Chemiluminescence flow injection analysis determination of sucrose

Jan 1, 1986 - Enzymic flow injection determination of lactose in milk with on-line dialysis. Rosa Puchades , Angel Maquieira , Luis Torró. The Analys...
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Anal. Chem. 1986, 58,116-119

Registry No. Morphine, 57-27-2;codeine, 76-57-3;thebaine, 115-37-7;noscapine, 128-62-1. LITERATURE CITED (1) Bowen, J. M.; Purdle, N. Anal. Chem. 1980, 52,573. (2) Bowen, J. M.; Crone, T. A.; Hermann, A. 0.; Purdie, N. Anal. Chem. 1980, 52,2436. (3) Crone, T. A.; Purdle, N. Anal. Chem. 1981, 53, 17. (4) Bowen, J. M.; Crone, T. A.; Kennedy, R. K.; Purdle, N. Anal. Chem. 1982, 54,66. (5) Bowen, J. M.; Purdie, N. Anal. Chem. 1981, 53,2237. (6) Bowen, J. M.; McMorrow, H. A.; Purdle, N. J . Forensic Sci. 1982, 27,

882. (7) Atkinson, W. M.; Bowen, J. M.; Purdle, N. J . Pharm. Sc;. 1984, 73, 1827. (8) Han, S. M.; Purdie, N. Anal. Chem. 1985, 57,2068. (9) Atkinson, W. M.; Han, S. M.; Purdie, N. Anal. Chem. 1984, 56,1947.

(IO) (11) (12) (13) (14) (15) (16) (17)

(18) (19)

Clarke, E. 0. C. "Isolation and Identification of Drugs"; The Pharmaceutical Press: London, 1978. Bechtel, A. Chromatographla 1972, 5 , 404. Smlth, R. M. J . Forenslc Sci. 1973, 18, 327. Wheals, B. B. J . Chromafogr. 1976, 122,85. Doner, L. W.; Hsu, A. F. J . Chromafogr. 1982, 253, 120. Hutln, M.; Cave, A.; Foucher, J. P. J . Chromafogr. 1983, 268, 125. Edlund, P. 0. J . Chromafogr. 1983, 279,615. Fell, A. F.; Scott, H. P.; GIiI, R.; Moffat, A. C. J . Chromatogr. 1983, 282, 123. Sperling, A. R. J . Chromatogr. 1984, 294,297. Mathers, A. P.; Butler, W. P. "Methods of Analysis for Alkaloids, Opiates, Marihuana, Barbiturates, and Miscellaneous Drugs"; Internal Revenue Service Publication No. 341, 1967; pp 45-76.

RECE~VED for review June 14,1985. Accepted August 29,1985.

Chemiluminescence Flow Injection Analysis Determination of Sucrose Using Enzymatic Conversion and a Microporous Membrane Flow Cell Cathy A. Koerner and Timothy A. Nieman*

Department of Chemistry, University of Illinois, 1209 West California Street, Urbana, Illinois 61801

Sucrose Is determlned In a flow lnjectlon system Involving converslon to glucose (vla reactlon wlth Invertase and mutarotase), generation of H202from the glucose (vla reaction wlth glucose oxldase), and chemllumlnescence determination of the H202(vla reaction wlth lumlnol). The glucose oxldase reaction and chemiluminescence reactlon occur In a mlcroporous membrane flow cell. Use of both dlssolved and Immobilized Invertase and mutarotase Is Investlgated. Optlmum performance results from use of pH 6 phosphate buffer carrier stream through columns of lmmoblllzed Invertase and mutarotase, lumlnol at pH 10.5 catalyzed by hemin and horseradlsh peroxldase, and glucose oxldase In pH 5 acetate buffer. Working range Is 5 pM-I mM wlth preclslon of 2-3%. Analysis time Is 2 mln. To determlne sucrose In food products, one must dlfferentlate between glucose orlglnally present and that formed from sucrose. Thls can be done on-line elther by separate determlnatlon of glucose or by catalytlc destructon of glucose In the sample prlor to sucrose determlnatlon. Results are presented for glucose and sucrose assays of soft drlnks, breakfast cereal, and cake mlx.

