Journal of Chromatographic Science Advance Access published April 23, 2015 Journal of Chromatographic Science 2015;1– 5 doi:10.1093/chromsci/bmv044
Article
Determination of Sucrose in Honey with Derivatization/Solid-Phase Microextraction and Gas-Chromatography/Mass Spectrometry Haijing Wang1, Helmut Geppert1*, Thomas Fischer1, Wolfgang Wieprecht2 and Detlev Mo¨ller2 1
Faculty of Environmental Sciences and Process Engineering, Central Analytical Laboratory, Brandenburg University of Technology Cottbus-Senftenberg, Konrad-Wachsmann-Allee 6, Cottbus 03046, Germany, and 2Brandenburg University of Technology CottbusSenftenberg, Volmerstr. 13, Berlin 12489, Germany *Author to whom correspondence should be addressed. Email: [email protected] Received 15 August 2014; revised 5 March 2015
A new method for the determination of sucrose in honey with derivatization solid-phase microextraction and gas chromatography/mass spectrometry (D-SPME –GC/MS) was developed. The method incorporates a sample derivatization with acetic anhydride using N-methylimidazole as the catalyst and the subsequent enrichment of the analyte in a Polyacrylate-SPME fiber. Results show that 100 mL N-methylimidazole and 800 mL acetic anhydride were sufficient to complete the acetylation for sucrose in 100 mL aqueous sample at room temperature. For SPME, an enrichment time of 30 min was sufficient. SPME was performed by immersing the fiber into the solution with additional vibration. Then, the analyte was desorbed for 5 min at 28088 C in the GC/MS injection port with splitless mode. The present method exhibits good linearity at a concentration range of 0.3– 8% of sucrose in honey with excellent regression (R 5 0.9993). The method has been successfully applied to the control of sucrose adulteration in honey. Introduction Honey is a supersaturated natural solution of sugars, in which the monosaccharide fructose and glucose are the most abundant (typically 80 –85% of the solids in the honey). Other disaccharides (i.e., sucrose, trehalose, isomaltose, etc.) and higher sugars (trisaccharides and oligosaccharides) are also present, although in quite small quantities (1). Sucrose (saccharose) is present in honey at a concentration of 1% of dry weight. However, this level can be increased if the beekeeper has over-fed the bees with sugar during spring (2, 3). According to British and German honey regulations the sucrose content should generally not exceed 5%. The sucrose content in honey can be determined by spectroscopic, electrophoretic and chromatographic methods: Fourier transform infrared spectroscopy (FTIR) (4), thin layer chromatography (1, 5), capillary electrophoresis (6), high performance liquid chromatography (HPLC) (7), gas chromatography (8– 12) and gas chromatography –mass spectrometry (GC –MS) (13, 14). FTIR has been proven to provide reliable results for fructose, glucose, sucrose, turanose, maltose, trehalose, isomaltose, erlose, as well as for a number of physical properties of honey using multivariate partial least squares (PLS) calibration (4). However, it is necessary for PLS calibration to hold numerous calibration and verification samples all with known concentrations of its constituents. For example, Lichtenberg-Kraag et al. (4) used 1600 FTIR spectra of honey samples for PLS calibration, which had been characterized by HPLC and other methods previously. Although HPLC does not necessarily require derivatization of
