Eur Food Res Technol (2010) 230:701706 DOI 10.

1007/s00217-009-1208-8

ORIGINAL PAPER

A comparative study of high-performance liquid chromatography and colorimetric method for inulin determination
Hao Wang Zesheng Zhang Liya Liang Shengping Wen Chunyan Liu Xiaojun Xu

Received: 23 June 2009 / Revised: 17 December 2009 / Accepted: 23 December 2009 / Published online: 6 January 2010 Springer-Verlag 2010

Abstract Inulin and oligofructose exhibit valuable nutritional and functional attributes, and so the utilization of inulin and its derivatives in the food industry has been continuously increasing. Compared with high-performance liquid chromatography (HPLC) which presents improved sensitivity and precision, colorimetric method is more widely used and accepted due to the advantages of its simplicity and economy. In order to determine inulin for the purpose of labeling, we develop an easy and effective HPLC method after extraction of inulin. HPLC conditions include a Shim-pack SCR 101C column (Shimadzu), deionized water at 80 C as the mobile phase, and a refractive index detector (RID). Moreover, we compared the HPLC method and colorimetric method on tested foods including claimed commercial inulin products such as inulin, inulin oligomers, and commercial food products. The obtained results demonstrated that the HPLC method could avoid the overestimation and inaccuracy caused by colorimetric method in the case where the samples contain glucan or sucrose, and be regarded as an approach which is easier, faster, and more accurate than colorimetric method for the routine quantication of inulin and inulin oligomers in commercial products. Keywords Inulin Fructose High-performance liquid chromatography Colorimetric method

Introduction Inulin is a natural storage carbohydrate mainly found in roots of chicory (Cichorium intybus), Jerusalem artichoke tubers (Helianthus tuberosus), and dahlia tubers (Dahlia variabilis). It is a mixture of polysaccharides composed of fructose unit chains (linked by b-(2 ? 1) D-fructosylfructose bonds) of various lengths, terminated generally by a single glucose unit (linked by an a-D-glucopyranosoyl bond), which can be hydrolyzed to fructose and glucose by inulinase [1]. The application of inulin and its derivatives in the food industry are on constant increase, and the main benets and nutritional interests of these products are broadly discussed in the literatures [2]. Owing to high nutritional value of natural inulin and its cost which is much higher than that of any other sweeteners, inulin becomes the target of adulteration for economic gains. Some inulin frauds involve the addition of preparations based on simple and complex sugars which can be modied to simulate the natural carbohydrate prole of inulin. The low-priced sweetening products such as glucan have been found to be employed with that aim. There have been many methods reported to determine inulin, which include colorimetric methods and chromatographic methods. Most of the colorimetric methods for inulin determination are based on acid hydrolysis of inulin [3] and the reaction of fructose with several compounds, such as phenolsulphuric acid [4], resorcinol, diphenylamine, anthrone [5], and indole-3-acetic acid, which produce a photometric reaction [6]. As for chromatographic procedures, inulin is enzymatically hydrolyzed to fructose and glucose, followed by measurement of these sugars by high-performance anion-exchange

H. Wang Z. Zhang (&) L. Liang S. Wen C. Liu X. Xu Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of Science and Technology, 300457 Tianjin, Peoples Republic of China e-mail: zhangzesheng@tust.edu.cn

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chromatography with pulsed amperometric detection (HPAECPAD) [7, 8], high-performance liquid chromatography with refractive index detection (HPLCRID) [9], with electrochemical detection, and with light-scattering detection (HPLCLCD) [10]. Compared with HPLC method which presents improved sensitivity and precision, colorimetric method is more widely used and accepted due to the advantages of its simplicity and economy; however, inaccuracy is mainly caused by interfering compounds, such as glucans, sucrose, and hexoses [5]. So far as we know, no one has made the researches on comparing the two methods on inulin determination. In this article, the colorimetric method is compared with enzyme-HPLC method for the determination of inulin, for the purpose of labeling, in which inulinase enzyme and RID is applied.

