Analysis of Sugars in Honey

APPLICATION NOTE

Liquid Chromatography

Authors: Chi Man Ng Wilhad M. Reuter PerkinElmer, Inc. USA

Analysis of Sugars in Honey Using the Introduction Honey consumption has grown PerkinElmer Altus HPLC significantly during the last few decades due to its high nutritional System with RI Detection value and unique flavor. The price of natural bee honey is much higher than other sweeteners making it susceptible to adulteration with cheaper sweeteners, primarily sucrose. Besides lower levels of non- sugar ingredients, natural honey primarily consists of glucose and fructose and may contain low levels of sucrose and/or maltose.1, 2 However, according to the international regulations, any commercially available “pure”-labeled honey products that are found to have in excess of 5% by weight of sucrose or maltose are considered to be adulterated.3 With the focus on possible honey adulteration, this application highlights the LC separation of various sugars found in honey and the analysis of these components in four store- bought honey samples. Method conditions and performance data, including linearity and repeatability, are presented. Experimental Solvents, Standards and Samples Hardware/Software All solvents and diluents used were HPLC grade and filtered via For all chromatographic separations, a PerkinElmer Altus™ 0.45-µm filters. HPLC system was used, including the Altus A-10 Solvent and The sugar standards were obtained from Supelco® (Irvine, CA) Sample Module, Column Module, integrated vacuum degasser/ and consisted of fructose, glucose, maltose and sucrose. Stock column oven and an Altus A-10 RI Detector. All instrument sugar standards were made using 65:35 acetonitrile/water as control, analysis and data processing was performed using the diluent. For the 1333 µg/mL (ppm) stock solution, the standards Waters® Empower® 3 CDS platform. were first dissolved in 17.5 mL of water before adding 32.5 mL Method Parameters of acetonitrile. The lower level standards were then prepared The HPLC method parameters are shown in Table 1 from this stock solution. All commercially available honey products were purchased at Table 1. HPLC Method Parameters. local stores. They were labeled Honey W, Honey X, Honey Y HPLC Conditions and Honey Z. Each honey was prepared by dissolving 2.5 g into PerkinElmer Brownlee™ Analytical Amino 3 µm, Column: 50 mL of 65:35 acetonitrile/water, followed by another 1:1 4.6 x 150 mm (Part# N9303505) dilution using the same solvent. Solvent A: 65:35 acetonitrile/water Solvent program: Prior to injection, all calibrants and samples were filtered through

Mobile Phase: Time Flow Rate 0.45-µm filters to remove small particles. %A %B %C %D Curve (min) (mL/min) Initial 1.000 100.0 0.0 0.0 0.0 Initial ResultsChro and DiscussionmatogramOnly Report Analysis Time 6 min. Figure 1 shows the chromatographic separation of the 1333-µg/mL Flow Rate: 1.0 mL/min. (2300 psi) (ppm) sugar standard containing the four target sugars using Oven Temp.: 25 ºC the optimized conditions described above. The analysis time was Detection: Altus A-10 RI; cell temp.: 35 °C under six minutes. Injection Volume: 5 µL Sampling (Data) Rate: 10 pts./sec

22.00

20.00 se to

18.00 uc Fr

16.00

14.00

12.00 IU e µR 10.00 os cr Su

8.00 e os

6.00 uc Gl e

4.00 os lt Ma 2.00

0.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 Minutes

Figure 1. Chromatogram of the 1333 µg/mL sugar standard.

Reported by User: System Project Name: Sugars in Honey with RI Report Method: ChromatogramOnly Report Date Printed: Report Method ID2160: 2160 1/30/2015 Page: 1 of 1 2:55:30 PM US/Eastern LC Calibration Report

Processi ng Method: Linearity 01152015_PM System: Acquity with RI Processi ng Method ID: 3183 Channel: RI Calibration ID: 3502 Proc. Chnl. Descr.: RI Date Calibrated: 2/3/2015 4:32:46 PM EST

Peak: Fructose Calc. % Sample Name Resu lt Id Peak Name Level X Value Resp onse Value Deviation 1 Sugar Std cal inj 1uL 3507 Fructose Level 1 133.000 1616.109 124.919 -6.08 2 Sugar Std cal inj 1uL 3514 Fructose Level 1 133.000 2546.947 144.291 8.49 3 Sugar Std cal inj 1uL 3516 Fructose Level 1 133.000 2721.115 147.901 11.20 4 Sugar Std cal inj 2uL 3518 Fructose Level 2 267.000 8715.301 269.327 0.87 5 Sugar Std cal inj 2uL 3520 Fructose Level 2 267.000 8087.928 256.863 -3.80 6 Sugar Std cal inj 2uL 3522 Fructose Level 2 267.000 7865.688 252.434 -5.46 7 Sugar Std cal inj 5uL 3524 Fructose Level 3 667.000 31054.842 682.181 2.28 LC Calibration Report 8 Sugar Std cal inj 5uL 3526 Fructose Level 3 667.000 29148.994 649.025 -2.69 9 Sugar Std cal inj 5uL 3528 Fructose Level 3 667.000 30110.834 665.801 -0.18

