Evaluation of Some Factors Influencing the o-Toluidine Reaction with Glucose Hugh Y . Y e e Department of Pathology. Hutzel Hospital. 432 E . Hancock A v e . , Detroit. Mich. 48201
Jesse F. Goodwin Genera/ Ciinicai Research Center. Children's Hospitai of Michigan. 3907 Beaubien. Detroit. Mich. 48207
The reaction of 0-toluidine with glucose has been investigated. Citric acid was found to be the most effective acid catalyst. Alcohol type solvents allowed for good dissociation of [ H f ] , maintained a high concentration of undissociated acid, and minimized the formation of protonated amine salt. Optimum reaction time is 6 to 8 minutes at a temperature of 85-95 "C. Thiourea and boric acid are the best non-acid catalysts of those that were examined. Spectrophotometric and thin layer chromatographic evidence indicates a complex series of reactions with the formation of at least four chromogens. A kinetic study shows that N-D-glucosyl-0-toluidine is a probable intermediate; however further reactions of the glucosylamine are necessary to generate the usual green colored solution.
The publication of the use of o-toluidine, by Hultman ( I ) , for assaying glucose concentrations in blood and urine afforded an excellent method with relatively high specificity. In recent years, increased interest has prompted several variations (2-4) of the original formulation to avoid the use of glacial acetic acid solutions or to decrease the acetic acid concentration. Previously, we had successfully improved the sensitivity of the o-toluidine-glucose reaction by the addition of boric and/or citric acids (4, 5 ) . In order to provide a more rational approach toward bettering the o-toluidine formulation for carbohydrate analysis, we have investigated some factors influencing the reaction. This information should be of value for other aromatic amine reagents which are used for carbohydrate detection and quantification. The chromogenic products of the o-toluidine-glucose reaction have been suggested to be an equilibrium mixture of a glycosylamine and its corresponding Schiff base (6). T o date, evidence has not been presented to substantiate this rationale, notwithstanding that these products are considered both logical and probable intermediates. The results of our investigation indicate that the initial condensation product, N-D-glucosyl-o-toluidine, does not show any color or visible light absorption. Additional reactions were necessary for obtaining the final green colored solution. Preliminary evidence shows that a t least four chromogenic products may be intermediates in producing this color. Based upon the assumption that the initial condensation of an amine with glucose follows a similar reaction mechanism such as that for the addition of a semicarbazide to a carbonyl group, several deductions may be made to describe the overall reaction. Evidence collected to date tends to substantiate the validity of this approach. (1) E. Hultrnan, Nature ( L o n d o n ; 183, 108 (1959). (2) J . Bierens de Haan and M Roth, 2 Kiln Chem 7. 624 (1969) (3) A . Hartel and H . Lang, A r z f i Lab 1 5 , 60 (1969). ( 4 ) H. Y . Yee, E. Jenest, and F. Bowles, Ciin Chem 17, 103 (1971). (5) J. F. Goodwin, C i i ~Chem . . 16, 85 (1970) (6) K . Dubowski, Ciin. Chem 8, 215 (1962).
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EXPERIMENTAL Reagents. o-Toluidine, redistill from a small amount of zinc dust, if it exhibits any orange coloration. Store the purified amine in an amber bottle under refrigeration. N-D-Glucosyl-o-toluidine was synthesized by two methods (7, 8). The crude material was recrystallized from ethanol to yield white needles; mp, 98-99 "C (uncorrected). All other chemicals used were either CP or AR grade. Instruments. A Coleman Model 124 spectrophotometer (Coleman Instruments Co., Maywood, 111.) equipped with a Model 165 recorder was used for absorbance measurements. Procedure. Chromogenic formation was carried out by heating a tube containing 5 ml of the reagent with appropriate amounts of a glucose standard for 8 min at 90 "C. Measurements were made at 635 nm after cooling.