There has been considerable recent interest in combining the advantages of chemiluminescence detection (low detection limits, wide dynamic range, simple instrumentation, and inexpensive reagents) with the specificity of enzyme reactions. Many oxidase enzymes react with their specific substrates to produce hydrogen peroxide as one of their products. This hydrogen peroxide can then be quantitated via chemiluminescence. Work in this area has been reviewed by Seitz (1)and includes the use of several different oxidase enzymes for the determination of substrates such as glucose (2-5), cholesterol (6), uric acid (7), amino acids (8), and aldehydes (9). Until now all analytes determined by combining the use of oxidase enzymes with chemiluminescence detection have been the immediate substrates of the oxidase reaction; this has limited the variety of substrates available for quantitation.

It is desirable to extend this approach to include other analytes for which no oxidase enzyme exists, but which can be enzymatically converted in one or more steps to a product for which an oxidase enzyme does exist. Much work has been done in the area of chemiluminescence determination of glucose by the use of glucose oxidase. As shown below, glucose reacts with glucose oxidase in the presence of molecular oxygen to produce hydrogen peroxide. P-D-glucose

+ 02

glucose oxidase

PH 5

D-gluconic acid

+ H202

. The hydrogen peroxide produced is then quantitated by reaction with luminol in the presence of a metal ion catalyst to produce light. luminol

+ H20z catalyst

3-aminophthalic acid

+ hv

As can be seen, these two reactions occur at very different pHs, which is the most serious problem in coupling these two reactions. In this laboratory the pH problem has been overcome by the use of a microporous membrane flow cell in which oxidase enzyme (buffered at its optimum pH) is contained in a reservoir and forced through a microporous membrane into the analyte side where an injected plug containing analyte and chemiluminescence reagents (buffered at the optimum pH for the chemiluminescence reaction) flows past the membrane (3, 6). This approach produces a stable pH gradient on the analyte side of the membrane so that the analyte can react with the oxidase enzyme at an appropriate pH in the region near the membrane, and the hydrogen peroxide produced can diffuse away from the membrane and react with luminol at a more alkaline pH to produce light. Use of this cell has been shown to have several advantages including extreme reagent conservation. This paper involves the extension of this approach to the determination of sucrose. Sucrose is an analyte for which no oxidase enzyme exists, but which can be enzymatically converted to glucose, which in turn can be determined by using glucose oxidase with chemiluminescence detection as de-

0003-2700/86/0358-0116$01.50/0@ 1985 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 58, NO. 1, JANUARY 1986

microporous membrane flow cell z

,

.

,injection valve

4

IMER

h

rto , I

Figure 1. Flow system.

scribed. Sucrose can be enzymatically hydrolyzed to produce glucose and fructose sucrose

+ H20

invertase pH 4.5

a-D-glucose

+ D-fructose

However, the glucose produced in this reaction is in the a form and glucose oxidase is specific for 0-glucose. Given enough time, the a-glucose will mutarotate to give an equilibrium mixture of a- and 0-glucose. However, this process is slow on the time scale of this experiment. This problem can be overcome by the use of mutarotase, an enzyme that catalyzes the interconversion of a- and 0-glucose a-D-glucose