sugars, they are not detectable by common UV/VIS and require refraction (RI) or light scattering detectors. On the other hand, conventional GC – MS of sugars necessitates time-consuming derivatization, bearing a high risk of error and requiring additional quality control measures. It was the aim of this study to develop a GC – MS method which overcomes these drawbacks and to make sugar analysis accessible to laboratories where the requirements for FTIR or HPLC are not available. In order to minimize the sample preparation time for GC–MS, we propose a new method with derivatization/solid-phase microextraction (D-SPME), which was firstly described in 1997 by Pawliszyn and coworkers (15), and was later used for detection of aldehydes, amines, phenols, steroids, thiols, organic acids and amino acids. To our knowledge, this is the first study to demonstrate the D-SPME method also for the determination of carbohydrates. Experimental Chemicals and reagents Sucrose, fructose, glucose, acetic anhydride and N-methylimidazole were purchased from Sigma-Aldrich (Steinheim, Germany). Mixtures of standard working solutions were prepared by dilution with water purified by a water purification system from Seral Reinstwassersyteme GmbH (Germany). GC–MS analysis GC – MS analysis was performed using a gas chromatograph VARIAN 450-GC coupled with a VARIAN 300MS TQ mass spectrometer. The GC was equipped with a VARIAN FactorFour capillary column (30 m 0.25 mm film thickness 0.25 mm i.d.). The injection was carried out in splitless mode. High purity helium gas (99.999%) was used as a carrier gas with a flow rate of 1.0 mL min21. The oven temperature programming was as follows: the initial oven temperature was held at 508C for 1 min, then increased to 3008C at a rate of 308C min21 and held for 4 min. The ion source and interface temperature were set to 2008C. All the samples were analyzed in selected ion monitoring mode (sucrose: RT ¼ 11.81 min, m/z ¼ 331 quantification ion, m/z ¼ 169, 221 confirmation ions).
Preparation of honey samples Honey was obtained from a local supplier and was harvested in summer 2014 in the Spreewald region in Germany. It was prepared by mixing 1 g honey with 20 mL pure water.
# The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Preparation of the sucrose standards The sucrose standards were prepared in an aqueous solution of fructose and glucose. Initially, 20 g fructose and 17.5 g glucose were dissolved in 1 L pure water. Then, 10 – 400 mg sucrose
was added to 100 mL portions of this solution. This corresponds to 0.2 –8% of sucrose in honey. SPME fibers Commercially available polydimethylsiloxane (PDMS)-, polyacrylate (PA)- and divinylbenzene-polydimethylsiloxane (DVB-PDMS-SPME) fibers were purchased from Supelco (Bellefonte, PA). Prior to use, the fibers were conditioned in the hot injector of the gas chromatograph in helium atmosphere according to the manufacturer’s directions.
Figure 2. Peak areas obtained by performing analyses in immersion modes (n ¼ 3) with three different fibers. PDMS, polydimethylsiloxane; PA, polyacrylate; DVB-PDMS, divinylbenzene-polydimethylsiloxane.
Figure 3. Effect of the enrichment time on peak area of sucrose octaacetate. Table I Specifications of the Method According to the German Standard DIN 32645 (17)
Figure 1. Effects of the derivatization conditions on peak area of sucrose octaacetate. (A) Effect of the volume of acetic anhydride, (B) effect of the volume of N-methylimidazole and (C) effect of reaction time.
2 Wang et al.
Regression equation
y ¼ 161667 x þ 8105
Correlation coefficient Method standard deviation LOD (%) Recovery (2% standard) Linear dynamic range (LDR) (%)
0.9993 0.084 0.3 0.91 0.3 –8
Optimized derivatization SPME procedure One hundred microliters of the aqueous honey sample or standard solution were added into 20 mL vials. Then 100 mL of N-methylimidazole and 800 mL of acetic anhydride were added. To ensure complete derivatization, the mixture was thoroughly mixed by hand and allowed to rest for another 10 min. Then 10 mL of pure water were added, and the vials were sealed with PTFE septa. SPME was performed by immersing the PAfiber into the solution for 30 min with additional vibration of the fiber with a Varian 8200 SPME agitation device. Desorption time and temperature were 5 min and 2808C, respectively.
Results Optimization of the derivatization procedures In order to obtain the best derivatization yields, the volume of acetic anhydride, the amount of N-methylimidazole and the derivatzation time were optimized.
