2018 Volume 24 Issue 3 Pages 501-508
The aim of this study was to develop an analytical method for the discrimination of reducing and non-reducing monosaccharides including rare sugars on a prototype hydrophilic interaction liquid chromatography (HILIC) column. The HILIC column was composed of a glycidyl methacrylate-ethylene glycol dimethacrylate copolymer attached to 8 wt% polyethyleneimine. Seven monosaccharides (reducing aldoses: D-xylose, D-allose, and D-glucose; non-reducing ketoses: D-fructose, D-psicose, D-sorbose, and D-tagatose) were targeted. In individual HPLC analysis of both aldoses and ketoses, elution with 90 v/v% acetonitrile permitted successful detection and separation only for the non-reducing ketoses. Besides, elution of a mixture of ketoses and aldoses with 85 v/v% acetonitrile containing 5 mmol/L sodium 1-octanesulfonate (OS) (pH 4.8) enabled the simultaneous separation of all seven monosaccharides within 40 min. The polyethyleneimine-attached HILIC column allowed discriminant HPLC analysis for non-reducing ketoses using elution with 90 v/v% acetonitrile, whereas elution with 85 v/v% acetonitrile containing 5 mmol/L OS simultaneously detected both ketoses and aldoses.
Rare sugars are a group of monosaccharides having an extremely low natural abundance. Purported effective health benefits of their intake include anti-oxidant (Hossain et al., 2015a; Sun et al., 2007), anti-hyperlipidemic, and anti-hyperglycemic effects (Hossain et al., 2015b; Matsuo and Izumori, 2004). Long-term intake of 5% D-psicose in water by an Otsuka Long-Evans Tokushima Fatty (OLETF) rat model of type 2 diabetes improved the impaired insulin sensitivity or glucose tolerance (Hossain et al., 2011). Therefore, interest has grown in the preventive effect of rare sugars on lifestyle-related diseases.
Hindering this interest, analytical evaluation of rare sugars or monosaccharides for food control is difficult owing to the compounds' similar chemical and structural features, low ionization efficiency in mass spectrometry (Hermes et al., 2016), and poor retention on reversed phase-high performance-liquid chromatography (RP-HPLC) columns (Young et al., 2016). To address these issues, several chemical derivatization techniques using anthranilic acid and 1-pheny-3-methyl-5-pyrazolone have been proposed for the successful HPLC-ultraviolet detection of common monosaccharides (Stepan and Staudacher, 2011). Meyer et al. (2001) also reported the enhanced HPLC-fluorescence detection of sugars at levels >50 ng/mL using p-aminobenzoic acid-derivatization. Despite the development of highly sensitive HPLC assays, conventional refractive index (RI)-HPLC using a hydrophilic interaction liquid chromatography (HILIC) column is still commonly used for sugar assays because of its convenience and lack of requirements for high sensitive detection in natural foodstuffs.
The selective RI-HPLC assay of a rare sugar, D-psicose, in various food products has been reported using a common HILIC column packed with amino group-induced resins (Oshima and Kimura, 2006). However, there have been no reports on the discriminant and simultaneous separation of typical rare sugars, and quaternary imine groups in the polyethyleneimine-attached resin of the prototype column were expected to interact with reducing aldoses rather than common HILIC columns, allowing a discriminant RI-HPLC analysis of non-reducing and reducing monosaccharides. In this work, a newly developed HILIC column packed with a polyethyleneimine-attached resin was used to gain insights into the separation characteristics of the prototype column for rare sugar assays.
Materials D-Glucose, D-xylose, tetrahydrofuran (THF), hexamethylenediamine (HMD), and sodium 1-octanesulfonate (OS) were purchased from Nacalai Tesque Inc. (Kyoto, Japan). D-Fructose was purchased from Wako Pure Chemical Ind. (Osaka, Japan). D-Psicose, D-sorbose, and sodium 1-propanesulfonate (PS) were purchased from Tokyo Chemical Ind. (Tokyo, Japan). D-Allose and D-tagatose were obtained from Sigma-Aldrich (St. Louis, MO, USA). Pyridine was obtained from Hayashi Pure Chemical Ind. (Osaka, Japan). Acetonitrile and N,N-dimethylformamide (DMF) were purchased from Kanto Chemical Co. (Tokyo, Japan). Deionized water was prepared using a Milli Q system (Millipore, Tokyo, Japan).
HPLC analysis HPLC analyses used an LC-10AD system (Shimadzu, Kyoto, Japan) connected with an RI-930 detector (JASCO, Tokyo, Japan). The prototype HILIC column (4.6 mm I.D × 150 mm, 5 µm) was a product of Mitsubishi Chemical Co. (Tokyo, Japan). The column was packed with glycidyl methacrylate-ethylene glycol dimethacrylate copolymer attached to 8 wt% polyethyleneimine.