RESULTS AND DISCUSSION Reaction Mechanism. Although the o-toluidine reaction has been used to assay glucose for some time, there has been little published evidence suggesting how the reaction proceeds. Dubowski (6) suggested that the chromogenic products were a mixture of a glucosylamine and its Schiff base. We found that the spectrum of N-D-glucosyl-o-toluidine does not exhibit any absorption in the visible portion of the spectrum (Figure 1).Extended heating of a glacial acetic acid solution of the glucosylamine ( 2 g/l.) yields a light yellow colored solution which shows a slight absorbance in the 350-nm region. In order to generate the usual chromogens, it is necessary to add o-toluidine to a concentration of 4-570v/v (Figure 2 ) . A kinetic study of chromogen formation from the reaction of 9% v/v o-toluidine reagent with equimolar solu(1 X tions of glucose and A~-~-glucosyl-o-toluidine mol/l.) showed that the glucosylamine generated more color and at a faster rate (Figure 3). Spectral scans and maxima of the resulting colored solutions were identical in all respects. Thin layer chromatographic analysis of the green, colored solution suggests the formation of a complex series of reaction products (Table I). At least four chromogenic products were revealed with maxima a t 350, 380, 480, and 630 nm. We conclude, from this evidence, that the glucosylamine and its Schiff base are probable intermediates and further reactions appear to be required to yield the chromogens commonly measured for glucose quantification. Kinetics. As suggested earlier (4),the condensation of glucose with 0-toluidine appears to follow a similar reaction mechanism as that for the addition of semicarbazide to a carbonyl compound (9). Based upon this assumption, the following may be deduced: ( a ) the most effective acid catalyst must have a K , K(Ar-NH3-), ( b ) an increase in the concentration of undissociated acid will increase ( 7 ) J. Irvine and R. J. Gilmour, J . Chem Soc 96, 1545 (1909). ( 8 ) F. Weygand, Ber 72, 1663 (1939). ( 9 ) W. P. Jencks, in "Progress In Physical Organic Chemistry. Vol. 2." S. G . Cohen e : ai. Ed.. Wiley-lnterscience. New York, N . Y . , 1964, pp 63-128.
A N A L Y T I C A L C H E M I S T R Y , V O L . 45, NO. 13, N O V E M B E R 1973
14
1
+
1.0
I
12
8
20
16
28
24
Time (minutes)
I
\
'
.+
150
M-0- glucosyl-o-toluidine
100
Figure 3. Rate of chromogen formation from glucose and N-Dglucosyl-o-toluidine (0.1 ml of a 1 X l o - ' mol/l. solution of each) with o-toluidine reagent
1t
151
Wavelength (nm) Figure 1. Absorption spectra of o-toluidine and N-D-glucosyl-o-
toluidine
14
4
,ecitric
I
t
T
// '\ I I
I I
I \
1.0
1
\ \
I I
I I I
I I
\
I
I
I
i
\ I
t
.
iolic
9 s o-Toluidine I
.
l
@o-toluidine*HCI
I
kcetic
I
t.ca,@' 1
Wavelength (nm) Figure 2. Absorption spectra showing the reaction of 0.050 ml of N-D-glucosyl-o-toluidine ( 2 g/l.) in acetic acid heated for 8 minutes with varying concentrations of added o-toluidine
the rate, (c) an increase in [H+] will increase the rate provided the free amine is not converted to its acid salt, and (d) if the condensation is a two-step type ( 9 ) , the pH-rate curve will be bell-shaped. The initial condensation and dehydration reactions were assumed to be major rate determining steps of the overall reaction. Several parameters were investigated to validate this hypothesis. Effect of pH (pKB).A 60% v/v 1-propanol solution containing 9% v/v o-toluidine and 0.15% w/v thiourea was adjusted to pH 5.0, 4.0, and 3.0 with glacial acetic acid. For a p H of 2.0, a 9% v/v o-toluidine solution in glacial
2
"3 " pH (pK)
'
' 4
Figure 4. Effect of pH (bottom curve -.-) ) on chromogen formation curve -.-._._._
5
6 '
and pK (upper
Table I . Thin Layer Chromatography of the Green Chromogenic Mixture from the o-ToluidineGlucose Reactiona Rf
Color
0.56-0.60 0.72-0.74
Blue Yellow Blue-gray
0.92-0.93
Yellow-orange
0.48-0.53
A m a x (nm)
350; v.sI. peak 480; 630 350; shoulder at 380
350; shoulder at 380; v.sI. peak at 610 350