HPLC analysis Instrumentation The chromatographic equipment consists of a Model LC-20AT pump system (Shimadzu, Japan), a 20-lL sample loop, and a LC solution system which acquires data from the refractive index detector (RID 10A, Shimadzu, Tokyo, Japan). The analytical column which is put to use in this case is Shim-pack SCR 101C from Shimadzu (Tokyo, Japan). Chromatographic conditions The chromatographic separation was carried out in 16 min, when ultrapure water was used as mobile phase at a ow-rate of 1.0 mL/min maintaining the column temperature and detector temperature at 80 C. The eluent was ltered through a 0.45-lm membrane lter and degassed. Peak areas were utilized for quantitative analysis. Calibration curves were prepared in six levels from 0.10 to 5.00 mg/mL of fructose or sucrose or glucose in water. Sample preparation All the samples were obtained from local market. About 100 g of the food product samples were milled in a hammer mill and properly mixed for the purpose of homogenizing the gradients. Then, the samples (1*5 g for each one) were accurately weighed and then extracted with 90-mL hot water in a shaking water-bath at 85 C during 25 min. Later, the beaker was removed from the bath and was cooled to 60 C. At this temperature, 100 lL of inulinase was added to the beaker and incubated in the shaking water-bath at 60 C for 30 min for total digestion of inulin or inulin oligomers. Then, the beaker was cooled to room temperature and was transferred quantitatively to a 100-mL volumetric ask, the volume of which was made up to 100 mL with water. After the processes of homogenization and centrifugation (10,0009g, 20 min), the solution was ltered through a 0.45-lm nylon lter before injection into the HPLC system. Calculation and expression of results The quantity of fructose obtained by HPLC (F1) represents the total amount of fructose in the sample. Simultaneously, a blank analysis was conducted, which is exactly similar to the procedure mentioned above except the addition of inulinase. The quantity of fructose obtained (F2) in this case represents the amount of free fructose in the sample. For samples containing sucrose, the blank analysis also enabled fructose quantication from sucrose (F3). All the calibration standards and experimental samples were prepared in duplicate and analyzed in triplicate. The amount of inulin is given by

Materials and methods Reagents and standards Commercial inulin (Beneo GR, average degree of polymerization 12) from chicory root was the product of Orafti (Belgium). The inulinase enzyme (Fructozyme LTM, which is a mixture of exo-inulinases EC 3.2.1.80 and endo-inulinases EC 3.2.1.7) used was from Aspergillus niger, provided by Novo Nordisk (Denmark). Fructozyme is a commercial liquid with a density of approximately 1.2 g/mL and an activity of 2,000 U/g. One Novo Nordisk inulinase unit (U) is the amount of enzyme which forms 1 lmol of reducing sugar (calculated as glucose) per minute under the conditions of 40 C and pH 5.0. Sweetening products such as claimed commercial Inulin and inulin oligomers, commercial food products (cookies, ice cream, chocolate bar, milk, and yoghurt) were purchased from local markets. All of the other chemicals were purchased from Merck (Darmstadt, Germany). Assay methods Colorimetric method Total carbohydrate was determined by the phenolsulphuric acid method of Dubois [11] taking inulin (Beneo GR) as standard. Reducing sugar was determined by the dinitrosalicylic acid method taking D (-)-Fructose (Mw = 180.16, Fluka) as standard [12]. The inulin or inulin oligomers content was measured from the difference between total carbohydrate and reducing sugars.

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%IN A F1 F2 =P 100 for a sample containing no sucrose, and %IN A F1 F2 F3 =P 100 for a sample containing sucrose, with F3 = S/B, where F1 is concentration of the total fructose in the sample (g/L); F2 is concentration of the free fructose (g/L); F3 is concentration of fructose from sucrose (g/L); S is concentration of sucrose (g/L); P is mean mass (g/L) of the duplicate test samples; A = 1.1 (empirical conversion factor for fructose to inulin or inulin oligomers); and B = 2.05 (empirical conversion factor for fructose to sucrose, obtained from different dilutions of sucrose hydrolyzed with inulinase).

linear relationship between the peak areas and concentration over a wide range of concentrations (0.2, 0.4, 0.8, 1.6, and 3.2 mg/mL). The regression equations of the calibration curve obtained from standards are given in Table 1. In this, y is the concentration (mg/mL), and x is the peak area. Each point was established from an average of six determinations. In order to check the sensitivity of the method under the working conditions proposed, the detection limits (signal-to-noise ratio equals 3) at the time when water was used as a blank were studied. These results showed that the high sensitivity was performed in the method while the detection limits for fructose, glucose, and sucrose were 0.02, 0.02, and 0.03 mg/mL, respectively. Precision

