when the chromatographic column had not been used for some time (ca. 15-30 min.), the first sample injected always gave a value t h a t was too low by 5-10%. Once the column had been pretreated with the first sample or a standard, quantitation of subsequent injections was both accurate and precise. We have no explanation for this observation. S t a t i o n a r y Phases. Several commercially available pellicular packing materials were evaluated during the course of this investigation and the significant variation of the retention of ascorbic acid is worthy of mention. Using the mobile phase and flow rate noted, Zipax SAX is a n excellent choice in t h a t ascorbic acid is eluted in ca. 4 min, giving adequate resolution from the void volume peak. This stationary phase was primarily used in the analysis of food products and biological fluids because of the probability of large void volume peaks due to nonretained oxidizable substances. Although a much less selective stationary phase, Vydac bonded phase SAX is useful in handling samples (e.g., multivitamin preparations) where the nonretained components are generally not detectable. In this case, the ascorbic acid is eluted shortly after the void volume and a
Fortified with Vitamin C It's a sign we often see But is it there just like they say, Or has it oxidized away?
ACKNOWLEDGMENT The authors thank Lloyd E. Fox (The Upjohn Company) for many helpful discussions.
LITERATURE CITED (1) V. R. White and J. M. Fitzgerald, Anal. Chem., 44, 1267 (1972). (2) J. E. Schlack, J . Assoc. Off. Anal. Chem., 57, 1346 (1974). (3) M. A. Eldawy, A. S. Tawfik, and S. R. Elshabouri, Anal. Chem., 47, 461 (1975). (4) 0. Pelletier and R. Brassard, J . Assoc. Off. Anal. Chem., 58, 104 (1975). (5) P. T. Kissinger, C. J. Refshauge, R. Dreiling, and R . N. Adams, Anal. Lett.. 6, 465 (1973). (6) R. M. Riggin, A. L. Schmidt, and P. T. Kissinger, J . Pharm. Sci., 64, 680 (1975). (7) R. M. Riggin. L.-D. Rau. R. L. Alcorn. and P. T. Kissinger. Anal. Lett., 7, 791 (1974). (8)M. Brezina and P. Zuman, "Polarography in Medicine, Biochemistry and Pharmacy", rev. English ed.. Interscience, New York, N.Y., 1958, pp 401-406. (9) J. Heyrovsky and P. Zuman, "Practical Polarography", Academic Press, New York, N.Y., 1968, p 102. (10) W. D. Mason, T. D. Gardner, and J. T. Stewart, J . Pharm. Sci., 61, 1301 (1972). (1 1) P. T. Kissinger. L. J. Felice, R. M. Riggin, L. A. Pachla, and D. C. Wenke, Clin. Chem., 20, 992 (1974). (12) K. V. Thrivikraman. C. Refshauge, and R. N. Adams, Life Sci,, 15, 1335 (1974). (13) L. A. Pachia and P. T. Kissinger, Anal. Chem.. 48, 237 (1976). (14) Pharmacopoeia of the United States, 18th ed., Mack Printing Co., Easton, Pa., 1970, p 51. (15) M. 2. Barakat, M. F. A. El-Wahab, and M. M. El-Sadr, Anal. Chem., 27, 536 (1955). (16) S.W. Johnson and S. S. Ziiva, Blochem. J., 28, 1393 (1934). (17) W. T. Caraway, "Fundamentals of Clinical Chemistry", N. W. Tietz. Ed., W. B. Saunders Company, Philadelphia, Pa., p 174. (18) G. N. Schrauzer and W. J. Rhead, lnt. J. Vitam. Nutr. Res., 43, 201 (1973). (19) J. H. 'Roe, M. B. Mills, M. J. Oesterling, and C. M. Damron, J . Biol. Chem., 174, 201 (1948).
RECEIVEDfor review September 22, 1975. Accepted November 10, 1975. This investigation was supported by grants from the National Science Foundation (GP42452X), the National Institutes of General Medical Sciences (GM-22713-Ol), and the Showalter Trust Fund. A portion of this work was carried out a t the Department of Chemistry a t Michigan State University.
ANALYTICAL CHEMISTRY, VOL. 48, NO.
2, FEBRUARY 1976