Chromatography on the HILIC column was carried out at 40°C using an isocratic elution mode. Elution conditions of flow rate and solvents were investigated and are described below. The concentration of each monosaccharide dissolved in deionized water was 1.0 wt%. Sample solution was injected in the RI-HPLC system with an injection volume of 10 µL. RI detection was performed in the positive signal mode at reference off; detection range, 4 × 10−5 relative index unit (RIU); temperature, 40°C. The retention of analyte on the column was calculated as a logarithmic retention factor (log k) (log k = log [(t − t0)/t0], where t is the retention time of analyte and t0 estimated by injection-shock peak is the dead time). Resolution (Rs) value was calculated as Rs = (tR2 − tR1)/0.5 (W1 + W2), where tR1, tR2 represents the retention time of each peak (tR2 > tR1) and W1, W2 is a peak width of each peak.
Effect of acetonitrile concentration on the retention of D-fructose Prior to examining the elution characteristics of the prototype HILIC column for seven monosaccharides, including five rare sugars (reducing aldoses: D-glucose, D-allose, and D-xylose; non-reducing ketoses: D-fructose, D-psicose, D-sorbose, and D-tagatose), the effect of acetonitrile concentration on the retention of D-fructose (a typical non-reducing ketose as analyte) on the prototype HILIC column was investigated. The preliminary experiment provided insight into the elution characteristics of the polyethyleneimine-attached glycidyl methacrylate-ethylene glycol dimethacrylate copolymer resin. As shown in Fig. 1, higher retention of D-fructose on the column was observed with increasing acetonitrile concentrations of 70 v/v% (log k: 0.07), 75 v/v% (log k: 0.18), 85 v/v% (log k: 0.71), and 90 v/v% (log k: 0.99) at a flow rate of 0.75 mL/min. The peak shapes broadened with increasing acetonitrile concentrations. There was no D-fructose peak at 95 v/v% acetonitrile owing to its high retention or broadened peak on the column. The elution behavior in different solvent concentrations on the prototype column agreed well with the typical HILIC elution, as the retention of analyte increases with hydrophilicity (Eksborg et al., 1973). Hence, the elution on the prototype polyethyleneimine-attached polymer resin column may depend mainly on HILIC characteristics.
HPLC elution profiles of D-fructose on the polyethyleneimine-attached HILIC column. RI-HPLC detection of 1.0 wt% D-fructose was performed on the prototype HILIC column packed with 8 wt% polyethyleneimine-attached copolymer resin (4.6 mm × 150 mm, 5 µm), with an isocratic elution using 70, 75, 85, 90, or 95 v/v% acetonitrile at a flow rate of 0.75 mL/min (40°C). The logarithmic retention factor (log k) of D-fructose was calculated by log k = log [(t − t0)/t0], where t is the retention time of analyte and t0 is the dead time. * indicates t0.
Elution behavior of reducing and non-reducing monosaccharides Elution with 90 v/v% acetonitrile at 0.75 mL/min, at which the highest retention of D-fructose was observed (Fig. 1), was investigated further to clarify the elution characteristics of three reducing and four non-reducing monosaccharides on the prototype HILIC column. Only non-reducing ketoses (D-fructose, D-psicose, D-sorbose, and D-tagatose) were detected by RI-HPLC on the HILIC column. The three reducing aldoses (D-glucose, D-allose, and D-xylose) were not detected by RI (Fig. 2). As expected from the individual log k values of 0.91, 0.67, 1.00, and 0.89 for D-fructose, D-psicose, D-sorbose, and D-tagatose, respectively (Fig. 2), an injection of their mixture onto the HILIC column resulted in an adequate separation using 90 v/v% acetonitrile (as compared to 80 v/v% and 85 v/v% acetonitrile) (Fig. 3A). To optimize the RI-HPLC separation of the four non-reducing ketoses on the prototype HILIC column, the effect of flow rate (0.75, 0.8, 0.85, and 0.9 mL/min) on their separation was also examined. As shown in Fig. 3B, a higher Rs value (Rs: D-psicose/D-tagatose: 7.51; D-tagatose/D-fructose: 0.91; D-fructose/D-sorbose: 1.81) was obtained at a flow rate of 0.9 mL/min compared to that at other flow rates (e.g., at 0.75 mL/min in Fig. 3A, Rs: D-psicose/D-tagatose: 3.73; D-tagatose/D-fructose: 0.44; D-fructose/D-sorbose: 1.19). Therefore, the prototype HILIC column could provide sufficient separation and detection of only non-reducing ketoses using elution with 90 v/v% acetonitrile at 0.9 mL/min and 40°C. A commercially available HILIC column with polyamine groups failed to achieve the discriminant detection of non-reducing ketoses (Fig. S1), indicating the advantage of polyethyleneimine-attached HILIC column for selective ketose detection.