Processi ng Method: Linearity 01152015_PM System: Acquity with RI 10 Sugar Std cal inj 7uL 3530 Fructose Level 4 933.000 47347.600 952.907 2.13 Processi ng Method ID: 3183 Channel: RI 11 Sugar Std cal inj 7uL 3532 Fructose Level 4 933.000 45187.067 918.215 -1.58 Calibration ID: 3502 Proc. Chnl. Descr.: RI 12 Sugar Std cal inj 7uL 3534 Fructose Level 4 933.000 46532.849 939.865 0.74 Date Calibrated: 2/3/2015 4:32:46 PM EST 13 Sugar Std cal inj 10uL 3536 Fructose Level 5 1333.000 72370.335 1332.293 -0.05 14 Sugar Std cal inj 10uL 3538 Fructose Level 5 1333.000 71911.214 1325.665 -0.55 15 Sugar Std cal inj 10uL 3540 Fructose Level 5 1333.000 72700.787 1337.057 0.30 Peak: Fructose Calc. % Peak: Fructose Sample Name Resu lt Id Peak Name Level X Value Resp onse Value Deviation Manual Ignore Manual Ignore Manual Ignore LC Calibration Report 1 Sugar Std cal inj 1uL 3507 Fructose Level 1 133.000 1616.109 124.919 -6.08 1 No No 6 No No 11 No No 2 Sugar Std cal inj 1uL 3514 Fructose Level 1 133.000 2546.947 144.291 8.49 Processi ng Method: Linearity 01152015_PM System: Acquity with RI 3 Sugar Std cal inj 1uL 3516 Fructose Level 1 133.000 2721.115 147.901 11.20 2 PrNoocessi ng NoMethod ID: 7318No3 No 12 No ChanneNol: RI 4 Sugar Std cal inj 2uL 3518 Fructose Level 2 267.000 8715.301 269.327 0.87 3 CaNolibrationNo ID: 8350No2 No 13 No Proc. ChNonl. Descr.: RI 5 Sugar Std cal inj 2uL 3520 Fructose Level 2 267.000 8087.928 256.863 -3.80 4 DaNote CalibrNoated: 92/3/No2015 4:32No:46 PM ES14T No No 6 Sugar Std cal inj 2uL 3522 Fructose Level 2 267.000 7865.688 252.434 -5.46 5 No No 10 No No 15 No No 7 Sugar Std cal inj 5uL 3524 Fructose Level 3 667.000 31054.842 682.181 2.28 Peak: Fructose 8 Sugar Std cal inj 5uL 3526 Fructose Level 3 667.000 29148.994 649.025 -2.69 Reported by User: System Project Name: Sugars in Honey with RI Calc. % Sample Name Resu lt Id Peak Name Level X Value Resp onse 9 Sugar Std cal inj 5uL 3528 Fructose Level 3 667.000 30110.834 665.801 -0.18 Report Method: LC Calibration Report Value DeDaviatteio Prn inted: Report Method ID2927: 2927 2/4/2015 10 Sugar Std cal inj 7uL 3530 Fructose Level 4 933.000 47347.600 952.907 2.13 Page:1 Su 165342gar of 6Std cal inj 1uL 3507 Fructose Level 1 133.000 1616.109 124.12:3918:927 -6PM.08 US/Eastern 11 Sugar Std cal inj 7uL 3532 Fructose Level 4 933.000 45187.067 918.215 -1.58 2 Sugar Std cal inj 1uL 3514 Fructose Level 1 133.000 2546.947 144.291 8.49 12 Sugar Std cal inj 7uL 3534 Fructose Level 4 933.000 46532.849 939.865 0.74 3 Sugar Std cal inj 1uL 3516 Fructose Level 1 133.000 2721.115 147.901 11.20 13 Sugar Std cal inj 10uL 3536 Fructose Level 5 1333.000 72370.335 1332.293 -0.05 4 Sugar Std cal inj 2uL 3518 Fructose Level 2 267.000 8715.301 269.327 0.87 14 Sugar Std cal inj 10uL 3538 Fructose Level 5 1333.000 71911.214 1325.665 -0.55 5 Sugar Std cal inj 2uL 3520 Fructose Level 2 267.000 8087.928 256.863 -3.80 Peak: Glucose 15 Sugar Std cal inj 10uL 3540 Fructose Level 5 1333.000 72700.787 1337.057 0.30 6 Sugar Std cal inj 2uL 