Anal. Chem. wag, 61,782-784
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TECHNICAL NOTES Sucrose Enzyme Electrode Y u a n h a n g X u a n d George G. Guilbault*
Department of Chemistry, University of N e w Orleans, N e w Orleans, Louisiana 70148, and Universal Sensors, Inc., 5258 Veterans Boulevard, Suite D, Metairie, Louisiana 70006 S h i a S. K u a n
Food and Drug Administration, 4298 Elysian Fields Avenue, N e w Orleans, Louisiana 70122 Americans have for a long time used sucrose extensively in most foodstuffs due to their love for sweets. Thus, it is rather important to have a method that is fast, simple, and sensitive and that could be used for the determination of sucrose in foods and agricultural products. The AOAC (Association of Official Analytical Chemists) method is accurate and precise, but very laborious and time-consuming. For some years, there has been a great deal of interest in the development of novel methods for routine analysis of sucrose (1-5). Several studies on the use of enzyme electrodes for the determination of sucrose have been reported (6, 12, 19-21); however, these sucrose electrodes suffer from lack of either sensitivity or stability. Lately, a flow injection method for sucrose assay has been described (13, 14). Although this method is rapid and precise, it is subject to interference originating from the sample matrix. Because immobilized enzyme electrodes offer many potential advantages, a sucrose enzyme electrode was constructed in this laboratory by using an oxygen electrode and three enzymes immobilized on pig intestine membranes (15). The electrode was workable and stable, but its narrow linear range and low sensitivity made it impractical for use in the food industry. Besides, the quality of the pig intestine membrane is difficult to control, thus resulting in poor reproducibility from batch to batch. Instead of a pig intestine membrane, we used more homogeneous artificial membranes, including Teflon, polyethylene, polypropylene, and cellulose acetate, for the construction of glucose and sucrose electrodes. The effects of these membranes and the amount of enzyme(s) immobilized were investigated. The applicability of the sucrose enzyme electrodes to the determination of sucrose in food and food products was evaluated. EXPERIMENTAL SECTION The sucrose electrode was constructed by mounting an internal membrane over the tip of an oxygen electrode jacket with an O-ring. Forty microliters of buffer solution containing 0.25 mg of bovine serum albumin (BSA) and various amounts of glucose oxidase, invertase, and mutarotase was placed on the membrane. Then, 10 pL of a 2.5% glutaraldehyde solution was added, with vigorous stirring for 30 s. The immobilized enzyme layer was allowed to form at room temperature for over 3 h. The immobilized enzyme layer was washed several times with phosphate buffer solution. Then an external membrane was placed on the enzyme layer with another O-ring. The excess membrane was cut away with a pair of scissors, and the assembly was tightly sealed with a strip of tape to prevent air from entering the enzyme layer. The completed sucrose electrode was stored in phosphate buffer (pH 6.88, 0.2 M) at 0-4 OC when it was not in use. A glucose electrode was constructed essentially by the same method as described above, except that invertase and mutarotase were not used. Two milliliters of internal filling solution (0.2 M 0003-2700/89/036 1-0782$01.50/0
phosphate buffer containing 0.1 M KCl, pH 6.88) was first placed inside an electrode jacket, which was covered with the immobilized enzyme layer. The oxygen electrode body was then carefully inserted into the jacket. Electrode measurements were made by immersing the electrode in 2 mL of phosphate buffer with constant stirring. A constant potential of -0.65 V (vs Ag/AgCl) was then applied to the electrode by using a Universal Sensors oxygen electrode adapter until a steady base-line Etmopotential was obtained. If there were any air bubbles between the internal membrane and oxygen electrode,a stable base-line potential could not be reached. One milliliter of standard working solution or food sample was then injected into the buffer solution. The potential change was monitored and recorded. All food samples containing high levels of sucrose must be properly diluted and cleaned with the use of alumina cream or neutral lead acetate solution before assay (16,17). Since the sucrose electrode measures both glucose and sucrose, the complete system for the analysis of sucrose in food samples is composed of a glucose electrode, which measures only glucose concentration, and a sucrose electrode. The