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Simultaneous chromatographic separation of enantiomers, anomers and structural isomers of some biologically relevant monosaccharides
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Journal of Chromatography A, 1188 (2008) 34–42
Simultaneous chromatographic separation of enantiomers, anomers and structural isomers of some biologically relevant monosaccharides Jo˜ao F. Lopes a , Elvira M.S.M. Gaspar b,∗ a
Department of Chemistry, Faculty of Science and Technology, New University of Lisbon, Quinta da Torre, 2825-114 Caparica, Portugal b Department of Chemistry, CQFB-Requimte, Faculty of Science and Technology, New University of Lisbon, Quinta da Torre, 2825-114 Caparica, Portugal Available online 15 December 2007
Abstract A one-step chiral high-performance liquid chromatography (HPLC) method was developed to separate anomers and enantiomers of some carbohydrates—glucose, fructose, arabinose, ribose, fucose, mannose, lyxose and xylose, using a Chiralpak AD-H column. The method allows the carbohydrate identification and determination of the absolute configuration (d or l) and simultaneously also determine the configuration of the anomeric center (␣ or ) of the monosaccharide. The method was applied to matrices involved in food chain and human health, as part of a general strategy for the structure determination of their constituents, due to the extreme importance of sugars since they are involved in food authenticity and nutritional characteristics and the biological role depends on the enantiomer and the anomeric form of a given monosaccharide. © 2007 Elsevier B.V. All rights reserved. Keywords: Monosaccharide; Glucose; Fructose; Arabinose; Ribose; Fucose; Mannose; Lyxose and xylose; HPLC; Carbohydrate analysis; Direct chromatographic stereodifferentiation of sugars; Direct anomeric separation of monosaccharides; Enantioselective chromatography of monosaccharides; Chiralpak AD-H
1. Introduction Carbohydrates are the most abundant biological molecules, and are widely distributed in nature. They can be isolated from plants, animals, bacteria and fungi as molecules of low molecular weight, oligosaccharides or as polymers. They are also building units of natural products, complex carbohydrates, molecules in which carbohydrates appear covalently bound to proteins, lipids or aglycons (alkaloids, steroids, etc.). Knowledge of the qualitative and quantitative distribution of sugars in materials, especially those related with food chain and human health, is extremely important because carbohydrates are one of the main food constituents and they are involved in food authenticity, nutritional characteristics (as flavour and sensory) and they are sometimes also responsible for some biological activity: for example, they are constituents of natural antibiotics (like streptomycin and puromycin) [1]. Carbohydrates and their derivatives are also important in all metabolisms of living organisms, in the storage and transport of energy, and are present as structural components.
∗
Corresponding author. E-mail address: [email protected] (E.M.S.M. Gaspar).
0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.12.016
Chemically monosaccharides are chiral polyhydroxy carbonyl compounds which often exist in cyclic hemiacetal form. Most of the cyclic isomers can be isolated in pure form. However, acyclic isomers have also been detected, but only as very minor components in solution, where they coexist in equilibrium with the cyclic forms (Fig. 1). Following the Rosanoff convention, the letter d and l at the beginning of the name indicate the absolute configuration of the chiral carbon most remote from the carbonyl group (C-5 in glucose, the most abundant monosaccharide). d- and l- isomers constitute a pair of enantiomers. The anomeric center is responsible for the diasterioisomeric ␣- and -anomers. Anomers are epimers that differ only in the configuration at the anomeric center. The ␣-isomer is the one that has the anomeric hydroxyl group (1-OH group in aldoses, 2-OH group in ketoses) on the same side as the hydroxyl group that determines the d or l configuration (Fig. 2). In the -isomer, both hydroxyl groups are on opposite sides. The magnitude of the anomeric effect depends, among other factors, on the electronegativity of the anomeric substituent. The more electronegative the substituent, the greater the tendency for its axial disposition. The configuration of the C-2 substituent also affects the magnitude of the anomeric effect: 2hydroxyl groups oriented axially increase the magnitude of the anomeric effect with respect to equatorial hydroxyl groups. The
J.F. Lopes, E.M.S.M. Gaspar / J. Chromatogr. A 1188 (2008) 34–42
Fig. 1. Furanose, open-chain and pyranose forms of glucose.
anomeric effect is larger in solvents of low dielectric constant [1,2]. There seems to be a correlation between the natural abundance of monosaccharides and their relative stabilities. dglucose has the most stable conformational ring and is the most abundant of the monosaccharides (␣-d-pyranose ([␣] = +113◦ , x = 0.36) and (-d-pyranose ([␣] = +19◦ , x = 0.64)) [1]. Basically it was postulated (reducing sugars) that an equatorial –OH group at the anomeric site produces a repulsive dipole–dipole interaction with the ring oxygen atom—the ␣-anomer is favoured. Since the effect is of an electrostatic nature, it is assumed to vary inversely with the dielectric permittivity of the solvent. In water the effect is small, the -anomers usually dominate [2]. Some aldohexoses exhibit complex mutarotation, where more than one tautomer is involved. The observations have shown that an axial –OH group on C(4) of a pyranose leads to appreciable proportions of furanose forms. The mutarotation of -d-fructopyranose in water produces a mixture of ␣- and -fructopyranose [2]. The analysis of underivatized carbohydrates is generally carried out by high-performance liquid chromatography (HPLC)
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[3–8]. Resolution difficulties arise from the myriad of different carbohydrate structures available. The mostly used chromatographic system employs amine-bonded silica gel column with water–acetonitrile as the eluent [8]. Recently, there was a report of a system employing a cation-exchange column and a mobile phase containing boric acid as additive to enhance the separation by complexation to vicinal cis hydroxyl groups on the sugar molecules [4]. Chromatographic separations of anomers and stereoisomers have been done [9,10]. However, they could only be achieved after derivatization ((+)-MNB derivatives of per-O-methyl d,l-monosaccharides) employing a Develosil column with n-hexane–AcOEt–THF as the eluent at 22 ◦ C [9] or employing an Aminex column in the Ca2+ -form in the ligandexchange mode with water as the eluent [10]. The latter method achieved enantiomeric and diasteriomeric separation. The present paper reports, for the first time, the enantioselective separation of some biologically relevant monosaccharides (arabinose, ribose, mannose, fucose, xylose, lyxose, glucose and fructose) and simultaneously the anomeric forms ␣- and -of each d- and l-enantiomer using chiral HPLC methodology. It represents an improved simple method to determine the absolute configuration of carbohydrate moieties and their prevalent anomeric forms present in different matrices, especially those involved in the food chain. 2. Experimental 2.1. Instrumentation and chromatographic conditions High-performance liquid chromatography separations were performed with Waters instruments, 501 and 600 pumps, equip-
Fig. 2. Enantiomeric and anomeric predominant pyranose forms of glucose. -d-glucose has all OH groups in axial position.