Effect of acetic anhydride volume Different volumes of acetic anhydride were added to 100 mL standard solution and 100 mL N-methylimidazole, the reaction was performed at room temperature for 10 min. As shown in Figure 1A, the peak area markedly increased at the volume of acetic anhydride ranging from 200 to 800 mL. When the volume was beyond 800 mL, the yields did not improve further. Thus, 800 mL of acetic anhydride were considered as the optimum amount.
Effect of N-methylimidazole volume Different volumes of N-methylimidazole were added to 100 mL standard solution and 800 mL acetic anhydride, and the reaction was performed at room temperature for 10 min. As shown in Figure 1B, the peak area was no further increased when the volume was greater than 100 mL. Thus, 100 mL of N-methylimidazole anhydride were considered as the optimum amount which also corresponds to the finding of Wu et al. (16).
Effect of reaction time One hundred microliters of standard solution, 100 mL N-methylimidazole and 800 mL acetic anhydride were mixed, the reaction was performed at room temperature from 5 to 15 min. As shown in Figure 1C, 10 min of reaction time was considered as sufficient.
Selection of the SPME fiber The commercially available fiber coatings PDMS, PA and PDMS/ DVB were evaluated. Best extraction efficiencies were obtained with DVB-PDMS as shown in Figure 2. To avoid possible discrimination effects due to the high concentration of fructose and glucose, the PA fiber was used for the further investigation.
Determination of the SPME enrichment time For the investigation of the influence of the SPME enrichment time it was varied from 15 to 75 min. To circumvent method errors associated with nonequilibrium states, 30 min were considered as sufficient (Fig. 3).
Calibration and validation Calibration and validation of the method were performed according to the German DIN 32645 (17). The specifications are given in Table I. The calibration curve is given in the Supplementary data. The positive increment of y at x ¼ 0 results from the fiber blank. The carryover of a blank run immediately following a SPME-injection of a 5% sucrose standard amounted to 2.9% of the initial peak height of the standard.
Discussion SPME is a rapid, portable, rugged and solvent-free extraction/ concentration technique widely used in analytical chemistry for almost 20 years, predominantly for the determination of nonpolar substances. The enrichment of polar metabolites in the relatively nonpolar commercially available SPME fibers is
Figure 4. Derivatzation of sucrose with acetic anhydride (AA) and N-methylimidazole as catalyst to sucrose octaacetate (chemical formulae retrieved 17.02.2014 from http://www.pubchem.com).
Determination of Sucrose in Honey with D-SPME –GC/MS 3
Figure 5. GC– MS chromatogram for a honey sample from the local market with a concentration of 0.5% (above) and the same sample to which additional 1% sucrose were added (below).
insufficient and requires the use of additional analytical derivatization techniques (15, 18 –20). Derivatization of sugars into volatile acetates is one of the most widely used method for GC analysis. Using N-methylimidazole as catalyst, the reaction is very rapid even at room temperature and the use of an aqueous sample is possible (16, 21, 22). Figure 4 shows the reaction equation for the derivatization of sucrose. With the help of 1H-NMR we have shown that the reaction takes place completely already after 10 min without side reactions (see Supplementary data 1). The EI-MS-spectrum of sucrose octaacetate showed no molecular peak (m/z ¼ 678). For the quantification of sucrose, therefore, we used the mass fragment m/z ¼ 331. An example of the mass spectrum of sucrose octaacetate recorded in this study is shown in Supplementary data 2. Figure 5 shows in the upper chromatogram the sucrose peak of a honey sample from the local market. The sucrose content was 0.5%. The lower chromatogram shows the same sample to which 1% sucrose were added.
sample was derivatized by N-methylimidazole catalyzed acetylation. Derivatization was completed rapidly at room temperature in an aqueous sample. For SPME, with a polyacrylate fiber an enrichment time of 30 min was sufficient. The method is sensitive enough to safely detect adulteration of honey by sucrose feeding. The proposed method may be adapted for the determination of other mono- and disaccharides in honey.