a Silica gel sheet, 20 X 20 cm, plain (Eastman); solvent system:
chlor0form:acetone:acetic acid (60:30:10); solvent front was allowed to r u n 8 c m (30 m i n ) ,
ANALYTICAL CHEMISTRY, VOL. 45, NO. 13, N O V E M B E R 1973
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Table II. Effect of Catalysts on the o-ToluidineGlucose Reaction
Compound
Ratio of absorbances. 635 nm
None (9% v / v o-toluidine in acetic acid) Thiourea
1 .oo 1.20
Thioacetamide
0.71
Thiosemicarbazide
0.47
Mercaptoacetic acid
0.00
Phenyl mercaptan
0.00
Stannous chloride Boric acid Cysteine hydrochloride
0.93 1.50 0.1 7
2- aethoay-ethanol
1-propanol
\ acetic acid acetic with additives
.It
i
p-dionnc IO
zb
o j
4b 5b 60 70 Dielectric Constant
60
Suppresses chromogen Amax 475 n m Suppresses chromogen A, 475 nm Forms chromogen A,,, 340 n m Forms chromogens A, 460 and 355 nm Forms chromogens Amax 460 and 355 nm
90
Figure 5. Effect of solvent on chromogen formation (the dtelectric constants are uncorrected)
acetic acid (0.15% w/v thiourea) was used. Glucose standard (1.5 g/l.), 0.050 ml, was added. The results shown in Figure 4 indicate a bell-shaped pH rate curve. T o examine the effect of pK. a 90% v/v 1-propanol solution containing 9% v/v o-toluidine, 0.15% w/v thiourea, 10% v/v acetic acid, and 1 mol/l. of each of the following acids: acetic, lactic, glycolic. citric, and trichloroacetic was used with 0.050 ml glucose standard ( 5 g/l.). o-Toluidine hydrochloride was added in a concentration of 0.33 mol/l. The alcohol solution without the added acids did not yield any measurable absorbance a t 635 nm. Results shown on the upper curve of Figure 4 substantiate the findings of the pH rate curve. Our data indicates that citric acid ( K 1 = 8.7 X 1 0 - 4 ; K2 = 1.8 x 10-5) was the most effective acid catalyst and the optimal pH range for color formation was 3.0-3.5. Compared to the other acids, citric acid has two available protons with K values near that of the o-toluidinium ion (4.04 x 10-5). Note that the hydrochloride salt of o-toluidine could also serve as a proton source by itself or in conjunction with a small amount of acetic acid as described. The data shown in Figure 4 support the deductions (a), (c), and (d). Effect of Solvent. Citric acid (10 g), 0.15 g thiourea, 1 g boric acid, and 20 ml acetic acid were dissolved with the aid of heat and stirring into each of the following solvents: water, acetic acid, 90% v/v ethanol, 90% v/v 1-propanol. 90% v/v ethylene glycol, 90% v/v 2-methoxyethanol, and 90% v/v 1,4 dioxane. After cooling, 9 ml o-toluidine was added and the final volume adjusted to 100 ml with their respective solvents. A solution of 9% v/v o-toluidine in glacial acetic acid (0.15% w/v thiourea) was also used. Glucose (2 g/l.), 0.050 ml, was added to a tube with each of these reagents and heated a t 90 "C for 8 minutes. A plot of the dielectric constant us absorbance is shown in Figure 5. The uncorrected dielectric constant was in each case similar to that calculated taking into account the contributions from the amounts of water and o-toluidine present. These data support the deduction ( b ) that an increase in the undissociated acid concentration will increase the reaction rate. We conclude that the best solvent for this reaction should have the following properties: (1) allow for good 2164
Remarks
dissociation of the acid catalyst while maintaining a relatively high concentration of undissociated acid. (2) suppress the formation of the protonated amine and be a good solvent for the amine, (3) have a boiling point of 100°C or more, (4) be available in high purity, and (5) have a dielectric constant in the range of 10 to 25. Other important considerations are viscosity, miscibility with water, and cost. Glacial acetic acid has been the solvent of choice, but benzyl alcohol ( 2 ) and various glycols and alcohols ( 3 ) have been reported to be suitable. Of the solvents we examined, 1-propanol and 2-methoxyethanol appeared to be the most satisfactory. The propanol formulation had a lower reagent blank, whereas