-

mutarotase

pH 1.4

P-D-glUCOSe

This paper describes a continuous flow method for the rapid determination of sucrose by on-line enzymatic conversion to glucose and detection by chemiluminescence based on the reactions given above. This method has been applied to the determination of sucrose and glucose in food products. EXPERIMENTAL SECTION Instrumentation. The instrumentation used is similar to that described previously (3, 6). The configuration used for most experiments is shown in Figure 1. A Rainin rabbit peristaltic pump was used to pump two channels at a flow rate of 1.0 mL/min. A Rheodyne Model 5020 sample injection loop was used to inject a 5OO-pL sample aliquot into a buffer stream. The sample was then carried through the appropriate immobilized enzyme reactor (IMER) packed with a wet slurry of the appropriate enzyme immobilized on controlled-pore glass (CPG). The IMER consists of a 21/4 in. long Plexiglas column with an inner diameter of 0.125 in. capped with nylon end fittings (with in.-28 threads) containing 20-pm stainless-steel frits. The sample in the buffer stream would then be combined in a mixing cell with the other stream containing chemiluminescence reagents. The analyte mixture then passed into a 15O-pLflow cell separated from a I-mL reagent reservoir by a microporous membrane. The reagent reservoir contained glucose oxidase, which was forced through the membrane at a flow rate of 5 pL/min by a Sage 355 syringe pump, A 1P28A photomultiplier tube biased at -980 V was placed directly in front of the flow cell and measured the light produced. The PMT anode current was amplified by a Pacific Precision Model 126 photometer, and the output was sent to a recorder. In previous experiments (3),a Celgard 5511 poly(propy1ene) membrane from Celanese, which had been coated with a nonionic surfactant to achieve wettability, was used in the microporous membrane flow cell. In this work a Rainin nylon 66 microporous membrane is used in the invertase flow cell (in the work with nonimmobilized invertase) because the Celgard membrane has a pore size (0.04 pm) and a molecular weight cutoff (200000) too low to allow passage of invertase, which has a molecular weight of 270000. The Rainin membrane has 0.2-pm pores and therefore a much larger molecular weight cutoff. The Rainin membrane was also used in the glucose oxidase flow cell (in all studies) because it gave better long-term reproducibility. Reagents. Glucose oxidase (EC 1.1.3.4) was obtained from Sigma Chemical Co. as type VI1 with a specific activity of 145000 units/g. The glucose oxidase reagent contained 2 mg of enzyme/ml of solution (290 units/mL) and was buffered at pH 5