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J.F. Lopes, E.M.S.M. Gaspar / J. Chromatogr. A 1188 (2008) 34–42
Table 1 Retention times for monosaccharide analysis in a carbohydrate analysis column at 25 ◦ C, at different flow rates Carbohydrate
d,l-Ribose d,l-Lyxose d,l-Xylose d,l-Arabinose d,l-Fucoseb d,l-Fructose d,l-Mannose d,l-Glucose
Retention time (min)a 0.5 mL min−1
1 mL min−1
2 mL min−1
12.5 14.3 14.6 17.2 17.4 20.0 21.3 22.5
6.3 7.2 7.4 8.8 8.6 10.2 11.0 11.8
3.3 3.7 3.8 4.5 4.0 5.2 5.6 6.2
a Mobile phase: water–acetonitrile (1:9, v/v); t = 8.1, 4.0 and 2.0 at 0.5, 1 m and 2 mL min−1 . b Broad or split peak.
ped with a Rheodine injector and connected to refractive index (RI) (Waters, model 2410) and diode array detection (DAD, model 996) and equipped with a Chiralpak AD-H column (Daicel) 0.46 cm × 25 cm or a carbohydrate analysis column ˚ 10 m, 3.9 mm × 300 mm. Separations were (Waters) 125 A, performed at room temperature (25 ◦ C), after performing studies at temperature range 15–40 ◦ C; column oven (Jet Stream, VDS Optilab, Berlin, Germany). For separation of carbohydrates, isocratic elution using as mobile phase water–acetonitrile (1:9, v/v) mixture was used. The flow rates were 0.5, 1 and 2 mL min−1 (Table 1). Stereoselective HPLC analysis of carbohydrates isomers was done using a Chiralpack AD-H column and the mobile phase for isocratic elution was a mixture of hexane–ethanol–TFA ((7:3):0.1, v/v); the flow was 0.5 mL min−1 (Table 2). 2.2. Chemicals and samples All HPLC solvents were obtained from Aldrich (Germany). Deionized and purified water (Millipore unit, USA) was used. All chemicals used in this study were of analytical-reagent grade. d-fructose, l- and d-arabinose, l- and d-lyxose, l- and d-fucose, d- and l-mannose, l-xylose, l- and d-glucose, ␣-d-glucose, dand l-ribose were purchased from Aldrich (Germany); d-xylose
was purchased from BDH Biochemicals (UK), d,l-arabinose from Fluka (Germany) and l-fructose from TCI Europe (Belgium). Standard solutions of each carbohydrate were prepared for each separation with a concentration of 100 mg L−1 , using vortex agitation and ultrasounds (5 min), due to low solubility of carbohydrates in the mobile phase for stereoselective separation. This is the unique drawback of this method. The samples: Portuguese honey (“Mel de Portugal”, M´ertola and “Mel Rosmaninho”, Serra da Malcata) and natural sweeteners (“Glucosine”, Diese, “Ac¸u´ car de frutos”, Provida and “Dieron”, Diese) were purchased in a Natural Products’ Pharmacy; Oporto wine (“Porto Ferreira”, 20 and 10 years old and “Ramos Pinto”, Tawny), were purchased in a local supermarket. All the samples were solved in the mobile phase mixture (according to separation) using vortex agitation and ultrasounds (5 min) and then filtered through a 0.45 m membrane filter. The standards and samples solutions were injected into the HPLC systems using a 50 L loop injector. 2.3. Chromatographic separations Each of the standard solutions was chromatographed on HPLC using a carbohydrate analysis column. The retention time for both enantiomers is the same (Table 1). Stereoselective HPLC analysis (Chiralpak AD-H column) of monosaccharide standards gave different retention times for each enantiomer (d and l) and also for the anomers (␣ and ) of each enantiomer (Table 2). As can be seen, it was possible to separate all the stereoisomers of each monosaccharide: the method allows enantiomeric and simultaneously the anomeric separation of the most stable conformational forms of each monosaccharide. The elution profile of glucose peaks was determined using standards. The elution order is -d-glucose, ␣d-glucose, -l-glucose and ␣-l-glucose respectively. For some of the other monosaccharides (mannose, ribose, lyxose) the elution order was deduced based on data described in the literature [10,11]. For xylose, arabinose and fucose, on the best of our knowledge, no data is available relative to anomeric equilibrium in (aqueous) solution. For fructose, according to the literature
Table 2 Retention times for monosaccharide separation in a Chiralpack AD-H column, at 25 ◦ C Carbohydrate
Arabinoseb Fructosec Fucoseb Glucose Lyxose Mannose Ribose Xyloseb a b c
Retention time (min)a l-Enantiomer
d-Enantiomer
␣/ anomer
␣/ anomer
23.0 (␣) 12.1 (furanose) 32.3 () 13.7 () 18.0 (␣) 12.9 (␣) 20.8 () 17.4 ()
35.9 () 19.8 (pyranose) 36.7 (␣) 14.3 (␣) 29.1 () 28.8 () 106.2 (␣) 19.3 (␣)
Mobile phase: hexane-ethanol-TFA ((7:3):0.1, v/v). Flow rate: 0.5 mL min−1 ; tm = 6.1 min. Anomeric elution profile based on relative OH configuration at C1 and C2. According to literature data [3].
27.7 (␣) 15.8 (furanose) 16.1 () 13.2 () 16.6 (␣) 12.5 (␣) 25.6 () 17.7 ()
31.6 () 19.5 (pyranose) 25.6 (␣) 13.6 (␣) 23.9 () 21.3 () 70.7 (␣) 20.9 (␣)
J.F. Lopes, E.M.S.M. Gaspar / J. Chromatogr. A 1188 (2008) 34–42
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Fig. 3. HPLC separation of lyxose at 25 ◦ C. Chromatogram A: (carbohydrate analysis column, flow rate: 0.5 mL min−1 ): (1) d,l-lyxose; chromatogram B (Chiralpak AD-H column, flow rate: 0.5 mL min−1 ); stereoselective analysis of the racemic d,l-lyxose: (2) ␣-d-lyxopyranose; (3) ␣-l-lyxopyranose; (4) -d-lyxopyranose; and (5) -l-lyxopyranose, respectively. Detection: refractive index.
Fig. 4. Chromatographic profiles of ␣- and -d-glucose at different temperatures: 15, 20, 25, 30, 35 and 40 ◦ C. Column: Chiralpak AD-H; eluent: hexane–ethanol–TFA ((7:3):0.1, v/v). Flow rate 0.5 mL min−1 , tm = 6.1 min; detection: refractive index.