sucrose concentration is computed by subtracting the total concentration of glucose obtained from the reading of the rate change, in current, of the glucose electrode from the total sucrose plus glucose concentration obtained from the reading of the sucrose electrode. The AOAC method, the Munson-Walker general method for the determination of sucrose in food samples (16, 17), was used for comparison study. The base-line potential (Eo) was determined by submerging an oxygen electrode mounted with a plain membrane into 2 mL of phosphate buffer with constant stirring. A constant potential of -0.65 V (vs Ag/AgCl) was then applied until a stable potential was reached. The stable potential, E,, was used as an oxygen permeability index of the membrane. RESULTS A N D DISCUSSION The permeability of oxygen through a membrane was investigated by using an oxygen electrode covered only with the membrane being studied. The amount of dissolved oxygen diffusing through the membrane to the electrode surface undergoes reduction until a steady base line, Eo, is reached. In general, the more oxygen diffusing through the membrane, the more negative is the steady base-line Eo potential (i.e. the larger the cathodic current). Our results suggest that the electrode that uses Teflon as the internal membrane gives the best sensitivity, i.e. the maximum steady-state current, because Teflon has the lowest E o among the tested membranes. As to the effect of external membranes on the properties of enzyme electrodes, two membranes, cellulose acetate ( E , = +240 mV) and Spectrapor 2 (Eo = -1920 mV), were used and compared. The results are given in Figure 1. The same phenomenon was observed. Spectrapor 2, having the lower Eo potential, gave much faster response and better sensitivity. This has led us to believe that for the construction of a sound oxygen sensing enzyme electrode, there is a simple and reliable way for the electrode designer to select the best $2 1989 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 7, APRIL 1, 1989
783
20
4
16
C
c C
12 L
3
0
e
4
+t 0
'
4
5
7
8
e
PH Figure 2. Effect of pH on sucrose electrode response. The steadystate measurement was performed In a 1 X lo4 M sucrose solution with (0)0.2 M acetate buffer and ( 0 )0.2 M phosphate buffer.
O
i
2
3
4
Themin.
5
6
7
6
200 1,1,
Figure 1. Typical response curves of the sucrose electrode with different external membranes: (A) Spectrapor 2, (B) cellulose acetate. Conditiins: sucrose 1 X lo3 M, in 0.2 M phosphate buffer, pH 6.88; amount of enzyme on the electrode, glucose oxidase, 24 units, ln-
vertase 500 units, mutarotase 580 units; Teflon as internal membrane.
membrane, i.e. by measuring the Eo of the membrane to be used. Similarly, the base-line E,,o potential of an enzyme electrode is the sum of the individual Eo values of the external and internal membranes and the enzyme layer. The lower the Et,o, the better the properties of an enzyme electrode, generally. The effect of various amounts of immobilized enzyme on the properties of the sucrose electrodes was examined. The sensitivity of the enzyme electrode decreases with the increase of glucose oxidase or mutarotase immobilized; in particular, the response time increases sharply with increased amount of mutarotase used. The use of enzymes with higher specific activity resulted in better sensitivity. The latter appears to be the governing factor; hence, too thick an enzyme layer gives the worst response and sensitivity, and longer response and recovery times. It was also found that the glucose oxidase played an important role in enhancing the upper limit of linearity of response. When the activity of the glucose oxidase was raised from 24 to 151 units, the upper limit of linearity M, indicating of the calibration curve was extended to 7 x that the sensitivity of a sucrose electrode increases with the increase of invertase activity. This is in agreement with the results obtained with soluble invertase (15). Both acetate and phosphate buffer solutions were used in this study. The effect of pH from 4 to 8 on the sucrose electrode response was investigated with 1 X M sucrose solution. The highest response for sucrose was obtained at pH 6.88 in phosphate buffer (Figure 2). A lower response was noted in acetate buffer. Several reducing mono- and disaccharides such as fructose, maltose, a-lactose, and &lactose were tested to study their possible interference. The results show that there was no
100
0
so
100
150
200
Time, days Flgure 3. Long-term stability of sucrose electrode. The steady-state measurement of the electrode response was performed in a 1 x lo4 M sucrose solution, pH 6.88, at room temperature.