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using a 0.1 M sodium acetate buffer. Invertase (EC 3.2.1.26) was obtained from Sigma Chemical Co. as grade VI1 with a specific activity of 500000 units/g. The invertase reagent contained 7 mg of enzyme/mL of solution (3500 units/mL) and was buffered at pH 5.65 with a 0.1 M potassium hydrogen phthalate buffer. Mutarotase (EC 5.1.3.3) was obtained from Sigma Chemical Co. as a suspension containing 5 mg of protein/mL of solution with a specific activity of 5700 units/mg of protein. The invertase/mutarotase reagent contained the invertase reagent plus 2.56 p L of mutarotase/mL of solution (73 units/mL). Catalase (EC 1.11.1.6) was obtained from Sigma Chemical Co. as a suspension containing 4.2 mg of protein/mL of solution with a specific activity of 10 700 units/mg of protein. Horseradish peroxidase (HRP) (EC 1.11.17)was obtained from Sigma Chemical Co. as type I with a specific activity of 90000 units/g. Hemin was obtained from Sigma Chemical Co. as type I. The chemiluminescence reagent contained 4 X lod M luminol (Aldrich),2 X lo4 M hemin, 0.8 units/mL HRP, and 0.1 M Tris (pH 10.5). This solution was allowed to stand at room temperature for several days prior to use. Glucose and sucrose solutions were prepared from a 0.1 M stock solution containing 10 mM sodium benzoate (buffered at pH 6) as a preservative. Samples of soft drinks were prepared by dilution of the degassed beverage to the appropriate concentration. Samples of breakfast cereal and cake mix were crushed and allowed to stand in water to extract the sugars. The suspensions were then centrifuged and filtered and diluted to the appropriate concentrations. All solutions were diluted with l mM phosphate buffered at pH 6. Reagents used in the enzyme immobilization procedure include (3-aminopropyl)triethoxysilane(Petrarch Systems) and glutaraldehyde (Eastman Kodak). Controlled-pore glass (CPG) used for the immobilization was obtained from ElectroNucleonics and . was 80/120 mesh with a 552-A average pore size. All other reagents used were reagent grade and were used without further purification. All solutions were prepared by the use of water from a Millipore/Continental water purification system. Immobilization of Enzymes. The immobilization procedure used is similar to one developed in this laboratory (IO). The CPG was cleaned with nitric acid then silanized with (3-aminopropy1)triethoxysilane in pH 5 acetate buffer at 90 "C for 2 h. Glutaraldehyde coupling was carried out in 0.1 M phosphate buffer at pH 7.4 for 2-3 h at room temperature. After the CPG was extensively rinsed, covalent attachment of enzymes (glucose oxidase 14500 units/g of CPG, invertase 10000 units/g of CPG, mutarotase 1140 units/g of CPG, and catalase 900 units/g of CPG) was carried out in 0.1 M phosphate buffer at pH 7.4 at 5 "C overnight. After the material was rinsed, the CPG-bound enzymes were stored at 5 "C in the appropriate buffer (glucose oxidase in pH 5.0 acetate buffer, invertase in pH 4.5 acetate buffer, mutarotase in pH 7.4 phosphate buffer, and catalase in pH 7.0 phosphate buffer). RESULTS AND DISCUSSION Nonimmobilized Enzymes. Initial work employed all enzymes in nonimmobilized forms. The experimental conditions employed were based on those used in earlier work done with this instrumentation (3) and differ considerably from those conditions given in the Experimental Section, Aqueous solutions of sucrose were injected into a water carrier stream (1 mL/min), which flowed through a first microporous membrane flow cell that contained both invertase and mutarotase in 0.1 M phthalate buffer (pH 5). The solution then passed through a 5-min delay loop (to allow sufficient time at near neutral pH for the invertase and mutarotase catalyzed reactions) and finally into a second microporous membrane flow cell that contained glucose oxidase in 0.1 M acetate buffer (pH 5). Just prior to entering the glucose oxidase flow cell, the analyte stream was combined with a second stream (also 1mL/min), which contained the chemiluminescence reagents in 0.1 M Tris (pH 10.5). Chemiluminescence emission was detected at this second cell. The percent conversion of sucrose

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ANALYTICAL CHEMISTRY, VOL. 58, NO. 1, JANUARY 1986

