[3]

A M P E R O M E T R I C D E T E C T I O N OF ASCORBIC ACID

15

[3] A n a l y s i s o f A s c o r b i c A c i d b y L i q u i d C h r o m a t o g r a p h y with Amperometric Detection By

LAWRENCE

A.

PACHLA and PETER T.

KISSINGER

Direct electrochemical methods for ascorbic acid have been available for many years. 1 These methods have not been popular owing to the inconvenience of the dropping mercury electrode, the tendency of electrodes to become fouled by adsorbed materials in biological samples, and the relatively poor resolution of electrochemical techniques in distinguishing between easily oxidized substances. All three of these difficulties can be overcome by the use of hydrodynamic thin-layer electrochemistry as a means of detection in high-performance liquid chromatography. The combined technique (LCEC) provides extraordinary sensitivity and selectivity while maintaining advantages in both cost and convenience. Principles The electrooxidation of ascorbic acid at carbon electrodes follows an irreversible EC type of electrode mechanism. 2 The electrochemical behavior of ascorbic acid (AA) and other easily oxidized compounds is most conveniently ascertained by linear sweep voltammetry. For example, Fig. 1 illustrates the typical behavior of ascorbic acid and its metabolite ascorbic acid 2-sulfate in acetate buffer at pH 5.25. An initial positive potential scan for an ascorbic acid solution yields an anodic peak, Eo,a at +0.45 V. The product of the electrochemical oxidation of AA is dehydroascorbic acid. Dehydroascorbic acid is then very rapidly hydrated to yield an electroinactive product. The equilibrium constant favors the hydrated form to the extent that for all practical purposes the reaction is irreversible. The electrooxidation of ascorbic acid 2-sulfate also follows a similar pathway. The sulfated analog is oxidized via a 2eprocess at a much higher potential (Ep,a = +0.88 V) and, therefore, unlike ascorbic acid, is a very poor reducing agent. Again, the hydrated form of dehydroascorbic acid is the principal product. An overview of these electrode processes is given in Figs. 2 and 3. As can be seen from the linear-sweep voltammograms, it would be possible to quantitate ascorbic acid in the presence of ascorbic acid 2-sulfate using an electroanalytical technique, because their oxidation J. Heyrovsky and P. Zuman, "Practical Polarography,'" p. 102. Academic Press, New

York, 1%8. 2 S. P. Perone and W. J. Keetlow,Anal. Chem. 38, 1760 (1966).

METHODS IN ENZYMOLOGY, VOL. 62

Copyright O 1979by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181962-(I

16

ASCORBIC ACID E

1.0

0.8

vs

[3]

Ag-AgC]

0.6

0.4

0.2

I

i

!2

A

3

L

£

I

I

I

3,

2 -3

Cg v ~J

-

4

-a

o -

Z

,(

-! --2 -3 --4

FiG. 1. Linear sweep voltammetry (0.03 V/sec) at the carbon paste electrode (dw = 3 mm) in 1.0 M acetate buffer (pH 5.25): (A) 1 mM ascorbic acid; (B) 1 mM ascorbic acid 2-sulfate; (C) 0.5 mM ascorbic acid, 0.5 mM ascorbic acid 2-sulfate.

potentials are distinctly different. If a sample to be assayed contains only compounds that oxidize at much greater potentials than ascorbic acid, then direct electroanalysis provides the desired specificity. Unfortunately, many " r e a l " samples contain a number of components whose oxidation potentials either overlap or are very close to that of ascorbic acid. Direct electroanalysis of such a sample results in positively biased values for the AA determination.

[3]

17

AMPEROMETRIC DETECTION OF ASCORBIC ACID

E

C 0

O

--2H+ ~2e--'

H

HO

"2O

)'

O

)'

HO CHOH I C H2OH

CHOH I CH2OH

CHOH I CH20H

FIG. 2. Mechanism for the electrochemical oxidation of ascorbic acid.