Conclusions A simple and rapid D-SPMEGC/MS method was developed for the determination of sucrose in honey. Prior to GC analysis, the
References
4 Wang et al.
Supplementary data Supplementary data are available at Journal of Chromatographic Science (http://chromsci.oxfordjournals.org).
Acknowledgements The authors thank Dr Heather Lord (Maxxam Analytics, Canada) for review of the text and the helpful hints.
1. Puscas, A., Hosu, A., Cimpoiu, C.; Application of a newly developed and validated high-performance thin-layer chromatographic method
2.
3.
4. 5.
6.
7.
8. 9.
10.
11.
12.
to control honey adulteration; Journal of Chromatography A, (2013); 1272: 132–135. Deifel, A., Gierschner, K., Vorwohl, G.; Saccharose in honey—saccharose and its transglycosidification products in natural and in sugar-feeding honeys; Deutsche Lebensmittel-Rundschau, (1985); 81: 356–362. Anklam, E.; A review of the analytical methods to determine the geographical and botanical origin of honey; Food Chemistry, (1998); 63: 549–562. Lichtenberg-Kraag, B., Hedtke, C., Bienefeld, K.; Infrared spectroscopy in routine quality analysis of honey; Apidologie, (2002); 33: 327–337. Pukl, M., Prosek, M.; Rapid quantitative TLC analysis of sugars using an improved commonly used solvent system; Journal of Planar Chromatography, (1990); 3: 173– 176. Rizelio, V.M., Tenfen, L., da Silveira, R., Gonzaga, L.V., Costa, A.C.O., Fett, R.; Development of a fast capillary electrophoresis method for determination of carbohydrates in honey samples; Talanta, (2012); 93: 62–66. Bugner, E., Feinberg, M.; Determination of mono-and disaccharides in foods by interlaboratory study: quanitation of bias components for liquid chromatography; Journal of AOAC International, (1992); 75: 443–464. Deifel, A.; Gas chromatographical determination of sugars in honey; Deutsche Lebensmittel Rundschau, (1985); 81: 209– 212. Mateo, R., Bosch, F., Pastor, A., Jimenez, M.; Capillary column gas chromatographic identification of sugars in honey as trimethylsilyl derivatives; Journal of Chromatography A, (1987); 410: 319–328. Low, N.H., Sporns, P.; Analysis and quantitation of minor disaccharides and trisaccharides in honey, using capillary gas chromatography; Journal of Food Science, (1988); 53: 558– 561. Low, N.H., South, W.; Determination of honey authenticity by capillary gas chromatography; Journal of AOAC International, (1995); 78: 1210– 1218. Kaskoniene, V., Venskutonis, P.R., Ceksteryte, V.; Carbohydrate composition and electrical conductivity of different origin honeys from Lithuania; Food Science and Technology, (2010); 43: 801–807.
