Mark E. Benvenuti, Gareth Cleland, and Jennifer Burgess Waters Corporation, Milford, MA, USA INTRODUCTION APPLICATION BENEFITS WATERS SOLUTIONS

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1 Profiling and Quantification of Mono and isaccharides and Selected lditols in Juice, eer, Wine, and Whiskey Using the QUITY rc System with Mass etection Mark. envenuti, Gareth leland, and Jennifer urgess Waters orporation, Milford, M, US PPLITION NFITS etection of sugars at lower levels than with Refractive Index (RI) or vaporative Light Scattering etection (LS). Minimum sample preparation required and samples can be diluted to reduce matrix effects. hromatographic separation of the difficult isomer pairs sorbitol and mannitol. INTROUTION Sugars and sugar alcohols are classes of carbohydrates that are important in human nutrition and natural constituents of foods. With the increasing incidence of obesity and diabetes across the developed world, interest in monitoring sugar intake has vastly increased in recent years. onsequently, there are now requirements to provide accurate information on product labeling in order to comply with increasingly stringent regulatory demands. Profiling the sugar content of products is also a useful tool in assessing product authenticity and potential adulteration. The analysis of sugars and sugar alcohols remains a challenging application, owing to the lack of chromophores and the similarity between these molecules. Many of these sugar compounds are isomers of one another, as can be seen in Figure 1, which illustrates the formulae and structures of the compounds analyzed in this study. ue to its separation power, accuracy, and speed of analysis, HPL has become the method of choice for the analysis of sugars. n alternative to RI and LS detection is the use of mass detection with electrospray ionization (SI). Mass detection is complementary to traditional detectors used for L. WTRS SOLUTIONS 6 H 12 6 H 12 H 1 O 5 12 H 22 O 11 QUITY rc System QUITY Qa etector Xridge H mide XP olumn 6 H 14 6 H 14 6 H 12 mpower 3 S Software KY WORS Saccharides, sugars, fructose, glucose, sucrose, maltose, mannitol, sorbitol, whisky, carbohydrate 18 H 32 O H 22 O 11 Figure 1. Structures and formulae for the sugar compounds analyzed. 1

2 X P R IM N T L L conditions L system: QUITY rc ata system: mpower 3 Runtime: olumn: 4 min olumn temp.: 85 Mobile phase : Mobile phase : Flow rate: Injection volume: 1 µl Time Flow rate Xridge XP H mide 2.5 µm, 3. x 15 mm 9% acetonitrile: 5% IP:5% water* 8% acetonitrile: 2% water*.8 ml/min (min) (ml/min) % % 1. Initial *oth containing 5 ppb guanidine hydrochloride and 5% diethylamine. MS conditions MS system: Ionization mode: apillary voltage: one voltage: QUITY Qa (Performance mode) SI-.8 V 5. V Probe temp.: 6 cquisition rate: Full scan: urve fit: 2 Hz 5 to 8 Hz Quadratic, 1/x weighting Smoothing: Mean filter, Level 7 SIR channels: nalyte Formula SIR (m/z) ([M+l] - ion) 5 H 1 O H 12 6 H 12 6 H 12 6 H 14 6 H 14 Sucrose 12 H 22 O H 22 O H 32 O Standard preparation 1 mg/l stock of the nine saccharides listed above was prepared in 1:1 acetonitrile-water. This stock was further diluted to produce nine individual levels (1, 2, 4, 5, 1, 2, 4, 5, and 1 mg/l). Sample preparation ll samples were purchased locally. The juice samples assessed included orange, apple, pineapple, pomegranate, and grape. The alcoholic beverages assessed included five beers, three lagers (one non-alcoholic), a lemon flavored beer, one hard cider, one sherry, one red wine, and four whiskeys. The beer samples were sonicated to remove carbonation. ll of the samples were filtered through a.22 µm PVF syringe filter and diluted in 1:1 acetonitrile-water. The dilution factors are listed in Table 1. Sample ilution factor Lager 1 and 2 5 Non-lcoholic 5 Hard ider 5 Lemon Flavored eer 5 Sherry Wine 5 Red Wine 5 Whiskey 2 Orange Juice 1 pple Juice 1 Pineapple Juice 1 Pomegranate Juice 1 Grape Juice 25 Table 1. ilution factors for the off-the-shelf samples studied. 2

3 It offers the opportunity to decrease detection limits and also to obtain mass spectral information on the components in the sample. The combination of both chromatographic retention time and mass information results in increased selectivity for the analysis of sugars and sugar alcohols. Here we show the application of the Waters QUITY Qa etector coupled to the QUITY rc System for the profiling and quantification of sugars in juice, wine, beer, and whiskey samples. RSULTS N ISUSSION Figure 2 shows the QUITY rc System with the QUITY Qa etector and a P etector. The P is shown for reference but was not used in this application. Figure 3 shows the SIR chromatograms for a mixed standard at 1 mg/l for each of the analytes listed above. xcellent separation of all of the standards was achieved. Initially, using isocratic conditions the lower mass saccharides were separated, including the difficult pair sorbitol and mannitol. fter 4.5 minutes a gradient was started which allowed timely separation of the larger molecular weight saccharides in the mix. Figure 2. QUITY rc System with the P and QUITY Qa detectors. 3.x x1 6 6x1 6 3x1 6 m/z 6x1 6 3x x1 6 Sucrose 8.x Figure 3. SIR chromatograms of the nine saccharide standards used in the study. The annnotated m/z represents the [M+l] - adducts. 3