3522 Fructose Level 2 267.000 7865.688 252.434 -5.46 Calc. % Sample Name Result Id Peak Name Level X Value Response Manual Peak: Fructose 7 Sugar Std cal inj 5uL 3524 Fructose Level 3 667.000 31054.842 682.Valu18e 1 2.De28viation LC Calibration Report 81 SuSugagarr StStdd ca cal lin inj 1uj 5uL L 35263507 FrGlucuctoossee Levell 13 667.133.000000 291481012.2.91594 649.124.310235 -2-6.6.539 No Manual Ignore Manual Ignore Manual Ignore 92 SuSugagarr StStdd ca cal lin inj 1uj 5uL L 35283514 FrGlucuctoossee Levell 13 667.133.000000 301101580.6.87534 665.143.698051 -08..1048 No 1 PrNoocessi ng NoMethod: 6LinearNoity 01152015_PMNo 11 No SystemNo: Acquity with RI 103 SuSugagarr StStdd ca cal lin inj 1uj 7uL L 35303516 FrGlucuctoossee Levell 14 933.133.000000 473471717.4.63800 952.148.339087 2.1113.53 No Processi ng Method ID: 3183 Channel: RI 2 No No 7 No No 12 No No 11 Sugar Std cal inj 7uL 3532 Fructose Level 4 933.000 45187.067 918.215 -1.58 Calibration ID: 3502 Proc. Chnl. Descr.: RI 4 Sugar Std cal inj 2uL 3518 Glucose Level 2 267.000 5406.417 270.777 1.41 No 3 No No 8 No No 13 No No Date Calibrated: 2/3/2015 4:32:46 PM EST 125 SuSugagarr StStdd ca cal lin inj 2uj 7uL L 35343520 FrGlucuctoossee Levell 24 933.267.000000 465324953.1.84249 939.256.018625 0.-474.12 No 4 No No 9 No No 14 No No 136 SuSugagarr StStdd ca cal lin inj 2uj 10uLL 35363522 FrGlucuctoossee Levell 25 1333.267.00000 0 723704842.8.35535 1332.252.402983 -0-5.0.475 No 5 No No 10 No No 15 No No 147 SuSugagarr StStdd ca cal lin inj 5uj 10uLL 35383524 FrGlucuctoossee Levell 35 1333.667.00000 0 7191119010..276142 1325.683.486635 -02..5475 No Peak: Fructose 158 SuSugagarr StStdd ca cal lin inj 5uj 10uLL 35403526 FrGlucuctoossee Levell 35 1333.667.00000 0 7270017801..767878 1337.648.900557 0.-230.71 No Calc. % ReporteSad bymp Usleer Na: Symestem Resu lt Id Peak Name Level X Value PrReojecspton Naseme: Sugars in Honey with RI 9 Sugar Std cal inj 5uL 3528 Glucose Level 3 667.000 18396.468 665.960 -0.16 No Value Deviation Peak: Fructose Report Method: LC Calibration Report Date Printed: 10 Sugar Std cal inj 7uL 3530 Glucose Level 4 933.000 28878.560 953.251 2.17 No Re1 porSut garMe thStodd ca IDl2927 :in 2927j 1uL 3507 Fructose Level 1 133.000 1616.109 124.919 -6.08 2/4/2015 Manual Ignore Manual Ignore Manual Ignore 11 Sugar Std cal inj 7uL 3532 Glucose Level 4 933.000 27489.203 916.509 -1.77 No Page:2 Su 132546gar of 6Std cal inj 1uL 3514 Fructose Level 1 133.000 2546.947 144.12:3298:127 8.PM49 US/Eastern 1 No No 6 No No 11 No No 12 Sugar Std cal inj 7uL 3534 Glucose Level 4 933.000 28363.229 939.667 0.71 No 3 Sugar Std cal inj 1uL 3516 Fructose Level 1 133.000 2721.115 147.901 11.20 2 No No 7 No No 12 No No 13 Sugar Std cal inj 10uL 3536 Glucose Level 5 1333.000 44065.493 1332.755 -0.02 No 4 Sugar Std cal inj 2uL 3518 Fructose Level 2 267.000 8715.301 269.327 0.87 3 No No 8 No No 13 No No 14 Sugar Std cal inj 10uL 3538 Glucose Level 5 1333.000 43724.767 1324.640 -0.63 No 5 Sugar Std cal inj 2uL 3520 Fructose Level 2 267.000 8087.928 256.863 -3.80 4 No No 9 No No 14 No No 15 Sugar Std cal inj 10uL 3540 Glucose Level 5 1333.000 44286.279 1338.005 0.38 No 6 Sugar Std cal inj 2uL 3522 Fructo Pese ak: LeGlveucl 2os267.e 000 7865.688 252.434 -5.46 5 No No 10 No No 15 No No 7 Sugar Std cal inj 5uL 3524 Fructose Level 3 667.000 31054.842 682.Calc18. 