One means of circumventing the poor specificity of electroanalytical techniques is to combine electrochemistry with high-performance liquid chromatography. Liquid chromatography with electrochemical detection (LCEC) has the resolving power of modern liquid chromatography but maintains the sensitivity of thin-layer electrochemistry, a'4 The specificity of LCEC is primarily due to the chromatographic separation, although considerable selectivity also results from the electrochemistry. Most components of a sample can be categorized into two classes. Those compounds that oxidize at much greater potentials than the analyte (e.g., ascorbic acid 2-sulfate) or compounds whose oxidation potential is lower or very close to that for the analyte of interest. The selectivity associated with the electrochemistry is apparent in the first case. Operation of the chromatographic detector in a potential region that would allow ascorbic acid to be oxidized, but would prevent the electrooxidation of these compounds, precludes concern for their separability by the chromatographic column. For those compounds that fall into the second class, the specificity of an assay for ascorbic acid would be entirely dependent on the o

E _

o

.+

+H2O

- 2.HO"

"~ CHOH CH2OH

C

-.

o

C o

"2 °

H 0 ~>

.o

o

-.2so4

CHOH

CHOH

CHOH

¢H2OH

CH2OH

CH2OH

F]G. 3. Mechanism for the electrochemical oxidation of ascorbic acid 2-sulfate.

a p. T. Kissinger, Anal. Chem. 49, 447A (1977). 4 p. T. Kissinger, C. S. Bruntlett, G. C. Davis, L. J. Felice, R. M. Riggin, and R. E. Shoup, Clin. Chem. 23, 1449 (1977).

18

ASCORBIC ACID

[3]

selectivity of the chromatographic separation because each compound would be detected. Ideal mobile phases for LCEC are aqueous buffers with or without a solvent (typ-methanol or acetonitrile). Because of this requirement, ionexchange or reverse-phase packing materials have been found to be the most compatible with the technique. Each class of stationary phase can be further subdivided according to structure and mean diameter. Pellicular packings have an average diameter between 20 and 60/zm whereas the more efficient microparticulates are in the range of 5-10 /xm. The authors' original work 5 involved the use of pellicular anion-exchange packing materials where the alkyl cationic sites were either chemically bonded to an inert core (e.g., Vydac SAX) or were mechanically coated onto a glass bead (e.g., Zipax SAX). The use of these pellicular packings provided an assay procedure with adequate selectivity while maintaining minimal cost. Since ascorbic acid exists as an anion in mildly acidic solution, it is readily retained by anion-exchange packing materials. Zipax SAX was found to be an excellent choice, since ascorbic acid was eluted in ca. 4 min; this gave adequate resolution from the void volume peak. This stationary phase is especially useful in the analysis of food products and biological fluids because of the probability of large void volume peaks due to nonretained oxidizable substances. A useful but less selective stationary phase is Vydac SAX, which was found to be useful for 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 therefore a higher sampling rate is achieved. The microparticulate reverse-phase packings are an important alternative to the pellicular anion-exchange packing materials. These stationary phases consist of chemically modified silica gel particles having alkyl side chains extending out into the mobile phase. The separation process for these packings primarily involves a partitioning of the analyte between the nonpolar stationary phase and the polar mobile phase due to hydrophobic forces. 6 The primary advantage of these stationary phases is the improved efficiency. In addition, microparticle reverse-phase packings are very versatile. Not only can nonpolar molecules be retained, but also cationic and anionic species if they are sufficiently hydrophobic and/or if the mobile phase composition is modified to include an ion-pair reagent. The use of a suitable ion-pair reagent enables the analyte to form an ion-pair complex, which can then partition onto the stationary phase. In addition to the partitioning process, the ion-pair reagent modifies the L. A. Pachla and P. T. Kissinger, Anal. Chem. 48, 364 (1976). C. Horvath, W. Melander, and I. Molnar, Anal. Chem. 49, 142 (1977).

[3]

AMPEROMETRIC DETECTION OF ASCORBIC ACID

19

packing material so that it appears to act as an ion exchanger. In most cases published to date, the latter process dominates. Ascorbic acid is not readily retained by reverse-phase packings, therefore a cationic ion-pair reagent must be added to the mobile phase. Careful selection of the column modifying reagent allows the development of a specific method for ascorbic acid. The relative retention time of AA and other components can be changed by the judicious choice of the ion-pair reagent, pH, and ionic strength. For example, AA is more strongly retained on the column if tridecylamine is used instead of a tert-butyl ammonium salt/Therefore, proper selection of the ion-pair reagent allows the chromatographic separation to be optimized for different sample types without changing the column. Materials and E q u i p m e n t Reagents