4 The mass spectra extracted from the SIR of each standard is shown in Figure 4. The use of guanidine chloride in the mobile phase ensured that the compounds were driven to their chloride adduct ([M+l] - ion). The smaller 37 l adduct response was also present. Figure 5 shows the calibrations curves for the compounds studied. n R 2 value >.995 was achieved for all of the analytes. 1.x1 6 5.x1 5 3.x x1 6 3.x x1 6 3.x x x1 6 6.x Sucrose m/z 539 Figure 4. Mass spectral information extracted from SIRs of the nine saccharide standards. The annotated m/z represents the [ M+ l ] - adducts. Figure 5. alibration curves for the nine saccharide standards analyzed showing R 2 values obtained for each analyte. 4

5 Figure 6 ( ) shows the SIR profiles of a lager beer. In Figure 6 (m/z 185) arabinose is present. Other peaks are also apparent, suggesting the presence of other pentose saccharides. In Figure 6 (m/z ) traces of fructose and glucose can be seen. The enhanced sensitivity of the QUITY Qa allows improved detection of these compounds, as opposed to less sensitive methods such as Refractive Index. 1 In Figure 6 (m/z ) traces of sorbitol and mannitol are present. We also saw small peaks representing the extraction of the l 37 adducts of fructose and glucose, which have the same molecular weight as sorbitol and mannitol. In Figure 5 and 5 (m/z and 539 respectively), we observed the P2 and P3 compounds maltose and maltotriose, along with isomers of the same mass, which would be expected for a beverage derived from grain x x1 6 m/z 1.4x1 6 7.x x1 6 6.x x1 6 6.x Figure 6. nnotated SIR profile of a lager beer annotated with saccharides found to be present. sherry wine profile is shown in Figure 7 ( ). The main analytes found to be present in sherry are fructose and glucose (Figure 7). small amount of arabinose was present ( Figure 7), along with trace levels of sorbitol and mannitol (Figure 7). was also apparent (Figure 7). The P3 compounds were absent (Figure 7), as would be expected, since wine is derived from grapes rather than from grains x x1 6 m/z 1.x1 6 5.x Figure 7. SIR profile of a sherry wine annotated with saccharides found to be present. 5

6 Figure 8 ( ) shows the SIR profile of a whiskey sample. The presence of arabinose (Figure 8), fructose and glucose (Figure 8) was evident. Of particular interest was an unknown saccharide apparent in Figures 8 (m/z ) and 8 (m/z ) at retention time 4.85 minutes. Using retention time alone with an RI or LS detector, this peak would most likely have been misidentified as mannitol. The presence of this peak at both m/z and m/z indicated that this component has the same mass as a monosaccharide, rather than an alditol. does not have an ion at m/z, as can be seen in Figures 2 and 3. Finally, the SIR chromatograms from an apple juice sample are shown in Figure 9 ( ). The presence of arabinose, fructose, glucose, sorbitol, and sucrose are highlighted. The quantification of various fruit juices is shown in Table 2., glucose, and sucrose were present in the orange, apple, and pineapple juices. The amounts and ratios of sugars in these juices are similar to those reported elsewhere. 2,3 Of particular interest was the detection of sorbitol in pomegranate juice. is not usually present in pomegranate juice 4 and its detection could be evidence of adulteration. second sample tested showed no sorbitol (Figure 1). The grape juice sample showed fructose, glucose, but no sucrose as expected. 2, x1 6 2x1 6 Unknown m/z 1.2x1 6 6.x Figure 8. SIR profile of a whiskey annotated with saccharides found, and an unknown saccharide found to be present at m/z x x1 6 m/z 1.4x1 6 7.x1 5 4 Sucrose Figure 9. SIR profile of an apple juice sample annotated with saccharides found to be present. 6

7 Juice Sucrose Orange N N 52.6 pple N 1.6 Pineapple N N 41.9 Pomegranate Grape N N N Table 2. alculated concentrations from the quantification studies of the various fruit juices profiled in this study (g/l). ONLUSIONS The QUITY Qa etector coupled to the QUITY rc System provides improved sensitivity and selectivity to analyze and quantify mono and disaccharide samples in a single injection. Mass detection is a viable alternative to Refractive Index (RI) or vaporative Light Scattering (LS) methods. This enhanced sensitivity allows the analysis * Pomegranate 1 of samples at higher dilution levels, which 1.4x1 6 * minimizes matrix effects. 7.x x1 6 7.x1 5 * Pomegranate 2 *M+ 37 l- dduct * Figure 1. Zoomed-in SIR chromatograms annotated with saccharides found to be present in two pomegranate juice samples. One sample showed the presence of sorbitol, indicating a potential adulteration. The combination of mass detection and chromatographic separation provides increased selectivity in identifying analytes of interest, while reducing false positives. References 1. M astellari et al. etermination of arboxylic cids, arbohydrates, Glycerol, thanol, and 5-HMF in eer by High-Performance Liquid hromatography and UV Refractive Index ouble etection. Journal of hromatographic Science. 39: , January M L Sanz et al. s and carbohydrates in different fresh fruit juices. Food hemistry. 87: , M envenuti. nalysis of Food Sugars in Various Matrices. Waters application note no N, May, R Jahromi Reference Guide for Pomegranate. Revision June 212. nalyte RT % RS mount %RS Sucrose Table 3. Reproducibility data for retention time (min) and amount (g/l) based on seven injections of orange juice. Waters, QUITY, Qa, mpower, Xridge, and The Science of What s Possible are registered trademarks of Waters orporation. rc is a trademark of Waters orporation. ll other trademarks are the property of their respective owners. 216 Waters orporation. Produced in the U.S.. February N G-PF Waters orporation 34 Maple Street Milford, M 1757 U.S.. T: F: