APPLICATION TN Materials and Methods Reagents and Chemicals

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1 TN-8 APPLICATION Fast Analysis of Sucrose,, and Composition in Fruit Juices and Processed Beverages using Simplified HPLC Methodology Brian Rivera Product Manager In addition to chromatography, Brian also has a passion for ice cream-making, and enjoys experimenting with bold, new flavors. Laura Snow, Matthew Trass, Brian Rivera, Michael Klein, and Allen Misa Phenomenex, 4 Madrid Avenue, Torrance, CA 95 USA Sucrose, glucose, and fructose are naturally found in fruit juices but are also added to various processed beverages. In this study, we present a fast and simple method for the analysis of sugar using Rezex HPLC columns evaluating a variety of commonly consumed beverages including sports/energy drinks, soda, fruit juices, and wine. Introduction The quantitation of sucrose and its monosaccharide constituents, glucose and fructose, is of critical importance for beverage manufacturers. High-fructose corn syrup (HFCS) is a sweetener composed primarily of glucose and fructose, which is ubiquitous in processed foods and beverages. There is a concern for HFCS content in processed foods because HFCS has been linked to metabolic disorders, including diabetes. There is also a need for quantitation of sugar in naturally sweetened beverages since sucrose may degrade during the manufacturing process. A number of HPLC methods have been used to quantitate sucrose, glucose, and fructose including HILIC and Ligand Exchange 4. However, HILIC methods will use high amounts of organic and analysis might require extensive method development. Previous Ligand Exchange methods provide linearity and limits of quantitation that are not practical for real-world applications. water. Although not performed in this study, the use of a.45 µm Phenex -NY (Nylon) syringe filter prior to injection is encouraged for beverages with high particulate content (e.g. fruit juice with pulp). Experimental Conditions HPLC analysis was performed using an Agilent 6 LC system (Agilent Technologies, Palo Alto, CA, USA) with an upper pressure limit of 6 bar, equipped with a binary pump, autosampler, and Refractive Index (RI) detector. Method conditions are below. Column: Rezex RCM-Monosaccharide Ca + Dimensions: x 7.8 mm Part No.: D--K Mobile Phase: Water Flow Rate:.4 ml/min Temperature: 8 ºC Detector: RI (Refractive 5 ºC Sample: Sucrose In this study, we demonstrate the use of Rezex RCM-Monosaccharide Ca + to develop a quick and quantitative method under 5 minutes for the analysis of the three main sugars in various beverages and fruit juices: sucrose, glucose, and fructose. Materials and Methods Reagents and Chemicals Sugar standards were purchased from Sigma Chemical (St. Louis, MO). Beverages were obtained from local grocers. A Sartorius arium comfort II was used for ASTM Type ultrapure water. Nine standards were prepared in water at concentrations from. mg/ ml to 5 mg/ml. Beverage samples were diluted : in ultrapure For additional technical notes, visit Page of 8

2 TN-8 Results and Discussion A nine-point standard curve was run for sucrose, glucose, and fructose from. mg/ml to 5 mg/ml (Table ). Retention times for standards are shown in Table. Using peak areas, a linear regression line was drawn for each sugar respectively, with correlation coefficients greater than.99 for each (Figures -). Figure. Standard curve for sucrose,.-5 mg/ml Column: Rezex RCM-Monosaccharide Ca + Dimensions: x 7.8 mm Part No.: D--K Mobile Phase: Water Flow Rate:.4 ml/min Temperature: 8 ºC Detector: RI (Refractive 5 ºC Sample: Sucrose Area ( ) Sucrose y = 474x + 95 R =.9984 Sucrose Table. Standard concentrations Standards Conc. (mg/ml) Area ( ) Concentration (mg/ml) Figure. Standard curve for glucose,.-5 mg/ml 6 y = 585x + 78 R = Concentration (mg/ml) Figure. Standard curve for fructose,.-5 mg/ml 6 5 y = 498x R =.9985 Area ( ) Concentration (mg/ml) Page of 8

3 TN-8 Typically in ion exclusion and ligand exchange, a minimum of a one minute retention time difference is required for baseline separation. Baseline separation is clearly observed in Figures 4 & 5, at. mg/ml and 5 mg/ml, respectively. Concentrations for each beverage were calculated using the calibration curve then multiplied by to adjust for the dilution factor (Table ). Figure 4.. mg/ml Sugar Standard, prepared in Water 6 Table. Standard Retention Times Sugar Retention Time (min). Sucrose min Figure 5. 5 mg/ml Sugar Standard, prepared in Water App ID 5 Column: Rezex RCM-Monosaccharide Ca + Dimensions: x 7.8 mm Part No.: D--K Mobile Phase: Water Flow Rate:.4 ml/min Temperature: 8 ºC Detector: RI (Refractive 5 ºC Sample: Sucrose App ID min For additional technical notes, visit Page of 8

4 TN-8 Percent deviations were determined by comparing the concentration of the three sugars combined to expected concentration of sugars according to the nutrition facts on the beverage label. Figures 6 &7 show sugar content in red and white wine, respectively. Red wine in particular can have fructose content which gives an undesirable sweetness 5. It has been reported that between -.5 % sugar content 6 can be detected by most palates and the linearity of the assay (i.e..-5 %) is appropriate for this analysis. Table. Analytes and Sugar Content in Comparison to Reported Nutrition Facts Beverages Sucrose (mg/ml) (mg/ml) (mg/ml) Total Sugars (mg/ml) Percent Deviation from Nutrition Facts Merlot (Red Wine)...6. N/A Moscato (White Wine) N/A Orange Juice % Orange Juice % Fruit Punch % Pineapple Orange Juice % Pineapple Mango Juice % Energy Drink % Cola % Sports Drink % Figure 6. Merlot (Red Wine) Figure 7. Moscato (White Wine) ETOH ETOH App ID 56 App ID min min Page 4 of 8

5 TN-8 Figures 8 & 9 show sugar content for orange juices from two separate sources. Both are similar in sugar content. Figure shows an artificially flavored fruit punch. Only 4. mg/ml sucrose is detected, indicating sweetening primiarly with HFCS. Figure 8. Orange Juice Figure 9. Orange Juice App ID 57 App ID min min Figure. Fruit Punch 5 Column: Rezex RCM-Monosaccharide Ca + Dimensions: x 7.8 mm Part No.: D--K Mobile Phase: Water Flow Rate:.4 ml/min Temperature: 8 ºC Detector: RI (Refractive 5 ºC Sample: Sucrose 5 5 App ID min For additional technical notes, visit Page 5 of 8

6 TN-8 The two samples Pineapple Orange Juice and Pineapple Mango Juice are reported to be % juice. These juices can also contain the natural sugar alcohol sorbitol. Note in this method, the sorbitol peak elutes at approximately minutes (Figures & ) Although not the focus of this study, this method could feasibly be used for sorbitol quantitation. Figure. Pineapple Orange Juice Figure. Pineapple Mango Juice The Energy Drink sample contained two unknown peaks eluting at approximately 7-8 minutes (Figure ). Further studies would need to be performed to confirm the identity of these peaks. Cola sample in Figure 4 shows almost no sucrose, indicating the beverage is sweetened primarily with HFCS. Conversely, Sports Drink sample showed it primarily being sweetened with sucrose (Figure 5). App ID 6 min min App ID 6 Figure. Energy Drink Figure 4. Cola App ID 6 App ID min min Page 6 of 8