1 2.28% Peak: Glucose Sample Name Result Id Peak Name Level X Value Response Manual Value Deviation % % 8 Sugar Std cal inj 5uL 3526 Fructose Level 3 667.000 29148.994 649.025 -2.69 Manual Ignore Manual Ignore 1 Sugar Std cal inj 1uL 3507 Glucose Level 1 133.000 1012.215 124.313 -6.53 No DeviatioRen ported by User:De Syvistatemion Project Name: Sugars in Honey with RI 9 Sugar Std cal inj 5uL 3528 Fructose Level 3 667.000 30110.834 665.801 -0.18 Report Method: LC Calibration Report Date Printed: 2 Sugar Std cal inj 1uL 3514 Glucose Level 1 133.000 1580.675 143.695 8.04 No -6.53 1 No No 0.38 15 No No 10 Sugar Std cal inj 7uL 3530 Fructose Level 4 933.000 47347.600 952.907 2.13 Report Method ID2927: 2927 2/4/2015 3 Sugar Std cal inj 1uL 3516 Glucose Level 1 133.000 1717.438 148.338 11.53 No 8.04 2 No No 11 Sugar Std cal inj 7uL 3532 Fructose Level 4 933.000 45187.067 918.215 -1.58 Page: 132546 of 6 12:38:27 PM US/Eastern 4 Sugar Std cal inj 2uL 3518 Glucose Level 2 267.000 5406.417 270.777 1.41 No 11.53 3 No No 12 Sugar Std cal inj 7uL 3534 Fructose Level 4 933.000 46532.849 939.865 0.74 5 Sugar Std cal inj 2uL 3520 Glucose Level 2 267.000 4953.142 256.012 -4.12 No 1.41 4 No No 13 Sugar Std cal inj 10uL 3536 Fructose Level 5 1333.000 72370.335 1332.293 -0.05 6 Sugar Std cal inj 2uL 3522 Glucose Level 2 267.000 4842.855 252.408 -5.47 No -4.12 5 No No 14 Sugar Std cal inj 10uL 3538 Fructose Level 5 1333.000 71911.214 1325.665 -0.55 7 Sugar Std cal inj 5uL 3524 Glucose Level 3 667.000 19010.762 683.483 2.47 No -5.47 6 No No 15 Sugar Std cal inj 10uL 3540 Fructose Level 5 1333.000 72700.787 1337.057 0.30 Peak: Glucose 8 Sugar Std cal inj 5uL 3526 Glucose Level 3 667.000 17801.678 648.905 -2.71 No 2.47 7 No No Calc. % Peak: Fructose Sample Name Result Id Peak Name Level X Value Response Manual 9 Sugar Std cal inj 5uL 3528 Glucose Level 3 667.000 18396.468 665.960 -0.16 No -2.71 8 No No Value Deviation 10 SugaManualr Std Igcanol inrej 7uL Ma3530nual IGlgnoucreose LeMavel n4ual933.Igno000re 28878.560 953.251 2.17 No -0.16 19 SugaNo r Std Nocal inj 1uL 3507 Glucose Level 1 133.000 1012.215 124.313 -6.53 No 2.17 10 No No 111 SugaNo r Std Nocal inj 7uL 6 No3532 NoGlucose 11LeNovel 4 933.No000 27489.203 916.509 -1.77 No 2 Sugar Std cal inj 1uL 3514 Glucose Level 1 133.000 1580.675 143.695 8.04 No -1.77 11 No No 122 SugaNo r Std Nocal inj 7uL 7 No3534 NoGlucose 12LeNovel 4 933.No000 28363.229 939.667 0.71 No 3 Sugar Std cal inj 1uL 3516 Glucose Level 1 133.000 1717.438 148.338 11.53 No 0.71 12 No No 133 SugaNo r Std Nocal inj 10uL 8 No3536 NoGlucose 13LeNovel 5 1333No.000 44065.493 1332.755 -0.02 No 4 Sugar Std cal inj 2uL 3518 Glucose Level 2 267.000 