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data [10], the retention times of the two main peaks of each enantiomer corresponds to the furanose and pyranose forms, respectively. 3. Results and discussion 3.1. Separation of glucose, fructose, arabinose, ribose, fucose, mannose, lyxose and xylose The standard solution of each enantiomer (d and l) of each monosaccharide, prepared as described in Section 2, was analyzed separately using both systems: achiral (carbohydrate analysis column) and chiral (Chiralpak AD-H column). The achiral separation showed only one peak for each enantiomer and allowed good separation for all analyzed monosaccharides, including the structural isomers and epimers; both enantiomers of each monosaccharide have the same retention time. No temperature effect was observed; so, room temperature (25 ◦ C) was chosen for separation. The results obtained are shown in Table 1. The chiral (Chiralpak) column allowed the separation of the diastereoisomeric pair (␣- and -anomers) for each enantiomer
d and l. Fig. 3 shows the separation of racemic d,l-lyxose: only one peak (1) in the achiral separation – chromatogram A and four peaks in the chiral analysis – chromatogram B, showing the separation of the main (four) isomeric forms (2,3,4,5). The sequence of elution on the chiral HPLC column for lyxose is 2 ␣-d-lyxopyranose, 3 ␣-l-lyxopyranose; 4 -d-lyxopyranose and 5 -l-lyxopyranose. The elution profiles of the saccharides depended on column temperature. The change was not uniform for all monosaccharides: for example, when the temperature was increased, the elution profile of lyxose became worse, while for glucose the best elution profile was achieved at 40 ◦ C (Figs. 4 and 5). At 25 ◦ C the monosaccharides tested showed reasonable elution profiles. Table 2 shows the retention times obtained by stereoselective HPLC analysis of the monosaccharides injected. The profile of each enantiomer of monosaccharides was checked, considering the ratios for anomers described in literature [2,11]: lyxose, ribose and mannose. It was observed that, using the chiral HPLC conditions mentioned above, the elution profiles are in good agreement with those obtained by Nishikawa et al.
Fig. 5. Chromatographic profiles of ␣- and -d,l-lyxose at different temperatures: 15, 20, 25, 30, 35 and 40 ◦ C. Column: Chiralpak AD-H; eluent: hexane–ethanol–TFA ((7:3):0.1, v/v). Flow rate 0.5 mL min−1 , tm = 6.1 min; detection: refractive index.
J.F. Lopes, E.M.S.M. Gaspar / J. Chromatogr. A 1188 (2008) 34–42
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Fig. 6. Stereoselective HPLC analysis of racemic mixture of d,l-lyxose (#) and d,l-mannose (*): (1) ␣-d-mannopyranose, (2) ␣-l-mannopyranose, (3) ␣-dlyxopyranose, (4) ␣-l-lyxopyranose, (5) -d-mannopyranose, (6) -d-lyxopyranose, (7) -l-lyxopyranose, (8) -l-mannopyranose and (9) minor form (probably furanose) of mannose. Flow rate: 0.5 mL min−1 ; tm = 6.1 min. Detection: refractive index.
[10], suggesting that the elution order in the Chiralpak column (using described parameters) may also be related with the vicinal OH configuration at positions C1 and C2 of the monosaccharide molecule enantiomer. Arabinose, fucose and xylose anomeric profiles were assigned based on this postulation. The profile of fructose was assigned based on the literature [2,10]. The method allowed excellent resolution of all monosaccharides that were tried. We obtained good separation for enantiomers of different monosaccharides (structural isomers) and good separation for the anomers of each enantiomer that has been analyzed (Fig. 6). The method reported here has also allowed a successful simultaneous stereodifferentiation of epimers (e.g. arabinose and ribose). For the first time a simple chromatographic method allows simultaneously enantiomeric and anomeric separation of several underivatized monosaccharides. The method was used with samples of Portuguese honey, Oporto wine and natural sugars marketed as nutritional supplements. The analysis made possible to assign unequivocally the absolute stereochemistry and prevalent anomeric forms of monosaccharides contained in the matrices. So, using the potentiality of this chiral HPLC technique, without derivatization and previous isolation, it was possible to determine the “normal” monosaccharide profiles of matrices. This is extremely important because carbohydrates are involved in food authenticity, nutritional characteristics and they are also responsible for some biological activity, since this depends on the ratios of isomeric (enantiomeric and anomeric) forms present.
3.2. Method performance The analytical method was evaluated to prove its applicability to the analysis of monosaccharides in Portuguese honey, Oporto wine and natural sugars marketed as nutritional supplements. Having the elution temperature determined (25 ◦ C), quality parameters of the stereoselective chromatographic method such as limits of detection (LODs) and quantitation (LOQs), linearity, repeatability, recovery, linearity range, precision and sensitivity were calculated [12]. Validation results are presented in Tables 3 and 4. 3.2.1. Recovery Recovery studies were performed in duplicate, by spiking the analyzed samples with two solutions of each monosaccharide with different concentration levels, 100 and 250 ppm (mg L−1 ). Quantification recoveries obtained for the target compounds ranged from 76 to 94%. Having in account the low solubility of carbohydrates in the mobile phase for stereoselective separation and the necessity of using samples in low concentration, the recoveries obtained were quite good, and therefore an acceptable quantitative estimation of the concentration of selected monosaccharides in samples could be obtained. 3.2.2. Linear dynamic range The linearity in the response was studied using matrixmatched calibration solutions prepared by spiking commercial products at seven concentration levels, ranging from 10, 20 or 30 to 750 ppm in the samples. The linear dynamic ranges,
Table 3 Analytical parameters obtained with the stereoselective HPLC method for the analysis of selected compounds in commercial products Carbohydrate
d-Fructofuranose d-Fructopyranose ␣ and -d-Glucose
Linearity Range (ppm)
Cal. Equation
R2
10–750 30–750 20–750
y = 93.248x + 214.59 y = 893.75x − 14463 y = 1148.3x + 63304
0.9974 0.9834 0.9949
LOD (ppm)
LOQ (ppm)
Repeatability (RSD, %), n = 5
Reproducibility (RSD, %), n = 5
0.7 5.1 2
2.3 16.9 6.2
7 3 3
6 4 5
RSD: relative standard deviation; LOQ: limit of quantification; LOD: limit of detection.
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Table 4 Recoveries of the optimized method for the analysis of selected commercial products Sample
Carbohydrate
Recovery (RSD, %) n = 5
Honey 1
d-Fructofuranose d-Fructopyranose ␣ and -d-Glucose
82 (8) 76 (6) 86 (4)
Honey 2
d-Fructofuranose d-Fructopyranose ␣ and -d-Glucose
89 (5) 82 (5) 85 (2)
Oporto wine 1
d-Fructofuranose d-Fructopyranose ␣ and -d-Glucose
92 (2) 94 (3) 89 (7)
Oporto wine 2
d-Fructofuranose d-Fructopyranose ␣ and -d-Glucose
88 (7) 88 (8) 87 (7)
RSD: relative standard deviation.