response or interference from any of these carbohydrates at concentrations up to 8 mM. On the basis of the data collected, the optimum glucose and sucrose electrodes were constructed by using the following enzyme levels: for the glucose electrode, glucose oxidase 4.8 units; for the sucrose electrode, glucose oxidase 24, invertase 500, and mutarotase 580 units. The Teflon and Spectrapor 2 dialysis membranes were used as the internal and external membranes, respectively. At room temperature, in a phosphate buffer (0.2 M, pH 6.88), calibration curves for glucose and sucrose, respectively, are linear in the range between 3.3 X lo4 to 3.3 X M and 3.3 X lo4 to 7 X M; the response times were 1-15 s and 1-6 min for the initial rate and steady-state methods, respectively. The long-term stability of the sucrose electrode is shown in Figure 3. The probe is very stable for a t least 300 days; some sucrose electrodes have maintained 100% relative activity for more than 11
Anal. Chem. 1989, 61 784-787
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Table I. Reproducibility of Sucrose and Glucose Electrodes in Quantifying Sucrose in a Soft Drinka
assay no. 1 2 3 4 5 6 7 8 9 10
meanb sd cv, %
current change (nA/min for rate; nA for steadv state) sucrose electrode glucose electrode rate steady state rate steady state 160 168 168 168 160 168 168 172 172 168
55 54 55 53 53 55 54 56 57 52
8.0 8.0 8.5 8.5 8.5 8.5 8.5 8.5 8.5 8.0
167 4.13 2.47
54.4 1.51 2.77
8.4 0.24 2.89
Dilution ratio = 3
X
400.
18.0 18.0 19.0 20.0 21.0 20.5 20.0 20.0 20.0
20.0 19.7 1.00 5.10
Mean of 10 determinations.
Table 11. Comparison Study between Enzyme Electrode and AOAC Method for the Determination of Sucrose in Food Products
food sample cola 1 cola 2 apple juice" apple grape juicen (baby food) mixed fruit juice" (baby food) honeyb
sucrose, mg AOAC electrode method rate steady state method 7.64 19.01 32.14 31.55 29.09 2.53
7.31 20.57 32.65 34.05 27.94 2.37
6.80 19.30 31.92 31.46 27.74 2.38
"For 1 mL. bFor 100 mg.
months and several hundreds of assays. Sucrose was determined in food products by using both the sucrose and glucose electrodes. The amount of sucrose present was calculated by subtracting the concentration of glucose obtained from the reading of the glucose electrode from the total sucrose plus glucose concentration determined from the reading of the sucrose electrode. Samples were prepared as mentioned in the Experimental Section. Table I shows the reproducibility of the sucrose and glucose electrodes when the sucrose content in a soft drink is measured. The standard AOAC method was used to run food samples for sucrose, and the results were compared with those obtained from the enzyme electrode (Table 11). The average correlation coefficients of the two methods for the analysis were 0.9989 and 0.9982,