5406.417 270.777 1.41 No -0.02 13 No No 144 SugaNo r Std Nocal inj 10uL 9 No3538 NoGlucose 14LeNovel 5 1333No.000 43724.767 1324.640 -0.63 No 5 Sugar Std cal inj 2uL 3520 Glucose Level 2 267.000 4953.142 256.012 -4.12 No -0.63 14 No No 155 SugaNo r Std Nocal inj 10uL 10 No3540 NoGlucose 15LeNovel 5 1333No.000 44286.279 1338.005 0.38 No 6 Sugar Std cal inj 2uL 3522 Glucose Level 2 267.000 4842.855 252.408 -5.47 No 7 Sugar Std cal inj 5uL 3524 Glucose Level 3 667.000 19010.762 683.483 2.47 No Peak: Glucose Reported by User: System Project Name: Sugars in Honey with RI % % Re8 portSuga Mer thStod:d ca LCl in jCa 5ulibL ration Report3526 Glucose Level 3 667.000 17801.678 648.905 -2.7Da1 te PrinteNod: ReporMatenuald by UsIgnoreer: System Manual Ignore Project Name: Sugars in Honey with RI Deviation Deviation Report Method: LC Calibration Report Date Printed: Re9 portSuga Mer thStodd ca IDl :in 2927j 5uL 3528 Glucose Level 3 667.000 18396.468 665.960 -0.16 2/4/2015No 12:38:27 PM US/Eastern -6.53 Re1 porNot MethodNo ID29270.: 292738 15 No No 2/4/2015 10 Sugar Std cal inj 7uL 3530 Glucose Level 4 933.000 28878.560 953.251 2.17 No Page: 132546 of 6 12:38:27 PM US/Eastern 8.04 2 No No 11 Sugar Std cal inj 7uL 3532 Glucose Level 4 933.000 27489.203 916.509 -1.77 No 11.53 3 No No 12 Sugar Std cal inj 7uL 3534 Glucose Level 4 933.000 28363.229 939.667 0.71 No 1.41 4 No No 13 Sugar Std cal inj 10uL 3536 Glucose Level 5 1333.000 44065.493 1332.755 -0.02 No -4.12 5 No No 14 Sugar Std cal inj 10uL 3538 Glucos Pee ak: LeSuvecrl 5os1333e .000 43724.767 1324.640 -0.63 No -5.47 6 No No Calc. % Peak: Glucose 15 SugaSar Stmpd leca Nal inmej 10uL Resu3540lt Id GlPeakucos Nae me LeLevevel 5l 1333X Va.0lu00e 44286.Respon27se9 1338.005 0.38 NoManual Value Deviation 2.47 7 No No Calc. % Sample Name Result Id Peak Name Level X Value Response Manual Value Deviation 1 Sugar Std caPel inakj 1u: GlL ucose3507 Sucrose Level 1 133.000 921.547 124.767 -6.19 No -2.71 8 No No % % 1 Sugar Std cal inj 1uL 3507 Glucose Level 1 133.000 1012.215 124.313 -6.53 No 2 MaSuganualr Std Igcanorel inj 1uL Manual3514 IgSunorecrose Level 1 133.000 1469.286 144.346 8.53 No -0.16 9 No No Deviation Deviation 2 Sugar Std cal inj 1uL 3514 Glucose Level 1 133.000 1580.675 143.695 8.04 No 3 Sugar Std cal inj 1uL 3516 Sucrose Level 1 133.000 1585.905 148.496 11.65 No 2.17 10 No No -6.53 1 No No 0.38 15 No No 3 Sugar Std cal inj 1uL 3516 Glucose Level 1 133.000 1717.438 148.338 11.53 No 4 Sugar Std cal inj 2uL 3518 Sucrose Level 2 267.000 5048.350 268.979 0.74 No -1.77 11 No No 8.04 2 No No 4 Sugar Std cal inj 2uL 3518 Glucose Level 2 267.000 5406.417 270.777 1.41 No 5 Sugar Std cal inj 2uL 3520 Sucrose Level 2 267.000 4668.514 256.011 -4.12 No 0.71 12 No No 11.53 3 No No 5 Sugar Std cal inj 2uL 3520 Glucose Level 2 267.000 4953.142 256.012 -4.12 No 6 Sugar Std cal inj 2uL 3522 Sucrose Level 2 267.000 4579.295 252.957 -5.26 No -0.02 13 No No 1.41 4 No No 6 Sugar Std cal inj 2uL 3522 Glucose Level 2 267.000 4842.855 252.408 -5.47 