Analysis of Functional Sugars in Foods Using HILIC Mode
Introduction
Recently, the effects of functional sugars in foods are gaining scientists’ interests, and thus their analytical methods are called for.
Shodex HILICpak VG-50 series used in this application is a set of polymer-based amino columns which effectively separate various saccharides. The packing material consists of a polyvinyl alcohol base with a hydrophilic functional group, a modified tertiary amine.
Fig. 1. Schematic diagram of VG-50 packing material
With some columns, reducing sugars form a Schiff base with the packing material and are retained in the column. This does not occur with the HILICpak VG-50 series columns, leading to the series’ ability to achieve a high recovery rate. Moreover, column bleeding, which is elution of column packing material related debris, is often observed with silica-based amino columns but is rarely found with the HILICpak VG-50 series columns. Consequently, the related problems of increased background and/or ion suppression in MS are less likely to occur.
Another advantage of this column over the silica-based amino column is that the HILICpak VG-50 series columns can be used under alkaline conditions, with a working pH range of 2 to 13.
In this work, analytical conditions were optimized for the analysis of functional sugars in foods using HILICpak VG-50 4E, 4.6 mm I.D. x 250 mm, particle size 5 µm, including the preparation methods for actual food products.
Results and Discussion
1. Analysis of Lactulose
Lactulose is a functional sugar found in dairy products and it regulates intestinal health of humans. Lactulose remains undigested and reaches the intestine unabsorbed, and then Bifidobacterium uses the sugar for its proliferation. The structures of lactulose, lactose, and epilactose are very similar, which makes their chromatographic separation challenging.
A mixture of acetonitrile and water, or buffer, eluent is generally used in HILIC mode separation. A mixture of acetonitrile and water was used as a starting eluent to separate the three disaccharides. Different ratios of acetonitrile and water were tested, but none were able to separate the three saccharides completely. Higher acetonitrile ratio made the retention time longer and increased the baseline noise. This resulted in lowering the signal to noise ratio, S/N.
Fig. 2. Optimization of LC conditions for lactulose analysis (1)
| Sample | 5 mg/mL each in H2O/CH3CN = 1/1, 5 µL |
| Column | Shodex HILICpak VG-50 4E |
| Eluent | H2O/CH3CN = 20/80 or 15/85 |
| Flow rate | 1.0 mL/min |
| Detector | RI |
| Column temp. | 40 °C |
As a next step, methanol was added to the eluent. Figure 3 shows the result of analyzing the three disaccharides using an eluent mixture of water, acetonitrile, and methanol. The addition of methanol to the eluent effectively separated the disaccharides that have very similar structures. Good linearity was obtained for the calibration curves of all sugars, showing the method’s applicability for the quantification of the three disaccharides.
Fig. 3. Optimization of LC conditions for lactulose analysis (2)
| Sample | 5 mg/mL each in H2O/CH3CN = 1/1, 5 µL |
| Column | Shodex HILICpak VG-50 4E |
| Eluent | H2O/CH3CN/CH3OH = 5/75/20 |
| Flow rate | 1.0 mL/min |
| Detector | RI |
| Column temp. | 40 °C |
Good linearity was obtained for lactulose, epilactose, and lactose calibration curves, with R2 values of 0.9995, 0.9994, and 0.9991, respectively.
To examine its feasibility for analyzing real samples, the method was applied to a commercial probiotic milk beverage that claims to contain lactulose. As sample pretreatment, 1 mL of the probiotic milk beverage was taken and 1 mL of acetonitrile was added. The mixture was centrifuged at 890 g for 10 minutes, and then the supernatant was filtered with a 0.45-µm membrane filter. The obtained solution was used for the analysis.
Lactulose was detected in the two test samples analyzed, and it was well separated from other saccharides. This demonstrated the effectiveness of the VG-50 for the analysis of lactulose in milk beverages.
Fig. 4. Analysis of lactulose in probiotic milk beverages
2. Analysis of Rare Sugars
Rare sugars slow down the blood sugar level elevation after a meal and inhibit the accumulation of visceral fat. The use of rare sugars in commercial food products has recently been increasing. Moreover, as rare sugars are known to provide various bioactive effects, their potential applications in medicinal products, functional foods, and cosmetics have been studied.
In this work, a method was developed for separating six rare sugars, fulfilling the International Institute of Rare Sugar Research and Education’s definition of rare sugars: L-ribose, D-psicose, xylitol, D-tagatose, D-allose, and L-glucose.