the methoxyethanol one had a higher color yield. However, ethers such as 2-methoxyethanol and 1,4-dioxane are very susceptible to peroxide formation, so that they are potentially less stable. The solutions made with the di-ether, 1,4-dioxane, were highly colored and unsuitable for analytical use. Solutions made in ethylene glycol also developed a coloration, although this did not impair their use. The formulation prepared in acetic acid or monohydric alcohols was found to be the most stable with respect to the development of background coloration. Effect of Temperature. The optimum temperature of reaction was from 85 to 95 "C with deterioration of the 635-nm absorbing chromogen occurring a t higher reaction temperatures. Effect of Time of Heating. Maximum color formation occurred a t 90 "C with a heating time of 6 to 8 minutes. This confirms earlier reports ( I , 6). Effect of Amine Concentration. The concentration of o-toluidine could be varied from 6-12% v/v with an absorbance difference of about 6%. The maximum color formation was obtained from a reagent which had a concentration of 9% v/v o-toluidine. From an amine concentration of 14-19% v/v, the absorbance was about 10% less than the maximum. At a 20% v/v concentration, the color formation was markedly reduced. Effect of Other Catalysts. A number of other catalysts were examined for their effect upon chromogen formation a t 635 nm; these data are given in Table 11. A concentration of 0.2% w/v of each substance was prepared in a solution of 9% v/v o-toluidine in glacial acetic acid. The amine reagent without any added material served as the reference solution.
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 13, N O V E M B E R 1973
Of the sulfur compounds tested, thiourea enhanced chromogen formation the most a t 635 nm, whereas the addition of mercaptoacetic acid or phenyl mercaptan resulted in complete suppression of this chromogen. Thiosemicarbazide addition decreased the chromogen formation a t 635 nm, but accelerated the formation of a chromogen with a maximum a t 340 nm that had an absorbance 8 times greater than that of the reference solution a t 635 nm. From this evidence, we conclude that these sulfur compounds alter the reaction pathway. Stannous chloride did not have any substantial effect on the color formation. This suggests that the action of the -SH group (mercaptoacetic acid and phenyl mercaptan) was probably not due to a reduction process. Boric acid caused an enhancement of absorbance of 1.5 times. We believe the action of this extremely weak acid may be through the formation of a borate complex ( 5 ) which causes a steric alignment of the hydroxyl groups of the carbohydrate. Based upon the information we have presented, many usable formulations for an o-toluidine or other aromatic amine reagent may be devised. Acetic acid, as a solvent and/or proton source, is not unique. Several other weak
acids may be substituted with the appropriate solvents. However, acetic acid formulations have been found superior in stability since there is no solvent reaction, such as that for glycols or alcohols (esterification), and the purity of acetic acid is better. In comparison, citric acid usually has residual amounts of carbohydrate material which may cause formulations containing high concentrations of this acid to achieve a dark green color on standing. Further advantages of acetic acid are its superior solubility in water and low cost. For continuous flow analysis of glucose with an o-toluidine reagent in glacial acetic acid, the pump tubings require frequent replacement (10). To circumvent this problem, the acetic acid content of the reagent may be reduced ( 4 ) . This modification should result in a wider use of this reagent for automated quantification of glucose. Received for review March 15, 1973. Accepted May 17, 1973. (10) H. Y . Yee and E. S. Jenest, in "Advances in Automated Analysis, Technicon 1969 International Congress." E. Barton et ai. Ed.. Mediad, White Plains, N.Y., 1970. pp 69-72.