Results and discussion Chemically speaking, the inulin molecule represented by the simple formula G-(F)n-G (where G denotes a glucose and n the number of fructose unites present in the molecule) is a linear b(2-1)-linked fructose polymer terminated by a glucose unite residue. High-performance liquid chromatography with refractive index detection (HPLC RID) has been widely used for the determination of sugars and small oligosaccharides. The inulin molecule can be hydrolyzed by inulinase enzyme to fructose and glucose. Figure 1 shows a typical chromatogram for different samples before and after being hydrolyzed by inulinase. Under the HPLC working conditions, glucose, fructose, and sucrose were separated completely. Linearity and sensitivity The linearity of the HPLC method was evaluated for glucose, fructose, and sucrose. The calibration curve shows a
Fig. 1 A typical chromatogram for different samples: (1) Blank (fructozyme), (2) glucan after hydrolyzation, (3) inulin after hydrolyzation, and (4) inulin before hydrolyzation

In order to evaluate the repeatability and reproducibility of the HPLC method, six replicate determinations on the same day and three determinations on three different days with the same sample were carried out respectively (Table 2). Moreover, we can see that the CV of repeatability was below 3.5%, and CV of inter-day was below 3.8%, which indicated that the method has good repeatability and reproducibility.

Table 1 Calibration curve, detection limit of fructose, glucose, and sucrose Regression equation Correlation coefcient (r) Detection limit (mg/mL) 0.02 0.02 0.03

Fructose y = 159858x ? 9550.1 0.999 Glucose y = 162026x ? 13700 0.998 Sucrose y = 164298x ? 9492.8 0.999

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704 Table 2 Precision of the HPLC method Repeatability (n = 3) Added 0.13 0.25 1.25 5.00 10.00 25.50 Found * 10
-2

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Reproducibility (n = 3) (mean SD) CV (%) 3.5 3.1 2.8 2.5 2.0 1.7 Added 0.13 0.25 1.25 5.00 10.00 25.50 Found * 10-2 (mean SD) 13.43 0. 43 25.15 0.95 124.55 4.21 499.93 17.34 1005.73 23.19 2549.47 45.51 CV (%) 3.2 3.8 3.5 3.4 2.3 1.9

13.25 0.49 25.31 0.85 124.96 3.37 501.76 12.54 1003.28 20.07 2550.51 43.36

Table 3 Recovery of the method (n = 3) Added (mg/L) 0.25 1.25 5.00 Mean Recovery (%) 103.78 103.52 101.93 102.68 1.18 102.65 102.36 99.97 103.42 103.27 103.21

Table 4 Inulin contents in claimed commercial inulin products (commercial inulin and inulin oligomers) with HPLC method and colorimetric method Product Claimed HPLC method (%) (%) 1 2 3 99.50 90.00 85.00 95.00 85.00 99.00 95.00 95.00 99.00 85.00 F2/P S/B * P F1/P 0 0 0.25 1.00 0.50 0 1.78 0.48 0.68 8.42 Inulin (%) Colorimetric method (%) 99.86 90.21 85.34 90.42 80.39 96.72 90.59 93.32 98.73 81.37

90.34 99.37 80.57 87.65 75.32 82.37 80.25 86.78 74.78 77.08 94.64 80.76 86.43 79.88 88.59 90.03 89.97 94.54 42.73 46.72

Analytical recovery The recovery was obtained by measuring inulin concentration in the samples before and after the addition. Three levels of standard concentrations of inulin were added to the samples. Furthermore, they were carried through the entire procedure including enzymatic hydrolysis with inulinase; After hydrolyzation, the samples were injected into the column. The details are given in Table 3. Before performing the HPLC analysis, the sample was extracted with hot water at 85 C and hydrolyzed at 60 C; all these permit the highly increased solubility of inulin and the use of higher inulinase enzyme activity. The method shows very good recoveries. The recoveries in all the concentrations were found to be more than 99.97%. Comparison of results by HPLC method and colorimetric method Colorimetric method is widely used and accepted. Fructose is derived from inulin by acid or enzymatic hydrolysis. It is then reacted with a substrate to produce a coloured reaction product that can be detected photometrically. Despite the discrepancy in the substrates, inaccuracy and overestimation are often caused by interfering compounds, such as glucan, sucrose, and hexoses [5]. Our research shows that samples containing glucan or sucrose can be hydrolyzed by inulinase enzyme to glucose and fructose, which one can be identied and quantied by HPLC. The free fructose (F2) and the fructose originated from sucrose (F3) can be thus assessed and subtracted from the amount of fructose (F1) obtained by