First, similar to the approach used for lactulose analysis, a water/acetonitrile eluent was used for the separation of six rare sugars. Increase of acetonitrile ratio again lowered the S/N, and the use of water/acetonitrile eluent could not provide complete separation of all six rare sugars.
Fig. 5. Optimization of LC conditions for rare sugar analysis (1)
| Sample | 5 mg/mL each in H2O/CH3CN = 1/1, 5 µL |
| Column | Shodex HILICpak VG-50 4E |
| Eluent | H2O/CH3CN = 20/80 or 15/85 |
| Flow rate | 1.0 mL/min |
| Detector | RI |
| Column temp. | 40 °C |
Next, an eluent containing water, acetonitrile, and methanol was tested. The addition of methanol to the eluent effectively separated the six rare sugars. Good linearity was obtained for the calibration curves of all rare sugars, indicating its feasibility for the quantification of those sugars.
Fig. 6. Optimization of LC conditions for rare sugar analysis (2)
| Sample | 5 mg/mL each in H2O/CH3CN = 1/1, 5 µL |
| Column | Shodex HILICpak VG-50 4E |
| Eluent | H2O/CH3CN/CH3OH = 5/75/20 |
| Flow rate | 1.0 mL/min |
| Detector | RI |
| Column temp. | 40 °C |
To examine its feasibility for analyzing real samples, the method was applied to a commercial syrup containing rare sugars. As sample pretreatment, 0.1 g of the syrup was taken into a test tube and 2.5 mL of ultra-pure water was added. The test tube was capped and manually shaken to dissolve the syrup. After the syrup was completely dissolved, 2.5 mL of acetonitrile was added. The tube was shaken again to mix the solutions and the mixture was filtered with a 0.45-µm membrane filter. The obtained solution was used for the analysis.
By comparing the chromatograms of the syrup and the standards, the syrup most likely contained D-psicose and D-allose. They were well separated from other saccharides. A peak was also detected at L-glucose’s position, but because D-glucose also has an identical retention time, its D or L configuration was not identified in this analytical condition.
Fig. 7. Analysis of rare sugar syrup
3. Analysis of Ethyl α-D-glucoside
Ethyl α-D-glucoside is an umami component specifically found in sake, Japanese rice wines. Recent studies show that it has a moisturizing effect, and the effects have been focused upon for cosmetic uses. In this study, a method was developed to simultaneously analyze ethyl α-D-glucoside and several saccharides generally present in sake.
A mixture containing four saccharides and ethyl α-D-glucoside was used as a standard. An eluent containing water/acetonitrile = 20/80 effectively separated the target compounds. Good linearity was obtained for all analytes, showing the method’s feasibility for the quantification of saccharides and ethyl α-D-glucoside.
Fig. 8. Optimization of LC conditions for ethyl α-D-glucoside analysis
| Sample | 1 mg/mL each in H2O/CH3CN = 1/1, 10 µL |
| Column | Shodex HILICpak VG-50 4E |
| Eluent | H2O/CH3CN = 20/80 |
| Flow rate | 1.0 mL/min |
| Detector | RI |
| Column temp. | 40 °C |
To examine its feasibility for analyzing real samples, this method was applied to a commercial sake. The sample was prepared as follows: 1 mL of sake was taken into a test tube and 1 mL of acetonitrile was added. The test tube was capped and manually shaken to mix the solutions, and then the mixture was filtered with a 0.45-µm membrane filter. The obtained solution was used for the analysis.
By comparing the chromatograms of the samples and the standards, ethyl α-D-glucoside and glucose were found in the sake. An unidentified peak was found after the ethyl α-D-glucoside peak. The LC/MS result showed that the unidentified compound consisted of two components having molecular weights of 138 and 252.
Fig. 9. Analysis of ethyl α-D-glucoside in sake
Conclusions
This article demonstrated the effectiveness of Shodex HILICpak VG-50 4E with its HILIC mode for the analysis of various functional sugars in foods. The eluent typically used in HILIC mode is a mixture of water and acetonitrile. However, the use of a mixture containing water, acetonitrile, and methanol improved the separation efficiencies of the VG-50 4E for some applications. The developed method was also proven effective for analyzing functional sugars in real food products.
Reference
Shodex Technical Article No. 3: LC/MS Analysis of Various Hydrophilic Compounds Using HILIC Mode and Alkaline Eluent. The article introduces an LC/MS analysis of nutritional components in a commercial energy drink using the HILICpak VG-50 2D column. The polymer-based packing materials allowed the use of alkaline eluent. The nutritional components included were saccharides, amino acids, caffeine, and water-soluble vitamins. All components were successfully and simultaneously analyzed using the method. Please read Technical Article No. 3 for details.