to glucose was 40%. A log-log working curve in the region of 0.04-2 mM sucrose was produced with a slope of 1.94 and a correlation coefficient of 0.9971. It is possible to simplify the setup such that all three enzymes are placed in a single microporous membrane flow cell. The sacrifice is that the percent conversion of sucrose to @-glucosedecreases to below 1% . This is due to insufficient time for the invertase and mutarotase reactions to occur. Once the enzymes diffuse away from the membrane and into the bulk solution they are at pH 10.5; at this pH invertase is inactivated (11)and the activity of mutarotase decreases to less than 10% of its maximum (12). Therefore, it is only for a few seconds, while enzymes and substrates are close to the membrane, that conversion of sucrose to glucose and glucose to peroxide occurs to any extent. Immobilized Enzymes. In an effort to increase the percent conversion of sucrose to glucose, and thus hopefully expand the linear working range for sucrose, the use of immobilized enzymes was investigated. Immobilization of invertase and mutarotase was carried out as described previously. The immobilized enzymes were packed into columns that were placed in the system shown in Figure 1so that an injected sucrose sample first passed through the invertase column where the sucrose is converted to a-glucose and then the mutarotase column where the a-glucose is converted to @-glucose;the @-glucosethen mixes with the CL reagents, and the resulting mixture enters the glucose oxidase microporous membrane flow cell where the @-glucosereacts with glucose oxidase that has been forced through the microporous membrane, and the generated peroxide is detected via chemiluminescence reaction with luminol. To preserve enzyme activity and help maintain a constant pH, the analyte carrier stream (which continuously flows through the enzyme columns) was changed from water to an appropriate buffer. Since invertase has a pH optimum at 4.5 and mutarotase at 7.4, solution pHs between 5 and 7 were investigated by using both 1mM phosphate and 1mM acetate buffers. The buffering capacity was kept low so that when the analyte stream mixed with the chemiluminescence reagent stream at pH 10.5 the resulting solution would remain near pH 10.5. Although both buffers at all pHs tried gave acceptable results and any of these combinations could have been used, the optimum appeared to be 1mM phosphate at pH 6. This buffer was used as the rinse solution for all experiments and was present in all analyte solutions. Several different analyte stream flow rates were examined, and the maximum chemiluminescence intensities were seen at a flow rate of 1 mL/min; this flow rate was used for all subsequent work. At slower flow rates, although the peak area remained constant, peak broadening caused peak intensity to decrease. At faster flow rates, peak intensities and peak areas decreased with increasing flow rate. This behavior is a result of reduced residence time in the enzyme columns and in the flow cell where detection occurs. With an analyte stream flow rate of 1 mL/min, the flow rate of the glucose oxidase solution was varied from 0.5 to 12 pLlmin. Below 2.5 pL/min the chemiluminescence intensity increases as the flow rate increases, probably due to the increased production of hydrogen peroxide (from glucose) achieved from the presence of additional glucose oxidase on the analyte side of the membrane. Above 8 pL/min the chemiluminescence intensity decreases as the flow rate increases, probably due to a change in the pH profile within the cell resulting in less than optimal performance from the chemiluminescence reaction. The optimal flow rate is between 2.5 and 8 pL/min. Not only is maximum chemiluminescence intensity obtained but also minor fluctuations in flow rate will not alter the chemiluminescence emission intensity because

l0,OOO .

1,000 -

100

-

'ii IO

-

-4, -

a

-e J V

10-5

[~ucrose]

10-4

IC

I

( M)

Flgure 2. Working curve for sucrose.

To Cell CL Reagents

Flgure 3. Arrangement of IMER columns for sucrose measurements via destruction of original glucose (upper channel) and for glucose measurements (lower channel): GO, glucose oxidase; MUT, mutarotase; CAT, catalase: INV invertase.

the chemiluminescence intensity is constant over this region. Based on these observations, a flow rate of 5 pL/min was chosen for all subsequent work. Sample injection volumes from 100 pL to 1 mL were investigated. Both the peak intensity and peak width decrease as the sample volume is decreased. From 100 to 500 pL the peak intensity increases markedly with injected volume while the peak width remains essentially constant. Above 500 pL there is significant increase in peak width with only slight increase in peak intensity. Therefore, for maximum peak intensity (for good sensitivity) and minimum peak width (for short assay time and high throughput) a 500-pL sample injection volume was selected and used for all work. Under these conditions 100% of the sucrose present is converted to glucose. A log-log working curve from 5 pM to 1mM sucrose with a slope of 1.98 (a = *0.03) and a correlation of 0.9997 was produced and is shown in Figure 2. As anticipated, working curves for sucrose and glucose are identical over this concentration range. Measurements of replicate injections over the range of 10-100 pM give a relative standard deviation of 2-3% and from 0.2 to 1 mM give a relative standard deviation of less than 1% The limit of detection for this method is 3.5 pM (based on the working curve leveling out below this point), and the time for analysis is 2 min. Since the use of immobilized invertase and mutarotase is obviously superior to the use of nonimmobilized invertase and mutarotase in working range, detection limit, sensitivity, and time of analysis, this approach was used for all following work. For the determination of sucrose, this method compares extremely well to other methods currently in use. These methods include the use of enzyme electrodes (13-17), HPLC (18-22), and other methods such as fluorescence (23). Methods that involve the use of enzyme electrodes operate in the millimolar sucrose range and have assay times of about 6 min. HPLC methods operate in the 1-10 mM sucrose range and have assay times between 5 and 20 min. Other techniques, such as fluorescence, generally require a prior enzymatic step

.