No 7 Sugar Std cal inj 5uL 3524 Sucrose Level 3 667.000 18042.116 681.777 2.22 No -0.63 14 No No -4.12 5 No No 7 Sugar Std cal inj 5uL 3524 Glucose Level 3 667.000 19010.762 683.483 2.47 No 8 Sugar Std cal inj 5uL 3526 Sucrose Level 3 667.000 16838.012 645.739 -3.19 No -5.47 6 No No Re8 portedSuga rby St Usd caerl: in Syj 5ustLem 3526 Glucose Level 3 667.000 Proj17801.ect Na67me8 : 648.Sugars905 in Ho-2.7ne1y with RINo 9 Sugar Std cal inj 5uL 3528 Sucrose Level 3 667.000 17632.200 669.553 0.38 No Report Method: LC Calibration Report Date Printed: 2.47 7 No No 9 Sugar Std cal inj 5uL 3528 Glucose Level 3 667.000 18396.468 665.960 -0.16 No 10 Sugar Std cal inj 7uL 3530 Sucrose Level 4 933.000 27596.266 954.751 2.33 No Report Method ID: 2927 2/4/2015 -2.71 8 No No 10 Sugar Std cal inj 7uL 3530 Glucose Level 4 933.000 28878.560 953.12:3258:127 PM2.17 US/EasteNorn 11 Sugar Std cal inj 7uL 3532 Sucrose Level 4 933.000 26162.744 915.150 -1.91 No -0.16 9 No No 11 Sugar Std cal inj 7uL 3532 Glucose Level 4 933.000 27489.203 916.509 -1.77 No 12 Sugar Std cal inj 7uL 3534 Sucrose Level 4 933.000 27105.085 941.232 0.88 No 2.17 10 No No 12 Sugar Std cal inj 7uL 3534 Glucose Level 4 933.000 28363.229 939.667 0.71 No 13 Sugar Std cal inj 10uL 3536 Sucrose Level 5 1333.000 41976.033 1329.913 -0.23 No -1.77 11 No No 13 Sugar Std cal inj 10uL 3536 Glucose Level 5 1333.000 44065.493 1332.755 -0.02 No 14 Sugar Std cal inj 10uL 3538 Sucrose Level 5 1333.000 41769.145 1324.770 -0.62 No 0.71 12 No No 14 Sugar Std cal inj 10uL 3538 Glucose Level 5 1333.000 43724.767 1324.640 -0.63 No 15 Sugar Std cal inj 10uL 3540 Sucrose Level 5 1333.000 42392.579 1340.247 0.54 No Peak: Sucrose -0.02 13 No No 15 Sugar Std cal inj 10uL 3540 Glucose Level 5 1333.000 44286.279 1338Ca.0lc05. 0.38% No Sample Name Result Id Peak Name Level X Value Response Manual-0.63 14 No NoPeak: Sucrose Value Deviation Peak: Glucose % % Manual Ignore Manual Ignore 1 Sugar Std cal inj 1uL 3507 Sucrose Level 1 133.000 921.547 124.767 -6.19 No Deviation Deviation % % Reported by User: System Project Name: Sugars in Honey with RI Manual Ignore Manual Ignore Deviation2 Sugar Std cal inDej 1uviatL ion 3514 Sucrose Level 1 133.000 1469.286 144.346 8.53 No -6.19 Re1 portNo Method:No LC0. Ca54libra15tion ReNoport No Date Printed: Report Method ID: 2927 2/4/2015 -6.53 1 No No 0.38 15 No No 3 Sugar Std cal inj 1uL 3516 Sucrose Level 1 133.000 1585.905 148.496 11.65 No 8.53 2 No No 12:38:27 PM US/Eastern 8.04 42 SugaNo r Std Nocal inj 2uL 3518 Sucrose Level 2 267.000 5048.350 268.979 0.74 No 11.65 3 No No 11.53 53 SugaNo r Std Nocal inj 2uL 3520 Sucrose Level 2 267.000 4668.514 256.011 -4.12 No 0.74 4 No No 1.41 64 SugaNo r Std Nocal inj 2uL 3522 Sucrose Level 2 267.000 4579.295 252.957 -5.26 No -4.12 5 No No -4.12 75 SugaNo r Std Nocal inj 5uL 3524 Sucrose Level 3 667.000 18042.116 681.777 2.22 No -5.26 6 No No Peak: Sucrose -5.47 86 SugaNo r Std Nocal inj 5uL 3526 Sucrose Level 3 667.000 16838.012 645.739 -3.19 No 2.22 7 No No Calc. % Sample