calibration equations and correlation coefficients (R2 ) obtained are shown in Table 3. Good linearity was observed in the studied range with R2 values higher than 0.98. 3.2.3. Sensitivity Limits of detection and quantitation were evaluated on the basis of the signal obtained with the analysis of unfortified samples (n = 5), taking into account the solubility of the target monosaccharides in the mobile phase. Values obtained ranged from 0.7 to 5.1 ppm and 2.3–16.9 ppm, respectively, values quite good for a method using RI detection. 3.2.4. Precision In order to evaluate the precision of the proposed method, repeatability and reproducibility were estimated. The repeata-
bility, expressed as percent relative standard deviation (RSD), varied between 3 and 7% for repeatability and 4 and 6% for the reproducibility. 3.3. Application of the method to real samples Adulterated or degraded honey may show a different sugar profile; so this method allows a quick and efficient test to determine the authenticity, quality or inadequate storage conditions of honeys. The typical monosaccharide profile of authentic Portuguese (good) honey is shown in Fig. 7. As expected, commercial Portuguese honey consists mostly of the monosaccharides glucose and fructose, containing more fructose than glucose (average ratio 1.2/1) [13]. The application of the method established the ratios of anomeric and tautomeric forms; results are summarized in Table 5. “Oporto wine” is a designation of controlled origin. It’s a Portuguese product recognized worldwide, economically very important to the country. Its monosaccharide profile has been established to detect fraudulent designations and adulteration; it is shown in Fig. 8. The results obtained are summarized in Table 5. As can be seen d-fructose and d-glucose are the main monosaccharide constituents. The prevalent forms were shown to be -d-glucose and d-fructopyranose. Sugar supplements/substitutes are used as alternative sweeteners by people who have metabolic disorders (e.g. diabetes) or under regimen (e.g. obesity). It is also used by athletes because of a need of higher energy. More expensive than the common sugar (mainly composed by sucrose, a disaccharide), they are easily subjected to adulteration or falsification. Natural monosaccharides like fructose, glucose and tagatose are the main substitutes recommended by physicians.
Fig. 7. Analysis of authentic Portuguese honey. Chromatogram A (carbohydrate analysis column, flow rate: 1.5 mL min−1 ; tm = 3 min): (1) fructose and (2) glucose; chromatogram B shows the stereoselective analysis: (3) glycerol; (4) -d-glucopyranose; (5) ␣-d-glucopyranose; (6) d-fructofuranose; and (7) d-fructopyranose. Flow rate: 0.5 mL min−1 . Detection: refractive index. Table 5 Average ratio of isomers present in authentic Oporto wine and Portuguese honey samples Monosaccharide
d-Glucose d-Fructose
Anomer or tautomer ratio
/␣ Pyranose/furanose
Samples Oporto wine (samples 1 to 9)
Portuguese honey (samples 1–4)
0.9–1.4 8.2–10.4
1.5–1.9 6.9–10.8
J.F. Lopes, E.M.S.M. Gaspar / J. Chromatogr. A 1188 (2008) 34–42
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Fig. 8. Authentic Oporto wine profile of monosaccharides. Chromatogram A (carbohydrate analysis column, flow rate: 1 mL min−1 ): (1) glycerol, (2) fructose and (3) glucose; stereoselective HPLC chromatogram (B): (4) -d-glucopyranose; (5) ␣-d-glucopyranose, (6) d-fructofuranose; and (7) d-fructopyranose. Flow rate: 0.5 mL min−1 . Detection: refractive index.
Fig. 9. Analysis of sugar substitute designated “Sugar from fruits”: (1) fructose (chromatogram A, carbohydrate analysis column): (2) d-fructofuranose; and (3) d-fructopyranose (chromatogram B, Chiralpak AD-H column). Flow rate: 0.5 mL min−1 . Detection: refractive index.
Fig. 10. Analysis of nutritional supplement “Glucosine”: (1) glucose (chromatogram A, carbohydrate analysis column); chromatogram B (Chiralpak AD-H column); (2) -d-glucopyranose and (3) ␣-d-glucopyranose. Flow rate: 0.5 mL min−1 . Detection: refractive index.
The method reported here made possible to determine the authenticity and nutritional characteristics of carbohydrates used as sweetener substitutes or nutritional supplements; Figs. 9 and 10 show the profiles of two of them. The results support the natural origin of these supplements. In fact, ␣-d-glucose is the most abundant natural form of this monosaccharide. d-fructose is the stereoisomer of natural origin. 4. Conclusions The stereodifferentiation of the enantiomers (d and l) and anomers (␣ and ) of some monosaccharides was carried out
by the use of a Chiralpak AD-H HPLC column. The one-step, no time consuming analysis of underivatized monosaccharides proved to be a simple, powerful, useful chromatographic method that allows the unequivocal determination of the absolute configuration and anomeric forms of sugar moieties present (even in trace amounts). The elution sequence was determined using standards (all enantiomers and one glucose anomer) and by comparison with literature data. The method is superior to others described in terms of the simplicity and stereoselectivity and allowed the determination of the absolute configuration and the anomeric prevalent forms of monosaccharide moieties contained in food chain matrices, without previous isolation. Authenticity and
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nutritional characteristics may be determined using the method; they are sometimes responsible for some anomalous biological activities/disorders. It has also potential to be applied to biological matrices and can be scaled-up. The method is patent pending. Acknowledgments The authors thank Eng. Jo˜ao Tiago and Mr. Pedro Marques for their technical informatics assistance. Special thanks to Professor Maria Margarida Gonc¸alves for her samples’ solubility assistance. Financial support from FCT – Portugal. References [1] M. Sznaidman, in: S.M. Hecht (Ed.), Bioorganic Chemistry Carbohydrates, Oxford University Press, Oxford, 1999, p. 1. [2] F. Franks, Pure Appl. Chem. 59 (1987) 1189 (and references cited therein).
[3] D. Gomis, D. Tamayo, J. Alonso, Anal. Chim. Acta 436 (2001) 173. [4] C. De Muynck, J. Beauprez, W. Soetaert, E. Vandamme, J. Chromatogr. A 1101 (2006) 115. [5] M. Little, J. Biochem. Biophys. Methods 11 (1985) 195. [6] F. Lamari, R. Kuhn, N. Karamanos, J. Chromatogr. B 793 (2003) 15. [7] A. Carmona, P. Borrachero, F. Cabrera-Escribano, M. Di´anez, M. Estrada, A. L´opez-Castro, R. Ojeda, M. G´omez-Guill´en, S. P´erez-Garrido, Tetrah. Asym. 10 (1999) 1751. [8] A. Clement, D. Young, C. Brecht, J. Liq. Chromatogr. 15 (1992) 805. [9] C. Bai, H. Ohrui, Y. Nishida, H. Meguro, Anal. Biochem. 246 (1997) 246. [10] T. Nishikawa, S. Suzuki, H. Kubo, H. Ohtani, J. Chromatogr. A 720 (1996) 167 (and references cited therein). [11] G.-J. Boons, in: G.-J. Boons (Ed.), Mono- and Oligosaccharides: Structure, Configuration and Conformation in Carbohydrate Chemistry, Blackie Academic & Professional, Thomson Science, London, 1998, p. 1. [12] J.C. Miller, J.N. Miller, Statistics for Analytical Chemistry, Ellis Horwood, Chichester, UK, 1988. [13] G. Rodr´ıguez, B. Ferrer, A. Ferrer, B. Rodr´ıguez, Food Chem. 84 (2004) 499 (and references cited therein).
Journal of Chromatography A, 1188 (2008) 34–42
Simultaneous chromatographic separation of enantiomers, anomers and structural isomers of some biologically relevant monosaccharides Jo˜ao F. Lopes a , Elvira M.S.M. Gaspar b,∗ a
Department of Chemistry, Faculty of Science and Technology, New University of Lisbon, Quinta da Torre, 2825-114 Caparica, Portugal b Department of Chemistry, CQFB-Requimte, Faculty of Science and Technology, New University of Lisbon, Quinta da Torre, 2825-114 Caparica, Portugal Available online 15 December 2007
Abstract A one-step chiral high-performance liquid chromatography (HPLC) method was developed to separate anomers and enantiomers of some carbohydrates—glucose, fructose, arabinose, ribose, fucose, mannose, lyxose and xylose, using a Chiralpak AD-H column. The method allows the carbohydrate identification and determination of the absolute configuration (d or l) and simultaneously also determine the configuration of the anomeric center (␣ or ) of the monosaccharide. The method was applied to matrices involved in food chain and human health, as part of a general strategy for the structure determination of their constituents, due to the extreme importance of sugars since they are involved in food authenticity and nutritional characteristics and the biological role depends on the enantiomer and the anomeric form of a given monosaccharide. © 2007 Elsevier B.V. All rights reserved. Keywords: Monosaccharide; Glucose; Fructose; Arabinose; Ribose; Fucose; Mannose; Lyxose and xylose; HPLC; Carbohydrate analysis; Direct chromatographic stereodifferentiation of sugars; Direct anomeric separation of monosaccharides; Enantioselective chromatography of monosaccharides; Chiralpak AD-H
1. Introduction Carbohydrates are the most abundant biological molecules, and are widely distributed in nature. They can be isolated from plants, animals, bacteria and fungi as molecules of low molecular weight, oligosaccharides or as polymers. They are also building units of natural products, complex carbohydrates, molecules in which carbohydrates appear covalently bound to proteins, lipids or aglycons (alkaloids, steroids, etc.). Knowledge of the qualitative and quantitative distribution of sugars in materials, especially those related with food chain and human health, is extremely important because carbohydrates are one of the main food constituents and they are involved in food authenticity, nutritional characteristics (as flavour and sensory) and they are sometimes also responsible for some biological activity: for example, they are constituents of natural antibiotics (like streptomycin and puromycin) [1]. Carbohydrates and their derivatives are also important in all metabolisms of living organisms, in the storage and transport of energy, and are present as structural components.
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Corresponding author. E-mail address: [email protected] (E.M.S.M. Gaspar).
0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.12.016
Chemically monosaccharides are chiral polyhydroxy carbonyl compounds which often exist in cyclic hemiacetal form. Most of the cyclic isomers can be isolated in pure form. However, acyclic isomers have also been detected, but only as very minor components in solution, where they coexist in equilibrium with the cyclic forms (Fig. 1). Following the Rosanoff convention, the letter d and l at the beginning of the name indicate the absolute configuration of the chiral carbon most remote from the carbonyl group (C-5 in glucose, the most abundant monosaccharide). d- and l- isomers constitute a pair of enantiomers. The anomeric center is responsible for the diasterioisomeric ␣- and -anomers. Anomers are epimers that differ only in the configuration at the anomeric center. The ␣-isomer is the one that has the anomeric hydroxyl group (1-OH group in aldoses, 2-OH group in ketoses) on the same side as the hydroxyl group that determines the d or l configuration (Fig. 2). In the -isomer, both hydroxyl groups are on opposite sides. The magnitude of the anomeric effect depends, among other factors, on the electronegativity of the anomeric substituent. The more electronegative the substituent, the greater the tendency for its axial disposition. The configuration of the C-2 substituent also affects the magnitude of the anomeric effect: 2hydroxyl groups oriented axially increase the magnitude of the anomeric effect with respect to equatorial hydroxyl groups. The
J.F. Lopes, E.M.S.M. Gaspar / J. Chromatogr. A 1188 (2008) 34–42
Fig. 1. Furanose, open-chain and pyranose forms of glucose.
anomeric effect is larger in solvents of low dielectric constant [1,2]. There seems to be a correlation between the natural abundance of monosaccharides and their relative stabilities. dglucose has the most stable conformational ring and is the most abundant of the monosaccharides (␣-d-pyranose ([␣] = +113◦ , x = 0.36) and (-d-pyranose ([␣] = +19◦ , x = 0.64)) [1]. Basically it was postulated (reducing sugars) that an equatorial –OH group at the anomeric site produces a repulsive dipole–dipole interaction with the ring oxygen atom—the ␣-anomer is favoured. Since the effect is of an electrostatic nature, it is assumed to vary inversely with the dielectric permittivity of the solvent. In water the effect is small, the -anomers usually dominate [2]. Some aldohexoses exhibit complex mutarotation, where more than one tautomer is involved. The observations have shown that an axial –OH group on C(4) of a pyranose leads to appreciable proportions of furanose forms. The mutarotation of -d-fructopyranose in water produces a mixture of ␣- and -fructopyranose [2]. The analysis of underivatized carbohydrates is generally carried out by high-performance liquid chromatography (HPLC)
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[3–8]. Resolution difficulties arise from the myriad of different carbohydrate structures available. The mostly used chromatographic system employs amine-bonded silica gel column with water–acetonitrile as the eluent [8]. Recently, there was a report of a system employing a cation-exchange column and a mobile phase containing boric acid as additive to enhance the separation by complexation to vicinal cis hydroxyl groups on the sugar molecules [4]. Chromatographic separations of anomers and stereoisomers have been done [9,10]. However, they could only be achieved after derivatization ((+)-MNB derivatives of per-O-methyl d,l-monosaccharides) employing a Develosil column with n-hexane–AcOEt–THF as the eluent at 22 ◦ C [9] or employing an Aminex column in the Ca2+ -form in the ligandexchange mode with water as the eluent [10]. The latter method achieved enantiomeric and diasteriomeric separation. The present paper reports, for the first time, the enantioselective separation of some biologically relevant monosaccharides (arabinose, ribose, mannose, fucose, xylose, lyxose, glucose and fructose) and simultaneously the anomeric forms ␣- and -of each d- and l-enantiomer using chiral HPLC methodology. It represents an improved simple method to determine the absolute configuration of carbohydrate moieties and their prevalent anomeric forms present in different matrices, especially those involved in the food chain. 2. Experimental 2.1. Instrumentation and chromatographic conditions High-performance liquid chromatography separations were performed with Waters instruments, 501 and 600 pumps, equip-
Fig. 2. Enantiomeric and anomeric predominant pyranose forms of glucose. -d-glucose has all OH groups in axial position.
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Table 1 Retention times for monosaccharide analysis in a carbohydrate analysis column at 25 ◦ C, at different flow rates Carbohydrate
d,l-Ribose d,l-Lyxose d,l-Xylose d,l-Arabinose d,l-Fucoseb d,l-Fructose d,l-Mannose d,l-Glucose
Retention time (min)a 0.5 mL min−1
1 mL min−1
2 mL min−1
12.5 14.3 14.6 17.2 17.4 20.0 21.3 22.5
6.3 7.2 7.4 8.8 8.6 10.2 11.0 11.8
3.3 3.7 3.8 4.5 4.0 5.2 5.6 6.2
a Mobile phase: water–acetonitrile (1:9, v/v); t = 8.1, 4.0 and 2.0 at 0.5, 1 m and 2 mL min−1 . b Broad or split peak.
ped with a Rheodine injector and connected to refractive index (RI) (Waters, model 2410) and diode array detection (DAD, model 996) and equipped with a Chiralpak AD-H column (Daicel) 0.46 cm × 25 cm or a carbohydrate analysis column ˚ 10 m, 3.9 mm × 300 mm. Separations were (Waters) 125 A, performed at room temperature (25 ◦ C), after performing studies at temperature range 15–40 ◦ C; column oven (Jet Stream, VDS Optilab, Berlin, Germany). For separation of carbohydrates, isocratic elution using as mobile phase water–acetonitrile (1:9, v/v) mixture was used. The flow rates were 0.5, 1 and 2 mL min−1 (Table 1). Stereoselective HPLC analysis of carbohydrates isomers was done using a Chiralpack AD-H column and the mobile phase for isocratic elution was a mixture of hexane–ethanol–TFA ((7:3):0.1, v/v); the flow was 0.5 mL min−1 (Table 2). 2.2. Chemicals and samples All HPLC solvents were obtained from Aldrich (Germany). Deionized and purified water (Millipore unit, USA) was used. All chemicals used in this study were of analytical-reagent grade. d-fructose, l- and d-arabinose, l- and d-lyxose, l- and d-fucose, d- and l-mannose, l-xylose, l- and d-glucose, ␣-d-glucose, dand l-ribose were purchased from Aldrich (Germany); d-xylose
was purchased from BDH Biochemicals (UK), d,l-arabinose from Fluka (Germany) and l-fructose from TCI Europe (Belgium). Standard solutions of each carbohydrate were prepared for each separation with a concentration of 100 mg L−1 , using vortex agitation and ultrasounds (5 min), due to low solubility of carbohydrates in the mobile phase for stereoselective separation. This is the unique drawback of this method. The samples: Portuguese honey (“Mel de Portugal”, M´ertola and “Mel Rosmaninho”, Serra da Malcata) and natural sweeteners (“Glucosine”, Diese, “Ac¸u´ car de frutos”, Provida and “Dieron”, Diese) were purchased in a Natural Products’ Pharmacy; Oporto wine (“Porto Ferreira”, 20 and 10 years old and “Ramos Pinto”, Tawny), were purchased in a local supermarket. All the samples were solved in the mobile phase mixture (according to separation) using vortex agitation and ultrasounds (5 min) and then filtered through a 0.45 m membrane filter. The standards and samples solutions were injected into the HPLC systems using a 50 L loop injector. 2.3. Chromatographic separations Each of the standard solutions was chromatographed on HPLC using a carbohydrate analysis column. The retention time for both enantiomers is the same (Table 1). Stereoselective HPLC analysis (Chiralpak AD-H column) of monosaccharide standards gave different retention times for each enantiomer (d and l) and also for the anomers (␣ and ) of each enantiomer (Table 2). As can be seen, it was possible to separate all the stereoisomers of each monosaccharide: the method allows enantiomeric and simultaneously the anomeric separation of the most stable conformational forms of each monosaccharide. The elution profile of glucose peaks was determined using standards. The elution order is -d-glucose, ␣d-glucose, -l-glucose and ␣-l-glucose respectively. For some of the other monosaccharides (mannose, ribose, lyxose) the elution order was deduced based on data described in the literature [10,11]. For xylose, arabinose and fucose, on the best of our knowledge, no data is available relative to anomeric equilibrium in (aqueous) solution. For fructose, according to the literature
Table 2 Retention times for monosaccharide separation in a Chiralpack AD-H column, at 25 ◦ C Carbohydrate
Arabinoseb Fructosec Fucoseb Glucose Lyxose Mannose Ribose Xyloseb a b c
Retention time (min)a l-Enantiomer
d-Enantiomer
␣/ anomer
␣/ anomer
23.0 (␣) 12.1 (furanose) 32.3 () 13.7 () 18.0 (␣) 12.9 (␣) 20.8 () 17.4 ()
35.9 () 19.8 (pyranose) 36.7 (␣) 14.3 (␣) 29.1 () 28.8 () 106.2 (␣) 19.3 (␣)
Mobile phase: hexane-ethanol-TFA ((7:3):0.1, v/v). Flow rate: 0.5 mL min−1 ; tm = 6.1 min. Anomeric elution profile based on relative OH configuration at C1 and C2. According to literature data [3].
27.7 (␣) 15.8 (furanose) 16.1 () 13.2 () 16.6 (␣) 12.5 (␣) 25.6 () 17.7 ()
31.6 () 19.5 (pyranose) 25.6 (␣) 13.6 (␣) 23.9 () 21.3 () 70.7 (␣) 20.9 (␣)
J.F. Lopes, E.M.S.M. Gaspar / J. Chromatogr. A 1188 (2008) 34–42
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Fig. 3. HPLC separation of lyxose at 25 ◦ C. Chromatogram A: (carbohydrate analysis column, flow rate: 0.5 mL min−1 ): (1) d,l-lyxose; chromatogram B (Chiralpak AD-H column, flow rate: 0.5 mL min−1 ); stereoselective analysis of the racemic d,l-lyxose: (2) ␣-d-lyxopyranose; (3) ␣-l-lyxopyranose; (4) -d-lyxopyranose; and (5) -l-lyxopyranose, respectively. Detection: refractive index.
Fig. 4. Chromatographic profiles of ␣- and -d-glucose at different temperatures: 15, 20, 25, 30, 35 and 40 ◦ C. Column: Chiralpak AD-H; eluent: hexane–ethanol–TFA ((7:3):0.1, v/v). Flow rate 0.5 mL min−1 , tm = 6.1 min; detection: refractive index.