4 5 6 7 8 9 10

7.80 26.84 4.62 18.26 3.36 1.86 0 6.74 4.77 0.25

the inulinase digestion. However, in the case that colorimetric method is conducted, inulin or inulin oligomers content was determined from the difference between total carbohydrates and reducing sugars, glucan or sucrose was not identied from fructan, then inulin or inulin oligomers contents determined would be much higher than that by HPLC method. The results obtained by both methods to analyze commercial products containing inulin or inulin oligomers are shown in Table 4. Although colorimetric method is widely used and accepted due to the advantages of its simplicity and economy, inaccuracy is mainly caused by interfering compounds, such as glucans, sucrose, and hexoses when claimed commercial inulin products containing adulteration. HPLC method compared with colorimetric method is more stably and presents improved sensitivity and precision provides precise and accurate information because it is immune to interfering of compounds which cause overestimation errors, as in the colorimetric method. Most commercially available inulin is extracted from chicory root (Cichorium intybus). The degree of polymerization (DP) varies as a function of the harvest date, but after extraction using a hot water diffusion process, the

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Fig. 2 Comparison of the results obtained by the described HPLC method with colorimetric method in commercial food products

resulting product has an average DP of 1012 units and contains 610% of free sugars, such as sucrose, fructose, and glucose. Inulin and inulin oligomers can be hydrolyzed by inulinase enzyme to produce fructose mainly and a little glucose, while a little fructose is hydrolyzed from samples which are rich in glucans or sucrose. Therefore, this method can result in an overestimation of the inulin content when commercial products containing glucans or sucrose are analyzed with colorimetric method instead of HPLC method. The comparison of the results of inulin content when commercial food products were analyzed by both methods is shown in Fig. 2. For this comparison, 60 commercial food samples including cookies, ice cream, chocolate bar, milk, and yoghurt from markets were analyzed. The solid sample was milled in a hammer mill and properly mixed for the purpose of homogenizing the gradients while the liquid sample was dispersed using a glass rod and then extracted with water. For samples containing sucrose, a correction has to be made since the inulinase enzyme also totally splits sucrose, producing approximately 50% glucose and 50% fructose. The fructose originating from sucrose (F3) can be thus assessed and subtracted from the amount of fructose (F1) obtained by inulinase enzyme digestion. Therefore, the linear correlation coefcient obtained by colorimetric method (R2 = 0.56) was less than the one obtained by HPLC method (R2 = 0.92) in the case of the samples that contain glucans or sucrose. Inulinase produced from A. niger strains have been shown to have pH and temperature optima in the range of 4.57.5 and 4560 C [1315]. We did not need to adjust the pH value to acidic situation because all of our samples met inulinase activity after fat was extracted if possible. Since inulin is a natural product, the ratio of fructose/ glucose uctuates somewhat in practice according to the origin of the raw product from which the inulin is

obtained [8]. Therefore, it is necessary to study the amount of fructose obtained from inulin by hydrolysis and the sample with inulinase to get the conversion factor of fructose to inulin. The mean conversion factor for fructose to inulin ranged from 1.0 to 1.2 for different bfructans. However, a correct conversion factor for routine analyses can give fairly accurate results, assuming a ratio of 8085% fructose and 15% glucose in inulin. Therefore, in practice, the mean conversion factor for fructose to inulin was calculated (A = 1.1), and the values of conversion factor which are very close to 1 stand for high purity of inulin. In the same way, the mean conversion factor for fructose to sucrose was calculated from different dilutions of sucrose hydrolyzed with inulinase (B = 2.05). Values of conversion factor, which are very close to 2, stand for the composition of sucrose of about 50% fructose and 50% glucose.

Conclusion The HPLC method with refractive index detection (RID) is a very sensitive and suitable one not only for verifying adulteration but also for the routine quantication of inulin and inulin oligomers as well as commercial food products containing such elements. This method, moreover, displayed good repeatability for the results and high applicability for routine analysis. However, most important is that HPLC method provides precise and accurate information because it is immune to effects from interfering compounds which cause overestimation errors, as in the colorimetric method.
Acknowledgments This study is partly supported by a grant from the National Natural Science Foundation (grant no. 30671766).

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