Name Result Id Peak Name Level X Value Response Manual 2.47 97 SugaNo r Std Nocal inj 5uL 3528 Sucrose Level 3 667.000 17632.200 669.553 0.38 No -3.19 8 No No Value Deviation -2.71 108 SugaNo r Std Nocal inj 7uL 3530 Sucrose Level 4 933.000 27596.266 954.751 2.33 No 0.38 19 SugaNo r Std Nocal inj 1uL 3507 Sucrose Level 1 133.000 921.547 124.767 -6.19 No -0.16 119 SugaNo r Std Nocal inj 7uL 3532 Sucrose Level 4 933.000 26162.744 915.150 -1.91 No 2.33 102 SugaNo r Std Nocal inj 1uL 3514 Sucrose Level 1 133.000 1469.286 144.346 8.53 No 2.17 1210 SugaNo r Std Nocal inj 7uL 3534 Sucrose Level 4 933.000 27105.085 941.232 0.88 No -1.91 113 SugaNo r Std Nocal inj 1uL 3516 Sucrose Level 1 133.000 1585.905 148.496 11.65 No -1.77 1311 SugaNo r Std Nocal inj 10uL 3536 Sucrose Level 5 1333.000 41976.033 1329.913 -0.23 No 0.88 124 SugaNo r Std Nocal inj 2uL 3518 Sucrose Level 2 267.000 5048.350 268.979 0.74 No 0.71 1412 SugaNo r Std Nocal inj 10uL 3538 Sucrose Level 5 1333.000 41769.145 1324.770 -0.62 No -0.23 135 SugaNo r Std Nocal inj 2uL 3520 Sucrose Level 2 267.000 4668.514 256.011 -4.12 No -0.02 1513 SugaNo r Std Nocal inj 10uL 3540 Sucrose Level 5 1333.000 42392.579 1340.247 0.54 No -0.62 146 SugaNo r Std Nocal inj 2uL 3522 Sucrose Level 2 267.000 4579.295 252.957 -5.26 No -0.63 14 No NoPeak: Sucrose 7 Sugar Std cal inj 5uL 3524 Sucrose Level 3 667.000 18042.116 681.777 2.22 No Reported by User: System Project Name: Sugars in Honey with RI % % 8 Sugar Std cal inj 5uL 3526 Sucrose Level 3 667.000 16838.012 645.739 -3.19 No ReportedManual by UsIgernore: System Manual Ignore Project Name: Sugars in Honey with RI Report Method: LC Calibration Report Date Printed: Deviation Deviation Report Method: LC Calibration Report Date Printed: Re9 portSuga Mer thStodd ca IDl :in 2927j 5uL 3528 Sucrose Level 3 667.000 17632.200 669.553 0.38 2/4/2015No -6.19 Re1 portNo MethodNo ID: 29270.54 15 No No 2/4/2015 10 Sugar Std cal inj 7uL 3530 Sucrose Level 4 933.000 27596.266 954.12:3758:127 PM2.33 US/EasteNorn 12:38:27 PM US/Eastern 8.53 2 No No 11 Sugar Std cal inj 7uL 3532 Sucrose Level 4 933.000 26162.744 915.150 -1.91 No 11.65 3 No No 12 Sugar Std cal inj 7uL 3534 Sucrose Level 4 933.000 27105.085 941.232 0.88 No 0.74 4 No No 13 Sugar Std cal inj 10uL 3536 Sucrose Level 5 1333.000 41976.033 1329.913 -0.23 No -4.12 5 No No 14 Sugar Std cal inj 10uL 3538 Sucros Pee ak:Le Mavellt 5os1333e .000 41769.145 1324.770 -0.62 No -5.26 6 No No Peak: Sucrose Calc. % 15 SugaSar Stmpd leca Nal inmej 10uL Resu3540lt Id SuPeakcros Nae me LeLevevel 5l 1333X Va.0lu00e 42392.Respon57se9 1340.247 0.54 NoManual 2.22 7 No No Calc. % Value Deviation Sample Name Result Id Peak Name Level X Value Response Manual Value Deviation Peak: Sucrose -3.19 8 No No 1 Sugar Std cal inj 1uL 3507 Maltose Level 1 133.000 604.361 126.202 -5.11 No % % 1 Sugar Std cal inj 1uL 3507 Sucrose Level 1 133.000 921.547 124.767 -6.19 No 2 MaSuganualr Std Igcanorel inj 1uL Manual3514 IgManoreltose Level 1 133.000 889.048 143.482 7.88 No 