ANALYTICAL CHEMISTRY, VOL. 58, NO. 1, JANUARY 1986 T a b l e I. P e r c e n t Error in D e t e r m i n a t i o n s M i x t u r e s of G l u c o s e and S u c r o s e

of P r e p a r e d

T a b l e 11. A s s a y R e s u l t s f o r Sugar D e t e r m i n a t i o n s in Food Products

% glucose

% error

[sucrose], ILM 20 50

100 200 500

100 100 100

100 100

[glucose], glucose, both ILM methods 100 100 100 100 100 20 59 100 200 500

sucrose destruction subtraction

-0.64

+1.03

-1.01 -1.80

+0.84

-0.59 +0.21 -3.17 +0.26

+0.11 -0.08 +1.17 +0.11

+5.05 +5.96 +7.42

+10.80 0.00

-1.80 +0.48

+0.11

+4.34 +8.60 +7.42

+0.98

+1.80

-0.20

+1,26

-6.43

0.93"

0.00

0.62"

5.60'

"Average I% errorl. and have detection limits in the range of 100 p M and analysis times on the order of 30 min to an hour. Sucrose Determination in Food Products. In order to apply this method to the analysis of food products that contain both sucrose and glucose, it is necessary to distinguish the glucose produced from the hydrolysis of sucrose from the glucose originally present in the sample. This can be accomplished either by prior determination of the amount of glucose present or by elimination of the glucose prior to the determination of sucrose. For the first approach, the concentration of glucose can be determined by passing the sample through only the mutarotase column and glucose oxidase microporous membrane flow cell. Since the sample does not contact invertase (to convert sucrose to glucose) and glucose oxidase does not react with sucrose, only the glucose originally present is detected. A second aliquot of the sample is then passed through both the invertase and mutarotase columns and the sum of the concentrations of sucrose and glucose is determined. The concentration of sucrose is then calculated by subtracting the concentration of glucose from the sum of the concentrations of sucrose plus glucose. Elimination of glucose from a sample containing glucose and sucrose can be accomplished on-line by the addition of a column containing mutarotase and glucose oxidase, which converts all of the glucose to hydrogen peroxide, and a column containing catalase, which destroys the hydrogen peroxide produced. The sample, which now contains only sucrose, then flows through the invertase and mutarotase columns and glucose oxidase microporous membrane flow cell as before. In this manner, only the 0-glucose that is produced from sucrose is detected in the glucose oxidase flow cell. The amount of glucose can be separately determined as described above using the mutarotase column, if desired. Figure 3 shows the arrangement of the sequence of enzyme columns in one channel for sucrose determination and in another channel for glucose determination. The accuracy of these two approaches was evaluated by using prepared mixtures of glucose and sucrose. These samples contained varying amounts of glucose in a fixed amount of sucrose and varying amounts of sucrose in a fixed amount of glucose; the glucose/sucrose ratio varies from 0.2 to 5. Results are given in Table 1. As can be seen, the subtraction and destruction methods give average errors of 5.6 and 0.6 %, respectively, for the determination of sucrose. For both methods the average error in determination of glucose is 0.9%. Although both approaches give acceptable results, the destruction approach is preferred in terms of both accuracy and convenience.