HPLC elution profiles of reducing and non-reducing monosaccharides on the polyethyleneimine-attached HILIC column. Individual RI-HPLC detection of each 1.0 wt% monosaccharide was performed on the column with 90 v/v% acetonitrile elution at 0.75 mL/min at 40°C. Monosaccharides (reducing aldose: D-glucose, D-allose, D-xylose; non-reducing ketose: D-fructose, D-psicose, D-sorbose, D-tagarose) were individually injected (10 µL) to the RI-HPLC. A number in parentheses indicates the log k of each monosaccharide. * indicates t0.
HPLC elution profiles of a mixture of reducing aldoses or a mixture of non-reducing ketoses on the polyethyleneimine-attached HILIC column (A). A mixture of each 1.0 wt% reducing aldose (D-glucose, D-allose, D-xylose) or a mixture of each 1.0 wt% non-reducing ketose (D-fructose, D-psicose, D-sorbose, D-tagarose) was injected to the RI-HPLC eluted with 80, 85, or 90 v/v% acetonitrile at a flow rate of 0.75 mL/min and a temperature of 40°C. For the separation of a mixture of non-reducing ketoses on the HILIC column, the effect of flow rate (0.8, 0.85, or 0.9 mL/min) was also examined with an elution using 90 v/v% acetonitrile at 40°C (B).
HPLC chromatograms of a mixture of reducing aldoses or non-reducing ketoses on either polyethyleneimine-attached or polyamine-attached commercially available HILIC column.
Detection of reducing aldoses It is reported that an amino group-induced HILIC column may often suffer from the formation of the Schiff-base with aldoses (Young et al., 2016). In order to detect the three reducing aldoses (D-glucose, D-allose, and D-xylose) on the HILIC column, the effect of additives (0.1 wt% pyridine, 0.1 wt% THF, 0.1 wt% DMF, 0.1 wt% HMD, 1 mmol/L PS, and 1 mmol/L OS) to the elution with 80 v/v% acetonitrile at 0.75 mL/min was examined (Fig. 4). The conditions reflected the observation of RI peaks of reducing aldoses with low intensity at 80 v/v% acetonitrile elution (Fig. 3A). Moreover, the six cationic and anionic additives used in this study could reduce the interaction of the analyte with the polyethyleneimine moiety of the prototype HILIC column by cationic ion pairing and suppress the formation of the Schiff-base with the moiety due to the anionic ion pairing. However, cationic ion-pair reagents (pyridine, THF, DMF, and HMD) did not show any significant RI-detection of the three targeted aldoses, suggesting that these reagents had less ability to reduce the strong interaction of aldoses with the HILIC column (Fig. 4). In contrast, significant improvement of RI-detection and retention of aldoses was evident on the column using the PS and OS anionic ion-pair reagents (pH 4.8, adjusted with 5 mmol/L NaH2PO4), both are potent ion-pair reagents for solid phase extraction (Carson, 2000). This suggested that anionic reagents weakened the strong retention of aldoses with the column by their counter-ionic effect. The improvement of Rs by OS was markedly better than by PS on the polyethyleneimine-attached HILIC column (Rs of PS: D-xylose/D-allose: 1.00, D-allose/D-glucose: 1.57; Rs of OS: D-xylose/D-allose: 1.63, D-allose/D-glucose: 1.37). The OS results agreed with the previously described improvement of HPLC separation of nucleoside reverse transcriptase inhibitors in the presence of 8 mmol/L OS (Fan and Stewart, 2002). The solution pH might affect the elution characteristics of ion-pair reagents (Brons and Olieman, 1983). The effect of pH of the OS solution on the retention behavior of aldoses will be discussed subsequently.
Effect of ion-pair additives on the detection and separation of reducing ketoses on the polyethyleneimine-attached HILIC column. A mixture of 1.0 wt% reducing aldose (D-glucose, D-allose, D-xylose) was injected to RI-HPLC eluted with 80 v/v% acetonitrile elution containing ion-pair reagents at 0.75 mL/min at 40°C. Ion-pair reagents used in this experiment were 0.1 wt% pyridine, 0.1 wt% THF, 0.1 wt% DMF, 0.1 wt% HMD, 1 mmol/L PS (pH 4.8), and 1 mmol/L OS (pH 4.8).