13. Horvath, K., MolnarPerl, I.; Simultaneous quantitation of mono-, diand trisaccharides by GC-MS of their TMS ether oxime derivatives. 2. In honey; Chromatographia, (1997); 45: S328–S335. 14. Sanz, M., Sanz, J., Martinez-Castro, I.; Gas chromatographic-mass spectrometric method for the qualitative and quantitative determination of disaccharides and trisaccharides in honey; Journal of Chromatography A, (2004); 1059: 143–148. 15. Pan, L., Pawliszyn, J.; Derivatization/solid-phase microextraction: new approach to polar analytes; Analytical Chemistry, (1997); 69: 196– 205. 16. Wu, J., Li, M.H., Lin, J.P., Wei, D.Z.; Determination of dihydroxyacetone and glycerol in fermentation process by GC after n-Methylimidazole catalyzed acetylation; Journal of Chromatographic Science, (2011); 49: 375–378. 17. DIN 32645: Chemical analysis—decision limit, detetion limit and determination limit under repeatability conditions—terms, methods evaluation. 2008– 11, Beuth publisher’s, http://www.beuth.de/ de/norm/din-32645/110729574. 18. Stashenko, E.E., Martinez, J.R.; Derivatization and solid-phase microextraction; TRAC-Trends in Analytical Chemistry, (2004); 23: 553–561. 19. Snow, N.H.; Sample preparation for gas chromatography using solidphase microextraction with derivatization. In Advances in Chromatography, Vol. 48. CRC Press, Boca Raton, FL, (2010), pp. 373– 378. 20. Rosenfeld, J.; Chemical derivatizations in analytical extractions. In Pawliszyn, J., Lord, H.L. (eds). Handbook of sample preparation, Chapter 13. Wiley, Hoboken, NJ, (2010), pp. 225– 245. 21. Connors, K.A., Pandit, N.K.; N-methylimidazole as a catalyst for analytical acetylations of hydroxy compounds; Analytical Chemistry, (1978); 50: 1542– 1545. 22. Wachowiak, R., Connors, K.A.; N-Methylimidazole-catalyzed acetylation of hydroxy compounds prior to gas chromatographic separation and determination; Analytical Chemistry, (1979); 51: 27– 30.
Determination of Sucrose in Honey with D-SPME –GC/MS 5
Article
Determination of Sucrose in Honey with Derivatization/Solid-Phase Microextraction and Gas-Chromatography/Mass Spectrometry Haijing Wang1, Helmut Geppert1*, Thomas Fischer1, Wolfgang Wieprecht2 and Detlev Mo¨ller2 1
Faculty of Environmental Sciences and Process Engineering, Central Analytical Laboratory, Brandenburg University of Technology Cottbus-Senftenberg, Konrad-Wachsmann-Allee 6, Cottbus 03046, Germany, and 2Brandenburg University of Technology CottbusSenftenberg, Volmerstr. 13, Berlin 12489, Germany *Author to whom correspondence should be addressed. Email: [email protected] Received 15 August 2014; revised 5 March 2015
A new method for the determination of sucrose in honey with derivatization solid-phase microextraction and gas chromatography/mass spectrometry (D-SPME –GC/MS) was developed. The method incorporates a sample derivatization with acetic anhydride using N-methylimidazole as the catalyst and the subsequent enrichment of the analyte in a Polyacrylate-SPME fiber. Results show that 100 mL N-methylimidazole and 800 mL acetic anhydride were sufficient to complete the acetylation for sucrose in 100 mL aqueous sample at room temperature. For SPME, an enrichment time of 30 min was sufficient. SPME was performed by immersing the fiber into the solution with additional vibration. Then, the analyte was desorbed for 5 min at 28088 C in the GC/MS injection port with splitless mode. The present method exhibits good linearity at a concentration range of 0.3– 8% of sucrose in honey with excellent regression (R 5 0.9993). The method has been successfully applied to the control of sucrose adulteration in honey. Introduction Honey is a supersaturated natural solution of sugars, in which the monosaccharide fructose and glucose are the most abundant (typically 80 –85% of the solids in the honey). Other disaccharides (i.e., sucrose, trehalose, isomaltose, etc.) and higher sugars (trisaccharides and oligosaccharides) are also present, although in quite small quantities (1). Sucrose (saccharose) is present in honey at a concentration of 1% of dry weight. However, this level can be increased if the beekeeper has over-fed the bees with sugar during spring (2, 3). According to British and German honey regulations the sucrose content should generally not exceed 5%. The sucrose content in honey can be determined by spectroscopic, electrophoretic and chromatographic methods: Fourier transform infrared spectroscopy (FTIR) (4), thin layer chromatography (1, 5), capillary electrophoresis (6), high performance liquid chromatography (HPLC) (7), gas chromatography (8– 12) and gas chromatography –mass spectrometry (GC –MS) (13, 14). FTIR has been proven to provide reliable results for fructose, glucose, sucrose, turanose, maltose, trehalose, isomaltose, erlose, as well as for a number of physical properties of honey using multivariate partial least squares (PLS) calibration (4). However, it is necessary for PLS calibration to hold numerous calibration and verification samples all with known concentrations of its constituents. For example, Lichtenberg-Kraag et al. (4) used 1600 FTIR spectra of honey samples for PLS calibration, which had been characterized by HPLC and other methods previously. Although HPLC does not necessarily require derivatization of
sugars, they are not detectable by common UV/VIS and require refraction (RI) or light scattering detectors. On the other hand, conventional GC – MS of sugars necessitates time-consuming derivatization, bearing a high risk of error and requiring additional quality control measures. It was the aim of this study to develop a GC – MS method which overcomes these drawbacks and to make sugar analysis accessible to laboratories where the requirements for FTIR or HPLC are not available. In order to minimize the sample preparation time for GC–MS, we propose a new method with derivatization/solid-phase microextraction (D-SPME), which was firstly described in 1997 by Pawliszyn and coworkers (15), and was later used for detection of aldehydes, amines, phenols, steroids, thiols, organic acids and amino acids. To our knowledge, this is the first study to demonstrate the D-SPME method also for the determination of carbohydrates. Experimental Chemicals and reagents Sucrose, fructose, glucose, acetic anhydride and N-methylimidazole were purchased from Sigma-Aldrich (Steinheim, Germany). Mixtures of standard working solutions were prepared by dilution with water purified by a water purification system from Seral Reinstwassersyteme GmbH (Germany). GC–MS analysis GC – MS analysis was performed using a gas chromatograph VARIAN 450-GC coupled with a VARIAN 300MS TQ mass spectrometer. The GC was equipped with a VARIAN FactorFour capillary column (30 m 0.25 mm film thickness 0.25 mm i.d.). The injection was carried out in splitless mode. High purity helium gas (99.999%) was used as a carrier gas with a flow rate of 1.0 mL min21. The oven temperature programming was as follows: the initial oven temperature was held at 508C for 1 min, then increased to 3008C at a rate of 308C min21 and held for 4 min. The ion source and interface temperature were set to 2008C. All the samples were analyzed in selected ion monitoring mode (sucrose: RT ¼ 11.81 min, m/z ¼ 331 quantification ion, m/z ¼ 169, 221 confirmation ions).
Preparation of honey samples Honey was obtained from a local supplier and was harvested in summer 2014 in the Spreewald region in Germany. It was prepared by mixing 1 g honey with 20 mL pure water.
# The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Preparation of the sucrose standards The sucrose standards were prepared in an aqueous solution of fructose and glucose. Initially, 20 g fructose and 17.5 g glucose were dissolved in 1 L pure water. Then, 10 – 400 mg sucrose
was added to 100 mL portions of this solution. This corresponds to 0.2 –8% of sucrose in honey. SPME fibers Commercially available polydimethylsiloxane (PDMS)-, polyacrylate (PA)- and divinylbenzene-polydimethylsiloxane (DVB-PDMS-SPME) fibers were purchased from Supelco (Bellefonte, PA). Prior to use, the fibers were conditioned in the hot injector of the gas chromatograph in helium atmosphere according to the manufacturer’s directions.
Figure 2. Peak areas obtained by performing analyses in immersion modes (n ¼ 3) with three different fibers. PDMS, polydimethylsiloxane; PA, polyacrylate; DVB-PDMS, divinylbenzene-polydimethylsiloxane.
Figure 3. Effect of the enrichment time on peak area of sucrose octaacetate. Table I Specifications of the Method According to the German Standard DIN 32645 (17)
Figure 1. Effects of the derivatization conditions on peak area of sucrose octaacetate. (A) Effect of the volume of acetic anhydride, (B) effect of the volume of N-methylimidazole and (C) effect of reaction time.
2 Wang et al.
Regression equation
y ¼ 161667 x þ 8105
Correlation coefficient Method standard deviation LOD (%) Recovery (2% standard) Linear dynamic range (LDR) (%)
0.9993 0.084 0.3 0.91 0.3 –8
Optimized derivatization SPME procedure One hundred microliters of the aqueous honey sample or standard solution were added into 20 mL vials. Then 100 mL of N-methylimidazole and 800 mL of acetic anhydride were added. To ensure complete derivatization, the mixture was thoroughly mixed by hand and allowed to rest for another 10 min. Then 10 mL of pure water were added, and the vials were sealed with PTFE septa. SPME was performed by immersing the PAfiber into the solution for 30 min with additional vibration of the fiber with a Varian 8200 SPME agitation device. Desorption time and temperature were 5 min and 2808C, respectively.
Results Optimization of the derivatization procedures In order to obtain the best derivatization yields, the volume of acetic anhydride, the amount of N-methylimidazole and the derivatzation time were optimized.
Effect of acetic anhydride volume Different volumes of acetic anhydride were added to 100 mL standard solution and 100 mL N-methylimidazole, the reaction was performed at room temperature for 10 min. As shown in Figure 1A, the peak area markedly increased at the volume of acetic anhydride ranging from 200 to 800 mL. When the volume was beyond 800 mL, the yields did not improve further. Thus, 800 mL of acetic anhydride were considered as the optimum amount.
Effect of N-methylimidazole volume Different volumes of N-methylimidazole were added to 100 mL standard solution and 800 mL acetic anhydride, and the reaction was performed at room temperature for 10 min. As shown in Figure 1B, the peak area was no further increased when the volume was greater than 100 mL. Thus, 100 mL of N-methylimidazole anhydride were considered as the optimum amount which also corresponds to the finding of Wu et al. (16).
Effect of reaction time One hundred microliters of standard solution, 100 mL N-methylimidazole and 800 mL acetic anhydride were mixed, the reaction was performed at room temperature from 5 to 15 min. As shown in Figure 1C, 10 min of reaction time was considered as sufficient.
Selection of the SPME fiber The commercially available fiber coatings PDMS, PA and PDMS/ DVB were evaluated. Best extraction efficiencies were obtained with DVB-PDMS as shown in Figure 2. To avoid possible discrimination effects due to the high concentration of fructose and glucose, the PA fiber was used for the further investigation.
Determination of the SPME enrichment time For the investigation of the influence of the SPME enrichment time it was varied from 15 to 75 min. To circumvent method errors associated with nonequilibrium states, 30 min were considered as sufficient (Fig. 3).
Calibration and validation Calibration and validation of the method were performed according to the German DIN 32645 (17). The specifications are given in Table I. The calibration curve is given in the Supplementary data. The positive increment of y at x ¼ 0 results from the fiber blank. The carryover of a blank run immediately following a SPME-injection of a 5% sucrose standard amounted to 2.9% of the initial peak height of the standard.
Discussion SPME is a rapid, portable, rugged and solvent-free extraction/ concentration technique widely used in analytical chemistry for almost 20 years, predominantly for the determination of nonpolar substances. The enrichment of polar metabolites in the relatively nonpolar commercially available SPME fibers is
Figure 4. Derivatzation of sucrose with acetic anhydride (AA) and N-methylimidazole as catalyst to sucrose octaacetate (chemical formulae retrieved 17.02.2014 from http://www.pubchem.com).