Acetate buffer, 1.0 M, pH 4.00. Prepare by diluting 83.5 ml glacial acetic acid and 44.5 g of anhydrous sodium acetate to a total volume of 2 liters. Actetate buffer, 1.0 M, pH 4.75. Prepare by diluting 58.0 ml of glacial acetic acid and 82.05 g of anhydrous sodium acetate to a final volume of 2 liters. Acetate buffer, 1.0 M, pH 5.25. Prepare by diluting 27.9 ml of glacial acetic acid and 124.05 g of anhydrous sodium acetate to 2 liters. Acetate buffer, 80 mM, plus 1 mM tridecylamine, and 15% methanol. This solution is prepared by appropriately mixing 1.0 M acetate buffer, pH 4.00, 100 mM tridecylamine and methanol to obtain the desired concentrations. The final pH of this solution is 4.5. Metaphosphoric acid, 3%; acetic acid, 8% Tridecylamine, 100 mM, in methanol Perchloric acid, 50 mM Acetate buffer, 70 mM, pH 4.75 Acetate buffer, 70 mM, pH 5.25 Instrumentation. All liquid chromatographic data were obtained using commercially available components and an amperometric detector (Bioanalytical Systems Inc., West Lafayette, Indiana, Model LC-50). Glass columns, 50 cm × 2 mm i.d., were dry-packed with the following pellicular high-performance, anion-exchange packing materials: Vydac SAX (The Separations Group, Hesperia, California, No. 301) and Zipax SAX (E. I. Dupont de Nemours and Co., Inc., Instrument Products Divi7 S. P. Sood, L. E. Sartori, D. P. Wittmer,and W. G. Haney,Anal. Chem. 48, 796 (1976).

20

ASCORBIC ACID

[3]

sion, Wilmington, Delaware, No. 820960005). A stainless steel column, 15 cm x 4.6 mm i.d., was slurry-packed with microparticulate Cls reversephase packing material (EM Laboratories Inc., Elmsford, New York, Type Lichrosorb RP-18). The potential of the chromatographic detector was set at 0.70 V versus a Ag/AgCI reference. An injection volume of 20 /zl is recommended for all solutions. To prevent clogging of the analytical column with particulate matter, a short plexiglas precolumn was placed in between the injection valve and the pellicular columns, s Samples to be analyzed on the microparticulate reverse-phase column were filtered using a centrifugal filter assembly (Bioanalytical Systems Inc., Model MF-1). Preparation of Solutions A stock 1 mg/ml solution in either the conventional extracting solution (3% metaphosphoric acid-8% acetic acid) or 50 mM HCIO4 is used to prepare calibration standards containing 0.1-5.0 mg/ml by diluting the stock solution with cold 50 mM HC104. These concentrations should be modified if an injection volume other than 20/zl is to be used. The amount of ascorbic acid to be introduced onto the chromatographic column should be within the range 2-100 ng. Tablets. Five tablets are accurately weighed and pulverized into a fine powder. A known amount of the total mass corresponding to 1 mg of ascorbic acid is transferred into a 100-ml volumetric flask and diluted to volume with 50 mM HC104 just prior to chromatographic analysis. Liquids. Liquid formulations are diluted with cold 50 mM HC104 to obtain a final assay solution with a nominal concentration of 1-3/zg/ml. Capsules. Five capsules are mechanically ruptured in the presence of the extracting solution, and the supernatant is quantitatively transferred into a 100-ml volumetric flask. This solution should then be diluted to yield an ascorbate concentration between 1 and 3 /~g/ml. Milk. Whole milk is diluted 20-fold with cold 50 mM HC104. The heterogeneous mixture is filtered using a centrifugal filter, and a 20-p,l aliquot of the clear supernatant is used for chromatographic analysis. Powdered milk samples are weighed and diluted with cold water to obtain a concentration of 20/zg/ml. The reconstituted sample is then worked up in the same manner as fresh milk samples. Powdered water-soluble food products, in appropriate amounts, are weighed into and transferred into dry 100-ml volumetric flasks. Each sample is then diluted to volume with cold 50 mM HCIO4, just prior to analysis, to yield a nominal 1-3 /zg/ml solution. s L. A. Pachla and P. T. Kissinger,Anal. Chem. 48, 237 (1976).