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data [10], the retention times of the two main peaks of each enantiomer corresponds to the furanose and pyranose forms, respectively. 3. Results and discussion 3.1. Separation of glucose, fructose, arabinose, ribose, fucose, mannose, lyxose and xylose The standard solution of each enantiomer (d and l) of each monosaccharide, prepared as described in Section 2, was analyzed separately using both systems: achiral (carbohydrate analysis column) and chiral (Chiralpak AD-H column). The achiral separation showed only one peak for each enantiomer and allowed good separation for all analyzed monosaccharides, including the structural isomers and epimers; both enantiomers of each monosaccharide have the same retention time. No temperature effect was observed; so, room temperature (25 ◦ C) was chosen for separation. The results obtained are shown in Table 1. The chiral (Chiralpak) column allowed the separation of the diastereoisomeric pair (␣- and -anomers) for each enantiomer
d and l. Fig. 3 shows the separation of racemic d,l-lyxose: only one peak (1) in the achiral separation – chromatogram A and four peaks in the chiral analysis – chromatogram B, showing the separation of the main (four) isomeric forms (2,3,4,5). The sequence of elution on the chiral HPLC column for lyxose is 2 ␣-d-lyxopyranose, 3 ␣-l-lyxopyranose; 4 -d-lyxopyranose and 5 -l-lyxopyranose. The elution profiles of the saccharides depended on column temperature. The change was not uniform for all monosaccharides: for example, when the temperature was increased, the elution profile of lyxose became worse, while for glucose the best elution profile was achieved at 40 ◦ C (Figs. 4 and 5). At 25 ◦ C the monosaccharides tested showed reasonable elution profiles. Table 2 shows the retention times obtained by stereoselective HPLC analysis of the monosaccharides injected. The profile of each enantiomer of monosaccharides was checked, considering the ratios for anomers described in literature [2,11]: lyxose, ribose and mannose. It was observed that, using the chiral HPLC conditions mentioned above, the elution profiles are in good agreement with those obtained by Nishikawa et al.
Fig. 5. Chromatographic profiles of ␣- and -d,l-lyxose at different temperatures: 15, 20, 25, 30, 35 and 40 ◦ C. Column: Chiralpak AD-H; eluent: hexane–ethanol–TFA ((7:3):0.1, v/v). Flow rate 0.5 mL min−1 , tm = 6.1 min; detection: refractive index.
J.F. Lopes, E.M.S.M. Gaspar / J. Chromatogr. A 1188 (2008) 34–42
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Fig. 6. Stereoselective HPLC analysis of racemic mixture of d,l-lyxose (#) and d,l-mannose (*): (1) ␣-d-mannopyranose, (2) ␣-l-mannopyranose, (3) ␣-dlyxopyranose, (4) ␣-l-lyxopyranose, (5) -d-mannopyranose, (6) -d-lyxopyranose, (7) -l-lyxopyranose, (8) -l-mannopyranose and (9) minor form (probably furanose) of mannose. Flow rate: 0.5 mL min−1 ; tm = 6.1 min. Detection: refractive index.
[10], suggesting that the elution order in the Chiralpak column (using described parameters) may also be related with the vicinal OH configuration at positions C1 and C2 of the monosaccharide molecule enantiomer. Arabinose, fucose and xylose anomeric profiles were assigned based on this postulation. The profile of fructose was assigned based on the literature [2,10]. The method allowed excellent resolution of all monosaccharides that were tried. We obtained good separation for enantiomers of different monosaccharides (structural isomers) and good separation for the anomers of each enantiomer that has been analyzed (Fig. 6). The method reported here has also allowed a successful simultaneous stereodifferentiation of epimers (e.g. arabinose and ribose). For the first time a simple chromatographic method allows simultaneously enantiomeric and anomeric separation of several underivatized monosaccharides. The method was used with samples of Portuguese honey, Oporto wine and natural sugars marketed as nutritional supplements. The analysis made possible to assign unequivocally the absolute stereochemistry and prevalent anomeric forms of monosaccharides contained in the matrices. So, using the potentiality of this chiral HPLC technique, without derivatization and previous isolation, it was possible to determine the “normal” monosaccharide profiles of matrices. This is extremely important because carbohydrates are involved in food authenticity, nutritional characteristics and they are also responsible for some biological activity, since this depends on the ratios of isomeric (enantiomeric and anomeric) forms present.
3.2. Method performance The analytical method was evaluated to prove its applicability to the analysis of monosaccharides in Portuguese honey, Oporto wine and natural sugars marketed as nutritional supplements. Having the elution temperature determined (25 ◦ C), quality parameters of the stereoselective chromatographic method such as limits of detection (LODs) and quantitation (LOQs), linearity, repeatability, recovery, linearity range, precision and sensitivity were calculated [12]. Validation results are presented in Tables 3 and 4. 3.2.1. Recovery Recovery studies were performed in duplicate, by spiking the analyzed samples with two solutions of each monosaccharide with different concentration levels, 100 and 250 ppm (mg L−1 ). Quantification recoveries obtained for the target compounds ranged from 76 to 94%. Having in account the low solubility of carbohydrates in the mobile phase for stereoselective separation and the necessity of using samples in low concentration, the recoveries obtained were quite good, and therefore an acceptable quantitative estimation of the concentration of selected monosaccharides in samples could be obtained. 3.2.2. Linear dynamic range The linearity in the response was studied using matrixmatched calibration solutions prepared by spiking commercial products at seven concentration levels, ranging from 10, 20 or 30 to 750 ppm in the samples. The linear dynamic ranges,
Table 3 Analytical parameters obtained with the stereoselective HPLC method for the analysis of selected compounds in commercial products Carbohydrate
d-Fructofuranose d-Fructopyranose ␣ and -d-Glucose
Linearity Range (ppm)
Cal. Equation
R2
10–750 30–750 20–750
y = 93.248x + 214.59 y = 893.75x − 14463 y = 1148.3x + 63304
0.9974 0.9834 0.9949
LOD (ppm)
LOQ (ppm)
Repeatability (RSD, %), n = 5
Reproducibility (RSD, %), n = 5
0.7 5.1 2
2.3 16.9 6.2
7 3 3
6 4 5
RSD: relative standard deviation; LOQ: limit of quantification; LOD: limit of detection.
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Table 4 Recoveries of the optimized method for the analysis of selected commercial products Sample
Carbohydrate
Recovery (RSD, %) n = 5
Honey 1
d-Fructofuranose d-Fructopyranose ␣ and -d-Glucose
82 (8) 76 (6) 86 (4)
Honey 2
d-Fructofuranose d-Fructopyranose ␣ and -d-Glucose
89 (5) 82 (5) 85 (2)
Oporto wine 1
d-Fructofuranose d-Fructopyranose ␣ and -d-Glucose
92 (2) 94 (3) 89 (7)
Oporto wine 2
d-Fructofuranose d-Fructopyranose ␣ and -d-Glucose
88 (7) 88 (8) 87 (7)
RSD: relative standard deviation.