and the regression equations for data were 3' = 1 . 0 0 0 9 ~+ 0.3755 and y = 1 . 0 4 0 1 ~+ 0.0819 (y = electrode result; x = AOAC method result; n = 6) for the initial rate and steady-state methods, respectively. ACKNOWLEDGMENT We are grateful to Xiangfang Xie for her assistance in setting up the apparatus needed for the quantitation of sucrose by the AOAC method and performing sucrose analysis in food products, as well as the Glenn J. Lubrano and Graham Ramsay for helpful discussions and for the supply of various membranes and electrodes. The financial assistance of the US.Department of Agriculture (SBIR Grant No. 86-SBIR8-0096) is gratefully acknowledged. Registry No. Sucrose, 57-50-1; D-glucose, 50-99-7; cellulose acetate, 9004-357; glucoee oxidase, 9001-37-0; invertase, 9001-57-4; mutarotase, 9031-76-9; Teflon, 9002-84-0. LITERATURE CITED (1) Guilbault, G. G. Analytical Uses of Imm06lked Enzymes: Marcel Dekker: New York and Basel; 1984, Chapter 3. (2) Kobos, R. K. TrAC, Trends Anal. Chem. (Pers. Ed.) 1987, 6(1), VIII-x. (3) Van Brunt, J. Bio/Technology 1887, 5(5), 437-441. (4) Murray, R. W.; Ewing, A. G.; Durst, R. A. Anal. Chem. 1987, 59(5), 379A. (5) Rechnltz, G. A. PAC, Trends Anal. Chem. (Pers. Ed.) 1886. 5(7), 172-174. (6) Cordonnier, M.; Lawny, F.; Chapot, D.;Thomas, D. FEBS Len. 1975, 59, 263. (7) Satoh, I.; Karube, I.; Suzuki, S. Bbfechnol. Bioeng. 1976, 18, 269-272. (8) Barker, A. S.; Somers, P. J. In Topics in Enzyme and Fermentation Blotechnolcgy; Wiseman, A., Ed.; Ellis Hotwood: Chichester, England, 1978; Vol. 2, p 120. (9) Bertrand, C.; Coulet, P. R.; Gautheron, D. C. Anal. Chim. Acta 1881, 126, 23-34. (10) Kulis. Yu. Yu.; Peslyakene, M. V. J . Anal. Chem. USSR (Engl. Transl.) 1880, 3 5 , 786-790. (11) Kulys, J. J. Anal. Len. 1981, 14, 377-397. (12) Scheller, F.; Renneberg. R. Anal. Chim. Acta 1883. 752, 265-289. (13) Masoom. M.; Towshend. A. Anal. Chim. Acta 1885, 171, 185-194. (14) Olsson, 6.; Stalbom, 6.; Johansson, G. Anal. Chlm. Acta 1986, 179, 203-208. (15) Nabi Rahni, M. A.; Lubrano, G. J.; Guilbauit, G. G. J . Agrlc. Food Chem. 1987, 35. 1001-1004. (16) Hart, F. L.; Flsher, H. J. Modem Food Analysis; Sprlnger-Verlag: New York, Heidelberg, Berlin, 1971. (17) Officlsl Methods of Analysis of the Association of OfHc&l Analytical Chemists, 14th ed.;WilUams, S., Ed.; Association of Official Analytical Chemists, Inc.: Washington. DC, 1984. (18) Carr, P. W.; Bowers, L. D. Immoblured Enzymes in Analytical and Clinical Chemlsfty; Wiley: New York, 1980; p 234. (19) Matsumoto, K.; Kamikado. H.; Matsubara, H.; Osajima, Y. Anal. Chem. 1888, 60, 147-151. (20) AWui Hamid. J.; Moody, G. J.; Thomas, J. D. R. Analyst 1888, 773(1), 81-85 -.
(21) Swindlehurst, C. A. (Koerner); Nieman, T. A. Anal. Chim. Acta 1988, 205, 195-205.
RECFJVED for review March 29,1988. Resubmitted November 1, 1988. Accepted December 2, 1988.
Liquid Chromatographic Determination of Theophytline Concentration with Syringe-Type Minicolumns for Direct Plasma Injection Masato Homma and Kitaro Oka*
Division of Clinical Pharmacology, Tokyo College of Pharmacy, Hachioji, Tokyo 192-03, J a p a n Noriyuki Takahashi
Department of Pharmacy, Tokyo Medical College Hospital, Shinjuku-ku, Tokyo 160, J a p a n INTRODUCTION A method for quantitative column extraction has been developed to determine very low concentrations of steroid hormones and certain drugs in biofluids by high-performance 0003-2700/89/0361-0784$01.50/0
liquid chromatography (HPLC) (1-7). This method, termed rapid-flow fractionation (RFF), involves the cleanup of biofluids using diatomaceous earth granules and an organic mobile-phase solvent of minimal polarity for extracting target 0 1989 American Chemical Society