Determination of Sucrose in Honey with D-SPME –GC/MS 3
Figure 5. GC– MS chromatogram for a honey sample from the local market with a concentration of 0.5% (above) and the same sample to which additional 1% sucrose were added (below).
insufficient and requires the use of additional analytical derivatization techniques (15, 18 –20). Derivatization of sugars into volatile acetates is one of the most widely used method for GC analysis. Using N-methylimidazole as catalyst, the reaction is very rapid even at room temperature and the use of an aqueous sample is possible (16, 21, 22). Figure 4 shows the reaction equation for the derivatization of sucrose. With the help of 1H-NMR we have shown that the reaction takes place completely already after 10 min without side reactions (see Supplementary data 1). The EI-MS-spectrum of sucrose octaacetate showed no molecular peak (m/z ¼ 678). For the quantification of sucrose, therefore, we used the mass fragment m/z ¼ 331. An example of the mass spectrum of sucrose octaacetate recorded in this study is shown in Supplementary data 2. Figure 5 shows in the upper chromatogram the sucrose peak of a honey sample from the local market. The sucrose content was 0.5%. The lower chromatogram shows the same sample to which 1% sucrose were added.
sample was derivatized by N-methylimidazole catalyzed acetylation. Derivatization was completed rapidly at room temperature in an aqueous sample. For SPME, with a polyacrylate fiber an enrichment time of 30 min was sufficient. The method is sensitive enough to safely detect adulteration of honey by sucrose feeding. The proposed method may be adapted for the determination of other mono- and disaccharides in honey.
Conclusions A simple and rapid D-SPMEGC/MS method was developed for the determination of sucrose in honey. Prior to GC analysis, the
References
4 Wang et al.
Supplementary data Supplementary data are available at Journal of Chromatographic Science (http://chromsci.oxfordjournals.org).
Acknowledgements The authors thank Dr Heather Lord (Maxxam Analytics, Canada) for review of the text and the helpful hints.
1. Puscas, A., Hosu, A., Cimpoiu, C.; Application of a newly developed and validated high-performance thin-layer chromatographic method
2.
3.
4. 5.
6.
7.
8. 9.
10.
11.
12.
to control honey adulteration; Journal of Chromatography A, (2013); 1272: 132–135. Deifel, A., Gierschner, K., Vorwohl, G.; Saccharose in honey—saccharose and its transglycosidification products in natural and in sugar-feeding honeys; Deutsche Lebensmittel-Rundschau, (1985); 81: 356–362. Anklam, E.; A review of the analytical methods to determine the geographical and botanical origin of honey; Food Chemistry, (1998); 63: 549–562. Lichtenberg-Kraag, B., Hedtke, C., Bienefeld, K.; Infrared spectroscopy in routine quality analysis of honey; Apidologie, (2002); 33: 327–337. Pukl, M., Prosek, M.; Rapid quantitative TLC analysis of sugars using an improved commonly used solvent system; Journal of Planar Chromatography, (1990); 3: 173– 176. Rizelio, V.M., Tenfen, L., da Silveira, R., Gonzaga, L.V., Costa, A.C.O., Fett, R.; Development of a fast capillary electrophoresis method for determination of carbohydrates in honey samples; Talanta, (2012); 93: 62–66. Bugner, E., Feinberg, M.; Determination of mono-and disaccharides in foods by interlaboratory study: quanitation of bias components for liquid chromatography; Journal of AOAC International, (1992); 75: 443–464. Deifel, A.; Gas chromatographical determination of sugars in honey; Deutsche Lebensmittel Rundschau, (1985); 81: 209– 212. Mateo, R., Bosch, F., Pastor, A., Jimenez, M.; Capillary column gas chromatographic identification of sugars in honey as trimethylsilyl derivatives; Journal of Chromatography A, (1987); 410: 319–328. Low, N.H., Sporns, P.; Analysis and quantitation of minor disaccharides and trisaccharides in honey, using capillary gas chromatography; Journal of Food Science, (1988); 53: 558– 561. Low, N.H., South, W.; Determination of honey authenticity by capillary gas chromatography; Journal of AOAC International, (1995); 78: 1210– 1218. Kaskoniene, V., Venskutonis, P.R., Ceksteryte, V.; Carbohydrate composition and electrical conductivity of different origin honeys from Lithuania; Food Science and Technology, (2010); 43: 801–807.
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Determination of Sucrose in Honey with D-SPME –GC/MS 5