calibration equations and correlation coefficients (R2 ) obtained are shown in Table 3. Good linearity was observed in the studied range with R2 values higher than 0.98. 3.2.3. Sensitivity Limits of detection and quantitation were evaluated on the basis of the signal obtained with the analysis of unfortified samples (n = 5), taking into account the solubility of the target monosaccharides in the mobile phase. Values obtained ranged from 0.7 to 5.1 ppm and 2.3–16.9 ppm, respectively, values quite good for a method using RI detection. 3.2.4. Precision In order to evaluate the precision of the proposed method, repeatability and reproducibility were estimated. The repeata-
bility, expressed as percent relative standard deviation (RSD), varied between 3 and 7% for repeatability and 4 and 6% for the reproducibility. 3.3. Application of the method to real samples Adulterated or degraded honey may show a different sugar profile; so this method allows a quick and efficient test to determine the authenticity, quality or inadequate storage conditions of honeys. The typical monosaccharide profile of authentic Portuguese (good) honey is shown in Fig. 7. As expected, commercial Portuguese honey consists mostly of the monosaccharides glucose and fructose, containing more fructose than glucose (average ratio 1.2/1) [13]. The application of the method established the ratios of anomeric and tautomeric forms; results are summarized in Table 5. “Oporto wine” is a designation of controlled origin. It’s a Portuguese product recognized worldwide, economically very important to the country. Its monosaccharide profile has been established to detect fraudulent designations and adulteration; it is shown in Fig. 8. The results obtained are summarized in Table 5. As can be seen d-fructose and d-glucose are the main monosaccharide constituents. The prevalent forms were shown to be -d-glucose and d-fructopyranose. Sugar supplements/substitutes are used as alternative sweeteners by people who have metabolic disorders (e.g. diabetes) or under regimen (e.g. obesity). It is also used by athletes because of a need of higher energy. More expensive than the common sugar (mainly composed by sucrose, a disaccharide), they are easily subjected to adulteration or falsification. Natural monosaccharides like fructose, glucose and tagatose are the main substitutes recommended by physicians.
Fig. 7. Analysis of authentic Portuguese honey. Chromatogram A (carbohydrate analysis column, flow rate: 1.5 mL min−1 ; tm = 3 min): (1) fructose and (2) glucose; chromatogram B shows the stereoselective analysis: (3) glycerol; (4) -d-glucopyranose; (5) ␣-d-glucopyranose; (6) d-fructofuranose; and (7) d-fructopyranose. Flow rate: 0.5 mL min−1 . Detection: refractive index. Table 5 Average ratio of isomers present in authentic Oporto wine and Portuguese honey samples Monosaccharide
d-Glucose d-Fructose
Anomer or tautomer ratio
/␣ Pyranose/furanose
Samples Oporto wine (samples 1 to 9)
Portuguese honey (samples 1–4)
0.9–1.4 8.2–10.4
1.5–1.9 6.9–10.8
J.F. Lopes, E.M.S.M. Gaspar / J. Chromatogr. A 1188 (2008) 34–42
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Fig. 8. Authentic Oporto wine profile of monosaccharides. Chromatogram A (carbohydrate analysis column, flow rate: 1 mL min−1 ): (1) glycerol, (2) fructose and (3) glucose; stereoselective HPLC chromatogram (B): (4) -d-glucopyranose; (5) ␣-d-glucopyranose, (6) d-fructofuranose; and (7) d-fructopyranose. Flow rate: 0.5 mL min−1 . Detection: refractive index.
Fig. 9. Analysis of sugar substitute designated “Sugar from fruits”: (1) fructose (chromatogram A, carbohydrate analysis column): (2) d-fructofuranose; and (3) d-fructopyranose (chromatogram B, Chiralpak AD-H column). Flow rate: 0.5 mL min−1 . Detection: refractive index.
Fig. 10. Analysis of nutritional supplement “Glucosine”: (1) glucose (chromatogram A, carbohydrate analysis column); chromatogram B (Chiralpak AD-H column); (2) -d-glucopyranose and (3) ␣-d-glucopyranose. Flow rate: 0.5 mL min−1 . Detection: refractive index.
The method reported here made possible to determine the authenticity and nutritional characteristics of carbohydrates used as sweetener substitutes or nutritional supplements; Figs. 9 and 10 show the profiles of two of them. The results support the natural origin of these supplements. In fact, ␣-d-glucose is the most abundant natural form of this monosaccharide. d-fructose is the stereoisomer of natural origin. 4. Conclusions The stereodifferentiation of the enantiomers (d and l) and anomers (␣ and ) of some monosaccharides was carried out
by the use of a Chiralpak AD-H HPLC column. The one-step, no time consuming analysis of underivatized monosaccharides proved to be a simple, powerful, useful chromatographic method that allows the unequivocal determination of the absolute configuration and anomeric forms of sugar moieties present (even in trace amounts). The elution sequence was determined using standards (all enantiomers and one glucose anomer) and by comparison with literature data. The method is superior to others described in terms of the simplicity and stereoselectivity and allowed the determination of the absolute configuration and the anomeric prevalent forms of monosaccharide moieties contained in food chain matrices, without previous isolation. Authenticity and
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nutritional characteristics may be determined using the method; they are sometimes responsible for some anomalous biological activities/disorders. It has also potential to be applied to biological matrices and can be scaled-up. The method is patent pending. Acknowledgments The authors thank Eng. Jo˜ao Tiago and Mr. Pedro Marques for their technical informatics assistance. Special thanks to Professor Maria Margarida Gonc¸alves for her samples’ solubility assistance. Financial support from FCT – Portugal. References [1] M. Sznaidman, in: S.M. Hecht (Ed.), Bioorganic Chemistry Carbohydrates, Oxford University Press, Oxford, 1999, p. 1. [2] F. Franks, Pure Appl. Chem. 59 (1987) 1189 (and references cited therein).
[3] D. Gomis, D. Tamayo, J. Alonso, Anal. Chim. Acta 436 (2001) 173. [4] C. De Muynck, J. Beauprez, W. Soetaert, E. Vandamme, J. Chromatogr. A 1101 (2006) 115. [5] M. Little, J. Biochem. Biophys. Methods 11 (1985) 195. [6] F. Lamari, R. Kuhn, N. Karamanos, J. Chromatogr. B 793 (2003) 15. [7] A. Carmona, P. Borrachero, F. Cabrera-Escribano, M. Di´anez, M. Estrada, A. L´opez-Castro, R. Ojeda, M. G´omez-Guill´en, S. P´erez-Garrido, Tetrah. Asym. 10 (1999) 1751. [8] A. Clement, D. Young, C. Brecht, J. Liq. Chromatogr. 15 (1992) 805. [9] C. Bai, H. Ohrui, Y. Nishida, H. Meguro, Anal. Biochem. 246 (1997) 246. [10] T. Nishikawa, S. Suzuki, H. Kubo, H. Ohtani, J. Chromatogr. A 720 (1996) 167 (and references cited therein). [11] G.-J. Boons, in: G.-J. Boons (Ed.), Mono- and Oligosaccharides: Structure, Configuration and Conformation in Carbohydrate Chemistry, Blackie Academic & Professional, Thomson Science, London, 1998, p. 1. [12] J.C. Miller, J.N. Miller, Statistics for Analytical Chemistry, Ellis Horwood, Chichester, UK, 1988. [13] G. Rodr´ıguez, B. Ferrer, A. Ferrer, B. Rodr´ıguez, Food Chem. 84 (2004) 499 (and references cited therein).