Yang Jinghe, Cao Xihui, Huang Fang, Wu Xia, Zhou Guangjun^#
(Department of Chemistry, Lab. Colloid & Interface Chemistry for Education Ministry, Shandong University, Jinan, 250100, ^#Shandong Supervision &Inspection Institute for Product Quality, China)

Received Oct. 21, 1999; Supported by the National Natural Science Foundation of China and Shandong Province, the Research Laboratory of SEDC of Analytical Science for Material and Life Chemistry

Abstract A new simple and sensitive fluorimetric method for the determination of carbohydrates is described. The method is based on the reaction of carbohydrates with 2,3-diaminonaphthalene (DAN) in the presence of sulfuric acid. Under optimum conditions, an excellent linear relationship was obtained between the fluorescence intensity and the concentration of carbohydrates. The detection limit is 1.1×10^-8mol/L for fructose and 9.7×10^-8mol/L for galactose and that of other carbohydrates lies between 1.1×10^-8mol/L and 9.7×10^-8mol/L.
Keywords carbohydrates, 2,3-diaminonaphthalene, Fluorimetric determination

Carbohydrates are very important in nature and they take part in almost all of the life processes. They are not only central to energy generation and storage but also offer us lots of information about other species and ourselves. So, they are also called as information molecules. The study of carbohydrates is very important and valuable in chemistry and bioscience.
    It is difficult to detect carbohydrates with usual means because they have no groups that could be detected sensitively. So, it is important to derive the carbohydrates and let them have some properties that could be detected sensitively in the determination. Recently, there are many reports about the derivatization of carbohydrates. The usual derivative reagents are 1,2-di(4-methoxyphenyl)ethylenediamine (DME)^[1], 1,2-phenulenediamine (PDM)^[2], malonamide^[3], guanidine^[4], arginine^[5], benzamidine^[6], 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS)^[7], 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate^[8] (AQC) and 2-metthyl-3-oxo-4-phenyl-2,3-dihydrofunan-2-yl acetate^[9] (PDFAc), etc, of which AQC and PDFAc are applied in the determination of the amino sugar. However, when they are applied to the determination of carbohydrates, they all have some drawbacks such as long reaction time, strong reaction conditions or low sensitivity and precision. Our research of finding new derivative reagents emphasizes on following points: a) rapid derivatization b) mild reaction conditions and c) high sensitivity and precision. The new derivative reagent 2,3-diaminonaphthalene answers above requirements well. Several carbohydrates were detected by the method of DAN. The limit of detection reaches 10^-8mol/L level and in the linear range the correlation coefficients are more than 0.99. The sensitivity of the proposed method is similar to that of the DME method, but 3-10 times higher than other analogous methods.
1. EXPERIMENTAL
1.1 Apparatus
All fluorescence intensities were measured on a Hitachi Model 850spectrofluorimeter and all absorption spectra were taken on a Shimadzu UV-240 spectrophotometer.
1.2 Reagents
The 2,3-diaminonaphthalene (DAN) solution was prepared by dissolving 0.995g DAN in 250ml 0.1 mol/L HCl, then treated with 50ml cyclohexane, shaken for about 1min, the was organic phase discarded, and the remaining aqueous phase treated twice more with cyclohexane according to the above procedure. The solution was stored in a brown vessel at room temperature.
    Glucose, fructose, galactose, glucosamine hydrochloride and N-acetylglucosamine were all obtained from Detecting Department of Medicines and Byproducts of China.Fucose and xylose were purchased from Medicine Company of China. a-Rhamnose was obtained from Chemical Reagents Company of Beijing.L-sorbose was obtained from Beijin Fangcao medicine and chemitechnology Research Company.D-arobinose was purchased from Medicine Store of Military Medical College of China.The carbohydrate solution was prepared with distilled water and the concentration is 1.00×10^-2 mol/L.Sulfuric acid solution is prepared by solving 75ml sulfuric acid to 25ml distilled water.
    All the other reagents were of analytical reagent grade and distilled deionized water was used throughout.
1.3 Procedure
To a 25ml test-tube were added 0.5ml of glucose standard solution, 2ml of Sulfuric acid solution and 2ml of DAN. This mixture was shaken and heated in boiling water bath for 150min, diluted to 25ml after cooled to room temperature. The fluorescence intensity of the system was measured in a 1cm-quart cell with excitation and emission wavelengths of 402 and 480nm, respectively.
2. RESULTS AND DISCUSSION
2.1 Fluorescence Spectra
Glucose H₂SO₄ have no fluorescence and DAN, when pH=1,has an emission peak at 400nm and two excitation peak at 292nm and 340nm respectively. When H₂SO₄ is added to DAN, the fluorescence was weakened evidently, and the maximum excitation peak is at 292nm.
    The fluorescence spectra of glucose-H₂SO₄-DAN are shown in Fig.1. It can be seen that the glucose- H₂SO₄-DAN system has two excitation peaks, both at 400 and 415nm respectively, and two emission peaks, at 480 and 498nm, respectively. From Fig. 1, one can also see that because the reagent blank is very low and stable, the redundant DAN do not affect the determination of carbohydrates when 400nm is selected as the excitation peak of glucose- H₂SO₄-DAN system.
    Many carbohydrates can react with DAN and give fluorescent compounds. The results are shown in table1. From table 1 we can see that except the a-Rhamnose, N-acetylglucosamin and HCl-glucosamine all the other carbohydrates react with DAN to give fluorescent compounds, the fluorescence spectra of which are similar to that of glucose- H₂SO₄-DAN although the intensity is different.