[3]

AMPEROMETRIC DETECTION OF ASCORBIC ACID

21

Food Products. Heterogeneous mixtures (e.g., fruits, baby foods) are centrifuged at 17,000 g and 5 ° for 10-15 min. The supernatant is decanted into a volumetric flask, and the residue is extracted using the extracting solution. This mixture is centrifuged, and the supernatants are pooled. The extraction procedure should be repeated twice. The supernatants are then diluted to volume. The final assay solution is prepared by diluting the above solution to a known volume with cold 50 mM HCIO4. Biological Samples. Aliquots (0.5 ml) of urine samples are diluted between 1 : 10 and 1 : 100 with cold water or 50 mM perchloric acid and analyzed immediately. Much smaller aliquots can be used for studies with tissue homogenates or fluids from laboratory animals. For these types of sample, the precolumn filter becomes an essential component of the chromatographic system. Replacement of the precolumn is generally needed after 100-1000 samples have been processed. Chromatographic Assay Acetate buffer (70 mM, pH 4.7) is employed in the assay using Zipax SAX columns, whereas acetate buffer (70 mM, pH 5.2) is used for columns packed with Vydac SAX. In each case the flow rate should be about 0.3 ml/min. When the Cla reverse-phase column is to be used, acetate buffer (80 mM, pH 4.0), 1 mM tridecylamine, and methanol (15%) is the mobile phase of choice. The flow rate for this column should be set at 1.26 ml/min. Twenty-microliter aliquots of calibration standards or samples with ascorbic acid concentrations in the range of 0.1 to 5.0/zg/ml are injected onto the chromatographic column. The concentration of AA in the samples are determined by comparing the area or peak height to that of an aqueous calibration standard or by using the standard addition technique. Because of the high acidity and ionic strength, it is not desirable to directly inject solutions prepared with the classical extracting solution (3% metaphosphoric acid-8% acetic acid). Samples prepared with this solution should be diluted with cold 50 mM perchloric acid prior to analysis. Discussion The retention times are such that assay of ascorbic acid can be performed at a sampling rate of 12-20 per hour depending on the chromatographic packing material chosen. Multivitamin formulations do not present any particular problems for iodimetric titration methods or LCEC except in those preparations containing ferrous sulfate. For these sample types, the chromatographic approach is superior. The selectivity and sensitivity of the LCEC approach becomes more apparent when it is applied

22

ASCORBIC ACID

[3]

to food products. Titrimetric methodology for food products yield positively biased values for such samples as limes. In several sample types, such as fortified cereals and milk products, titrimetric methods are unable to provide a distinct end point. To date, LCEC has been successfully applied to the analysis of AA in baby foods, fruit juices, artificial fruit drinks, fortified cereals, whole fruits (including limes), and milk products. The values obtained are lower than those obtained by nonspecific redox methods. ~ The precision of this chromatographic approach is ca. ___2% with a sampling rate of 15 per hour. The sensitivity of LCEC is clearly demonstrated in Fig. 4, which illustrates chromatograms obtained from homogenized milk diluted 1:20. In this case, quantitation was achieved by standard addition. The present scheme was originally developed for biological fluids. In samples from individuals ingesting popular dosages of ascorbic acid (0.2-1.0 g/day), it is usually possible to simply dilute the sample 100-fold with 50 mM perchloric acid and inject it onto the chromatographic column. Several well known stabilizing solutions were examined; these included (1) acetate buffer, pH 4.7; (2) 50 mM perchloric acid, 2 mM thiourea, and 1 mM EDTA; (3) 3% metaphosphoric acid-8% acetic acid; and (4) 50 mM perchloric acid. Standards prepared with acetate buffer exhibited significant decomposition within a period of hours and could not be used in the assay. The second solution containing EDTA proved to be a good candidate for stabilizing ascorbate solutions. However, a large void volume peak and-a broad peak eluting just past AA hindered its usefulness. The classical extracting solution, 3% metaphosphoric acid-8% acetic acid, afforded excellent stabilizing properties. When samples and standards were prepared with this solution, a noticeable decrease in retention time was observed. This effect was due to the plug of high ionic strength that passed through the column. Samples prepared with the extracting solution were diluted prior to injection on the anion-exchange column. Cold 50 mM perchloric acid was found to be the ideal medium to inhibit the oxidation of ascorbic acid. Samples and standards were frequently diluted with this solution and stored refrigerated for several hours. Approximately 99% of the ascorbic acid remained after 12 hr when prepared in cold dilute perchloric acid. When powdered samples are to be analyzed, the samples should be weighed into dry volumetric flasks and then diluted just prior to their analysis. Drying the flasks with a vacuum oven is necessary to remove extraneous moisture; otherwise decomposition of the vitamin will occur. By weighing all the samples at the same time, it is possible to prepare a set of samples that could be assayed the following day. This approach improves