119

food product

Pepsi" Coke ("old Coke") cerealb cake mixb

%

both methods

destruction

4.97 3.76 0.14 1.50

0.69 4.72 3.43 50.7

sucrose subtraction 1.11 5.42 3.56 53.0

" Percent weiehtlvolume. Percent weiahtlweieht, Determination of mixtures of sucrose and glucose in food products was performed by the use of these two methods. The food products analyzed include soft drinks (Coke, Pepsi), breakfast cereal (Cheerios), and cake mix (Duncan Hines Deluxe Yellow). Samples were prepared as indicated in the Experimental Section and were analyzed in the manner described except that cereal and cake mix samples were injected through a 0.2-pm prefilter to remove any particles that escaped filtration. Results are given in Table 11. The sugar composition of soft drinks can vary, but a typical composition for Pepsi is 6.3% fructose, 4.3% glucose, and 0.7% sucrose (24). Previous reports have given the sugar composition of Cheerios as 3-5% total sugar, with all the sugar being sucrose and no glucose or fructose (25); the manufacturer lists the sugar content as 1g/oz or 3.5%. Cake mixes are typically 40-55% sugar. Thus, our results are in good agreement with expected values. Registry No. Sucrose, 57-50-1; invertase, 9001-57-4; mutarotase, 9031-76-9; glucose oxidase, 9001-37-0; luminol, 521-31-3; glucose, 50-99-7.

LITERATURE CITED Seitz, W. R. I n "Methods in Enzymology"; DeLuca, M. A,, Ed.; Academic Press: New York, 1978; Voi. LVII, p 445. Bostick, D. T.; Hercules, D. M. Anal. Chem. 1975, 47, 447-451. Piiosof, D.; Nieman, T. A. Anal. Chem. 1982, 54, 1698-1701. Ridder, C.; Hansen, E. H.; Ruzicka, J. Anal. Lett. 1982, 15, 1751-1766. Hara, T.; Toriyama, M.; Imaki, M. Bull. Chem. SOC.Jpn. 1982, 5 5 , 1854-1857. Piiosof, D.; Malavolti, N.; Nieman, T. A. Anal. Chlm. Acta 1985, 170, 199-207. Gorus, F.; Schram, E. Arch. Int. Physiol. Biochlm. 1977, 85, 981-982. Lowrey, S. N.; Carr, P. W.; Seitz, W. R. Anal. Lett. 1977, 10, 931-943. Riggin, V. I . Zh. Anal. Khim. 1981, 36, 1582-1587. Klopf, L. L.; Nieman, T. A. Anal. Chem. 1985, 57, 46-51. Lampen, J. 0. I n "The Enzymes"; Boyer, P. D., Lardy, H., Myrback, K., Eds.; Academic Press: New York, 1971; Vol. 4, p 300. Amano Enzymes Catalog; Arnano International Enzyme Co., Inc.: Troy, VA, 1984; p 37. Satoh, I.;Karube, I.; Suzuki, S. Biotechnol. Bioeng. 1976, 78, 269-272. Nikoieiis, D. P.; Monola, H. A. Anal. Chem. 1978, 50, 1665-1670. Bertrand, C.; Coulet. P. R.; Gautheron, D. C. Anal. Chim. Acta 1981, 126, 23-34. Scheiler, F.; Renneberg, R. Anal. Chlm. Acta 1983, 152, 265-269. Scheiier, F.; Karsten, C H. Anal. Chlm. Acta 1983, 155, 29-36. DeVries, J. W.; Heroff, J. C.; Egberg, D. C. J . Assoc. Off. Anal. Chem. 1979. 62, 1292-1296. Wong-Chong, J.; Martin, F. A. J. Agrlc. Food Chem. 1979, 27, 927-929. Wong-Chong, J.; Martin, F. A. J. Agrlc. Food Chem. 1979, 2 7 , 9 2 9-93 2. Damon, C. E.; Penitt, B., Jr. J. Assoc. Off. Anal. Chem. 1980, 6 3 , 476-460. Ondrus, M. G.; Wenzel, J.; Zlmmerman, G. L. J. Chem. Educ. 1983, 60, 776-778. Guiibault, 0. G.; Brignan, P. J., Jr.; Juneau, M. Anal. Chem. 1968, 40, 1256-1 263. Pepsi, Technical Dept., Chicago, IL, personal communication, 1984. Ondrus, M. G.; Wenzel, J.; Zimrnerman, G. L. J . Chem. Educ. 1983, 6 0 , 776-778.

RECEIVED for review April 1, 1985. Resubmitted August 12, 1985. Accepted August 12,1985. This research was supported in part, by National Science Foundation Grant CHE-81-08816.