7 TN-8 Figure 5. Sports Drink Conclusion In this study, we demonstrated a method using Rezex RCM for the analysis of sucrose, glucose, and fructose content in processed sugary drinks and beverages. The method is quantitative, simple, and robust, being compatible with a variety of sample matrices, including wines, fruit juices, and sports drinks. Further studies could include the inclusion of sorbitol, as well as other sugar alcohols min Finally, it is is worth noting that both Orange Juice and Fruit Punch samples were outside the % discrepancy allowed by the FDA 7. App ID 64 References. Chan, Amanda L. (--7). Is There A Link Between High Corn Syrup And Diabetes Rates?. Huffington Post.. Shahidi, Fereidoon, Chi-Tang Ho, and Nguyen Van Chuyen. Process-induced Chemical Changes in Food. Vol. 44. S.l.: Springer-Verlag New York,. Print.. Ma, Chunmei, Zhen Sun, Changbao Chen, Lili Zhang, and Shuhua Zhu. Simultaneous Separation and Determination of, Sorbitol, and Sucrose in Fruits by HPLC ELSD. Food Chemistry (4): Print. 4. Mahmood, Tahir et al. Compositional Variation in Sugars and Organic Acids at Different Maturity Stages in Selected Small Fruits from Pakistan. International Journal of Molecular Sciences. (): 8 9. PMC. Web. 7 May Sugars in Wine. Wikipedia. Wikimedia Foundation. Web. 7 May Berthels, N., R. Corderootero, F. Bauer, J. Thevelein, and I. Pretorius. Discrepancy in and Utilisation during Fermentation by Wine Yeast Strains. FEMS Yeast Research (4): Print. 7. Bender, Mary M, Jeanne I. Rader, and Foster D. McClure. Guidance for Industry: FDA Nutrition Labeling Manual A Guide for Developing and Using Data Bases. US Dept of Health and Human Services, Food and Drug Administration Center for Food Safety and Applied Nutrition. Web. 7 May 5 Rezex Ordering Information Columns Guards SecurityGuard Cartridges (mm) Description Part No. Cross Linkage Ionic Form Size (mm) Part No. Size (mm) 4 x.* RCM-Monosaccharide D--K 8 % Calcium x 7.8 B--K 5 x 7.8 AJ-449 RCM-Monosaccharide F--K 8 % Calcium 5 x 7.8 B--K 5 x 7.8 AJ-449 RCM-Monosaccharide H--K 8 % Calcium x 7.8 B--K 5 x 7.8 AJ-449 RHM-Monosaccharide H--K 8 % Hydrogen x 7.8 B--K 5 x 7.8 AJ-449 RAM-Carbohydrate H--K 8 % Silver x 7.8 AJ-449 RSO-Oligosaccharide P--N 4 % Silver x. R--N 6 x. RNO-Oligosaccharide P-7-N 4 % Sodium x. R-7-N 6 x. RPM-Monosaccharide H-5-K 8 % Lead x 7.8 B-5-K 5 x 7.8 AJ-449 (for USP procedure) D-5-K 8 % Lead x 7.8 B-5-K 5 x 7.8 AJ-449 RNM-Carbohydrate H-6-K 8 % Sodium x 7.8 B-6-K 5 x 7.8 ROA-Organic Acid F-8-E 8 % Hydrogen 5 x 4.6 AJ-449 ROA-Organic Acid G-8-E 8 % Hydrogen 5 x 4.6 AJ-449 ROA-Organic Acid F-8-K 8 % Hydrogen 5 x 7.8 B-8-K 5 x 7.8 AJ-449 ROA-Organic Acid H-8-K 8 % Hydrogen x 7.8 B-8-K 5 x 7.8 AJ-449 RKP-Potassium H-5-K 8 % Potassium x 7.8 RFQ-Fast Acid D--K 8 % Hydrogen x 7.8 B--K 5 x 7.8 AJ-449 RCU-USP Sugar Alcohols G--D 8 % Calcium 5 x 4. AJ-449 for ID:.-8. mm *SecurityGuard Analytical Cartridges require universal holder Part No.: KJO-48 If Phenomenex products in this technical note do not provide at least equivalent separations as compared to other products of the same phase, size and dimensions, return the product with comparative data within 45 days for a FULL REFUND. For additional technical notes, visit Page 7 of 8

8 TN-8 APPLICATION Australia t: +6 () f: +6 () Austria t: +4 ()-9- f: +4 ()-9- Luxembourg t: + ()-487 f: + ()-8749 nlinfo@phenomenex.com Mexico t: f: tecnicomx@phenomenex.com Belgium t: + () 5 45 (French) t: + () (Dutch) f: + ()-8749 beinfo@phenomenex.com Canada t: + (8) f: + () info@phenomenex.com China t: +86 () f: +86 () 89-8 chinainfo@phenomenex.com Denmark t: f: nordicinfo@phenomenex.com Finland t: +58 () f: nordicinfo@phenomenex.com France t: + () 9 f: + () 9 franceinfo@phenomenex.com Germany t: +49 () f: +49 () anfrage@phenomenex.com India t: +9 ()4-4 f: +9 ()4-4 indiainfo@phenomenex.com Ireland t: +5 () f: eireinfo@phenomenex.com Italy t: f: italiainfo@phenomenex.com The Netherlands t: + ()-487 f: + ()-8749 nlinfo@phenomenex.com New Zealand t: +64 () f: +64 () nzinfo@phenomenex.com Norway t: f: nordicinfo@phenomenex.com Puerto Rico t: + (8) 54-HPLC f: + () info@phenomenex.com Spain t: f: espinfo@phenomenex.com Sweden t: +46 () f: nordicinfo@phenomenex.com United Kingdom t: +44 () f: +44 () ukinfo@phenomenex.com USA t: + () -555 f: + () info@phenomenex.com All other countries Corporate Office USA t: + () -555 f: + () info@phenomenex.com Phenomenex products are available worldwide. For the distributor in your country, contact Phenomenex USA, International Department at international@phenomenex.com Terms and Conditions Subject to Phenomenex Standard Terms and Conditions which may be viewed at Trademarks Rezex, SecurityGuard, and Phenex are trademarks of Phenomenex. Agilent is a registered trademark of Agilent Technologies, Inc. arium is a registered trademark of Sartorius AG. Disclaimer Dimensions and chromatographic conditions are the same for all columns unless otherwise noted. SecurityGuard is patented by Phenomenex, U.S. Patent No. 6,6,6 CAUTION: this patent only applies to the analytical-sized guard cartridge holder, and does not apply to SemiPrep, PREP or ULTRA holders, or to any cartridges. 5 Phenomenex, Inc. All rights reserved. TN55_W Page 8 of 8