[3]

23

AMPEROMETRIC DETECTION OF ASCORBIC AC|D

AA

nA AA

AA

A

C

B

t_ I'

I

I

I"

I

I

1

1

I

0

4

8

O

4

8

0

4

8

MINUTES

FIG. 4. Chromatogram for ascorbic acid (AA) in homogenized milk diluted 1:20. (A) Ten-nanogram standard injected. (B) Milk sample containing 6.8 /ag/ml (6.8 ng of AA injected). (C) Sample following standard addition of 10 ng (16.8 ng of AA injected). Merck Cla reverse phase, 15 cm × 4.6 mm, stainless steel column. Mobile phase: 80 mM acetate buffer, 1 mM tridecylamine, 15% methanol (pH of final solution = 4.5) at a flow rate of 1.26 ml/min. Applied potential = 0.7 V vs Ag/Ag/AgCl.

the overall rate of analysis. In some cases (e.g., blood) the stability of AA is particularly poor, and sample handling prior to work-up should be carried out in the shortest possible time. Another problem encountered is that when the pellicular anion-exchange column has not been used for some time (ca. 15-30 min), the first sample injected always gives a value that is too low by 5-15%. Once the column has been treated with the

24

ASCORBIC A C I D

[4]

sample or standard, quantitation of subsequent injections are both accurate and precise. Conclusion The LCEC approach affords convenience of sample preparation, sensitivity, and selectivity equal or superior to any method for ascorbic acid published to date. The sensitivity of LCEC is superior by two orders of magnitude when compared to liquid chromatography with ultraviolet detection. In addition, the selectivity associated with electrochemistry is a decided advantage. Although the use of pellicular, high-performance, anion-exchange packing materials is adequate for most sample types, increased selectivity is obtainable when microparticulte reverse-phase packings and different ion-pairing reagents are employed. A decided advantage of the latter approach is that an assay procedure can be optimized for maximum sample rate when repetitive analysis of a single sample type is desired.

[4]

l.,-Gulono-~/-lactone Oxidase (Rat and Goat Liver) By MORIMITSU N1SHIKIMI L-Gulono-3~-lactone + 02 ~ L-ascorbic acid + H20~

L-Gulono-y-lactone oxidase catalyzes the last step in the biosynthesis of L-ascorbic acid in animals. Humans, primates, and guinea pigs are unable to synthesize this vitamin because they lack this enzyme. The primary product of the above reaction is thought to be 2-keto-L-gulono-ylactone, which isomerizes spontaneously to give L-ascorbic acid. Assay Methods

Principle. The L-ascorbic acid formed can be determined colorimetrically by the 2,4-dinitrophenylhydrazine method I or by reduction of Fe 3+ to Fe 2+, followed by coupling of the Fe 2+ with c~,a'-dipyridyl,s These methods can be used to assay crude tissue preparations provided that corrections are made for endogenous k-ascorbic acid. The Fc2+-o~,a'dipyridyl method was used in the purification of the enzyme because it is rapid and simple. The dehydrogenase activity of the enzyme can be meal j. H. Roe and C. A. Kuether, J. Biol. Chem. 147, 399 (1943). 2 M. X. Sullivan and H. C. N. Clarke, J. Assoc. Off. Agric. Chem. 35~ 514 (1955). METHODS IN ENZYMOLOGY, VOL. 62

Copyright © 1979by Academic Press, Inc. All rights of reproduction in any formreserved. ISBN 0-t2-181962-0

Analysis of ascorbic acid by liquid chromatography with amperometric detection.

[3] A M P E R O M E T R I C D E T E C T I O N OF ASCORBIC ACID 15 [3] A n a l y s i s o f A s c o r b i c A c i d b y L i q u i d C h r o m a t o g...
631KB Sizes 0 Downloads 0 Views