If		If
Fig.1 Fluorescence Spectra (a) excitation spectra (l =480nm), (b) emission spectra (l=400nm) 1. the system of glucose-H2SO4 , 2. the system of DAN- H2SO4 ,3. the system of glucose-DAN- H2SO4

Table 1. Fluorescence Spectra of different carbohydrates

2.2 Factors Affecting the Fluorescence Intensity
2.2.1 Effect of the concentration of H₂SO₄
DAN is basic, and does not solve in neutral or basic solution. When pH<2, it reacts with some carbohydrates and forms fluorescent substance. The concentration of H₂SO₄ has an important effect on the fluorescence intensity of the carbohydrate- H₂SO₄-DAN system and the results are shown in Fig 2. From which, we can see that the fluorescence intensity increases with the increasing of the concentration of the H₂SO₄. The fluorescence intensity of the system reaches the maximum and retains constant when the volume of H₂SO₄ solution is 1.5-4ml. In this paper, 2ml of H₂SO₄ solution were selected.
2.2.2 Effect of the concentration of DAN
The effect of the concentration of DAN was studied and the results were shown in Fig3. From which one can clearly see that a final concentration of 0.002mol/L gave the maximum fluorescence under the conditions defined.
2.2.3Effects of reaction temperature and heating time
The effects of different reaction temperature were studied and the results were given in Fig 4. It shows that under 80°C the fluorescence is very weak and the balance time is very long, but under 100°C the fluorescence is quite strong and the balance time is very short. 100°C was selected in this paper. The results also show that the fluorescence increases with the increasing of the heating time. The fluorescence intensity reaches the maximum after the system is heated in boiling water bath for 100-150min, in which the concentration of glucose is 5.0×10^-4 mol/L (Fig 4). Other carbohydrates are similar to glucose. In this paper the heating time is 150min.

    Compared with the analogous derivative reagents, DAN has the properties of mild reaction conditions and rapid derivatization. It is reported that the reaction temperature is usually between 120°C (1,2-phenylenediamine method) to 160°C (guanidine method 150°C, DME method 150°C and arginine method 160°C). At the same time this proposed method is more rapid than other methods, such as the 1,2-phenylenediamine method, which requires180 min.
2.3 The stability
It is discovered that the fluorescence intensity of this system retained stable for two hours after it reaches the maximum.
3. ANALYTICAL APPLICATION
3.1 Calibration curve and detection limit
Under the optimum conditions defined, a linear relationship was obtained between the fluorescence intensity and the concentration of carbohydrates such as glucose, fructose, galactose, xylose, fucose, L-sorbose, D-arabinose. The results are shown in table 2, from which we understand that in the linear ranges all kinds of carbohydrates above have an excellent correlation coefficient, and their limits of detection could reach 10^-8 mol/L (S/N=2).
Table 2. the linear range and detection limit of different carbohydrates

3.2 Recovery tests and sample determination
Tests of the recovery efficiency for known amounts of glucose, fructose and xylose solutions were made. The results are listed in table 3 which indicate that about 94.8% can be recovered.

Table 3. Recovery tests

The proposed method was also used to determine glucose in glucose injection solution (Jinan Mingshui LiMing Pharmaceutical, China). The satisfied results are shown in table 4.
Table 4. Sample determination

S.D.: standard deviation

REFERENCES
[1] Umegae Y. Anal. Sci., 1989, 5: 371.
[2] Towne J C, Spikner J E. Anal. Chem., 1963, 35 (2): 211.
[3] Honda S, Matsuda Y, Terao M et al. Anal. Chim. Acta, 1979, 108: 421.
[4] Yamauchi S, Nimure N, Kinoshita T et al. Analyst, 1993, 118 (7): 773.
[5] Mikami H, Ishida Y. Bunseki Kagaku, 1983, 32: E207.
[6] Coquet A, Venthey J L, Haerdi W. J. Chromatogr., 1991, 553 (1-2): 255.
[7] Stefansson M, Novotny M. Anal. Chem., 1994, 66: 1134.
[8] Diaz J, Lliberia J L, Comellas L et al. J.Chromatogr., 1996, 719 (1): 171.
[9] Chen P, Novotny M V. Anal. Chem., 1997, 69 (14): 2806.

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