Simultaneous separation of reducing and non-reducing monosaccharides The eluent of 80 v/v% acetonitrile containing 1 mmol/L OS (pH 4.8) enabled successful detection and separation of three reducing aldoses on the prototype HILIC column (Fig. 4). However, the monosaccharides (i.e., a mixture of three reducing aldoses and four non-reducing ketoses) were not individually separated at the elution condition (Fig. 5). Thus, further experiments on simultaneous separation of the mixture on the prototype HILIC column (0.75 mL/min, 40°C) examined acetonitrile and OS concentrations and pH of the solution. Increasing concentrations of acetonitrile (70 v/v% to 85 v/v%) greatly improved the OS-aided separation of the seven monosaccharides on the polyethyleneimine-attached HILIC column, while a simultaneous separation of each analyte in the mixture was not fully achieved at 85 v/v% acetonitrile containing 1 mmol/L OS (pH 4.8) (Fig. 5). Increasing the OS concentration to 5 mmol/L resulted in complete separation and RI detection of all seven monosaccharides. Owing to the poor solubility of 5 mmol/L OS into >90 v/v% acetonitrile solution, we concluded that the elution composed of 85 v/v% acetonitrile containing 5 mmol/L (pH 4.8) was an optimal separation condition for non-reducing and reducing monosaccharides when the prototype HILIC column was used. Fig. 5 also shows that at the elution condition of 90 v/v% acetonitrile without additives, the HILIC column detected only the four non-reducing monosaccharides in the mixture of reducing and non-reducing monosaccharides. The results agreed with the results in Fig. 3. In another experiment, the effect of pH on the separation of the mixture under optimal elution conditions was examined. At higher pH values of 7.5 and 9.5 (adjusted with NaOH) each separated peak at pH 4.8 was broadened and disappeared (Fig. 6). The ability of ion-pair reagents is known to vary with pH (Bidlingmeyer et al., 1979). Thus, the cationic status of the (poly) ethyleneimine group at pH 4.8 (pKa 7.0; Suh et al., 1994) may enhance the ion-pair interaction with anionic OS to prevent a strong reaction of the reducing aldoses on the polyethyleneimine-attached HILIC column through the Schiff-base formation.
HPLC elution profiles of a mixture of reducing aldoses and non-reducing ketoses on the polyethyleneimine-attached HILIC column. Each 1.0 wt% reducing aldose (D-glucose, D-allose, D-xylose) and non-reducing ketose (D-fructose, D-psicose, D-sorbose, D-tagarose) were mixed and injected to the RI-HPLC at 0.75 mL/min at 40°C. Elution experiments were performed at each isocratic elution condition of 70, 80, or 85 v/v% acetonitrile containing 1 mmol/L OS (pH 4.8), or 85 v/v% acetonitrile containing 5 mmol/L OS (pH 4.8). Elution with 90 v/v% acetonitrile without additives was also performed for the separation of the mixture.
The influence of pH on the separation of a mixture of four non-reducing ketoses and three reducing aldoses by the polyethyleneimine-attached prototype HILIC column. The HPLC chromatograms of a mixture of seven monosaccharides (1.0 wt% D-psicose, 1.0 wt% D-xylose, 1.0 wt% D-fructose, 1.0 wt% D-tagatose, 1.0 wt% D-allose, 1.0 wt% D-sorbose, and 1.0 wt% D-glucose) were obtained using the following conditions: 5 mmol/L OS containing 85 v/v% acetonitrile adjusted to pH 4.8 by 5 mmol/L NaH2PO4, original pH 7.5, and adjusted to pH 9.6 by 0.1 mol/L NaOH at a flow rate of 0.75 mL/min. The chromatogram at pH 4.8 was the same as that at 85 v/v% acetonitrile/5 mmol/L OS shown in Fig. 5.
In this study, we demonstrate that a HILIC column with attached polyethyleneimine (8 wt%) enables the discriminant RI-HPLC analysis of non-reducing ketoses in a monosaccharide mixture including five rare sugars, using elution with 90 v/v% acetonitrile at a flow rate at 0.9 mL/min at 40°C without any derivatization. The lack of detection of reducing aldoses was due to the Schiff-base formation of the reducing moiety in aldoses with the quaternary imine group in the polyethyleneimine-attached resin, since the addition of the PS or OS anionic ion-pair reagent to acetonitrile elution allowed the significant detection of reducing aldoses. Aiming at the simultaneous detection and separation of reducing and non-reducing monosaccharides, elution with 85 v/v% acetonitrile containing 5 mmol/L OS (pH 4.8) at 0.75 mL/min was considered optimal for the polyethyleneimine-attached HILIC column. Thus, the prototype polyethyleneimine-attached HILIC column allows the simultaneous analysis of reducing and non-reducing monosaccharides, or discriminant analysis of non-reducing monosaccharides with the aid of an anionic ion-pair reagent. Thus, the prototype polyethyleneimine-attached HILIC column must be profitable for convenient discriminant analysis of dairy monosaccharides including rare sugars in the coming future.
Acknowledgments The authors thank Ms. Tomomi Saiki, Ms. Jocelyn R. Sato, and Ms. Kaori Miyazaki at Kyushu University for their technical support on HPLC experiments.