Ion-Electrode Based Automatic Glucose Analysis System R . A. Llenado a n d G. A. Rechnitz Department of Chemistry, State University of New York. Buffalo. N. Y. 14214
An automated analysis system is described which utilizes a novel flow-through type ion-selective membrane electrode for the enzymatic determination of glucose. The system functions well in conjunction with aqueous, protein-loaded, and serum samples containing glucose in the physiological concentration range at sampling rates of up to 7 0 determinations per hour. The proposed methodology eliminates the need for color development or dialysis steps and successfully overcomes protein interference at membrane electrodes via improved electrode design.
There has been a tremendous growth of interest in the analytical applications of ion-selective membrane electrodes during the past few years ( I ) , especially to bioanalytical problems (2-5). In a recent paper (j),we demonstrated an automated system capable of enzyme determinations based on the continuous flow technique. We have now extended this principle to substrate determinations by taking advantage of enzyme specificity and ion-electrode selectivity to devise a precise and accurate assay procedure for glucose in synthetic and clinical samples. The proposed approach is made possible, in part, by the development of a novel flow-through electrode which appears to be free of protein-poisoning effects. (1 j R . P. Buck. Anal. Chem.. 44 ( S i , 270R (1972). (2) G. G. Guilbault and J. G. Montalvo, J . Amer. Chem. SOC.. 92, 2533 (1970). (3) R . A . Llenado and G. A. Rechnitz, Ana/. Chem.. 43, 1457 (1971). (4) M . M. Fishman and H. F. Schiff. Anal. Chem.. 44 ( 5 ) , 543R (1972). (5) R . A . Llenado and G. A. Rechnitz, Ana/. Chem.. 45, 826 (1973).
The recognized diagnostic value of glucose has fostered intense development of possible assay procedures. ( 4 , 6-8) Both optical (9-12) and electrometric (13-18) methods have been used for the enzymatic glucose determination. Normally, optical procedures use the coupled glucose oxidase-peroxidase enzyme system to catalyze the transfer of oxygen from the hydrogen peroxide produced from the glucose oxidation to a chromogenic oxygen acceptor such as o-toluidine or o-dianisidine. While such methods have proved satisfactory in many applications, there is the problem of day-to-day variation due to the instability and nonspecificity of the color indicators. Electrometric methods (13-18), on the other hand, are indifferent to optical parameters and, hence, provide certain advantages. The potentiometric method we propose (6) G. G . Guilbault, "Enzymatic Methods of Analysis." Pergamon Press, Oxford, 1970, p 114. (7) R . J. Henry, "Clinical Chemistry: Principles and Technlques," Hoeber, New York, N. Y., 1965, p 620. (8) "CRC Handbook of Cllpical Laboratory Data," The Chemical Rubber Company. Cleveland, Ohio, 1968, p 272. (9) L. L. Solomon and J. E. Johnson, Anal. Chem.. 31, 453 (1959). (10) F. W. Sunderman, J r . , and F. W. Sunderman, Amer. J Ciin Patho/.. 36, 75 (1961). (11) W. J. Blaedel and G. P. Hicks. Anal. Chem.. 34, 388 (1962). (12) H. V. Malmstadt and S. I . Hadjiioannou, Anal Chem.. 34, 452 ( 1962). (13) H. V. Malmstadt and H. L. Pardue, Clin. Chem.. 8, 607 (1962). (14) W. J. Blaedel and C. Olson, Anal. Chem.. 36, 343 (1964) (15) S. J. Updike and G. P. Hicks. Nature (London). 214, 986 (1967) (16) R . K. Simon, G. D Christian, and W. C. Purdy, Ciin Chem., 14, 463 (1968). (17) C. J. Sambucetti and G. W. Neff, "Methods in Clinical Chemistry," University Park Press, Baltimore, Md., 1969, p 118. (18) G. G. Guilbault and G. J. Lubrano, Anal. Chim. Acta. 60, 254 (1972).
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