0.38 9 No No Deviation Deviation 2 Sugar Std cal inj 1uL 3514 Sucrose Level 1 133.000 1469.286 144.346 8.53 No 3 Sugar Std cal inj 1uL 3516 Maltose Level 1 133.000 983.771 149.205 12.18 No 2.33 10 No No -6.19 1 No No 0.54 15 No No 3 Sugar Std cal inj 1uL 3516 Sucrose Level 1 133.000 1585.905 148.496 11.65 No 4 Sugar Std cal inj 2uL 3518 Maltose Level 2 267.000 2963.948 266.023 -0.37 No -1.91 11 No No 8.53 2 No No 4 Sugar Std cal inj 2uL 3518 Sucrose Level 2 267.000 5048.350 268.979 0.74 No 5 Sugar Std cal inj 2uL 3520 Maltose Level 2 267.000 2844.989 259.151 -2.94 No 0.88 12 No No 11.65 3 No No 5 Sugar Std cal inj 2uL 3520 Sucrose Level 2 267.000 4668.514 256.011 -4.12 No 6 Sugar Std cal inj 2uL 3522 Maltose Level 2 267.000 2724.392 252.166 -5.56 No -0.23 13 No No 0.74 4 No No 6 Sugar Std cal inj 2uL 3522 Sucrose Level 2 267.000 4579.295 252.957 -5.26 No 7 Sugar Std cal inj 5uL 3524 Maltose Level 3 667.000 10845.862 687.319 3.05 No -0.62 14 No No -4.12 5 No No 7 Sugar Std cal inj 5uL 3524 Sucrose Level 3 667.000 18042.116 681.777 2.22 No 8 Sugar Std cal inj 5uL 3526 Maltose Level 3 667.000 9908.221 640.276 -4.01 No -5.26 6 No No Re8 portedSuga rby St Usd caerl: in Syj 5ustLem 3526 Sucrose Level 3 667.000 Proj16838.ect Na01me2 : 645.Sugars739 in Ho-3.1ne9y with RINo 9 Sugar Std cal inj 5uL 3528 Maltose Level 3 667.000 10421.724 666.129 -0.13 No 2.22 7 No No Report Method: LC Calibration Report Date Printed: 9 Sugar Std cal inj 5uL 3528 Sucrose Level 3 667.000 17632.200 669.553 0.38 No 10 Sugar Std cal inj 7uL 3530 Maltose Level 4 933.000 16482.005 956.178 2.48 No Report Method ID: 2927 2/4/2015 -3.19 8 No No 10 Sugar Std cal inj 7uL 3530 Sucrose Level 4 933.000 27596.266 954.12:3758:127 PM2.33 US/EasteNorn 11 Sugar Std cal inj 7uL 3532 Maltose Level 4 933.000 15595.366 915.334 -1.89 No 0.38 9 No No 11 Sugar Std cal inj 7uL 3532 Sucrose Level 4 933.000 26162.744 915.150 -1.91 No 12 Sugar Std cal inj 7uL 3534 Maltose Level 4 933.000 16195.141 943.018 1.07 No 2.33 10 No No 12 Sugar Std cal inj 7uL 3534 Sucrose Level 4 933.000 27105.085 941.232 0.88 No 13 Sugar Std cal inj 10uL 3536 Maltose Level 5 1331.767 -0.09 No -1.91 11 No No 1333.000 25146.237 13 Sugar Std cal inj 10uL 3536 Sucrose Level 5 1333.000 41976.033 1329.913 -0.23 No 14 Sugar Std cal inj 10uL 3538 1322.233 -0.81 No 0.88 12 No No Maltose Level 5 1333.000 24914.904 14 Sugar Std cal inj 10uL 3538 Sucrose Level 5 1333.000 41769.145 1324.770 -0.62 No 15 1340.124 Peak: Maltose -0.23 13 NoSugar Std Nocal inj 10uL 3540 Maltose Level 5 1333.000 25349.509 0.53 No 15 Sugar Std cal inj 10uL 3540 Sucrose Level 5 1333.000 42392.579 1340Ca.2lc47. 0.54% No Sample Name Result Id Peak Name Level X Value Response Manual-0.62 14 No No Peak: Maltose Value Deviation Peak: Sucrose % % 1 Sugar Std cal inj 1uL 3507 Maltose Level 1 133.000 604.361 126.202 -5.11 No Manual Ignore Manual Ignore % % DeviatioRen ported by User: DeSyvistatemion Project Name: Sugars in Honey with RI Manual Ignore Manual Ignore Overlay Report Report Method: LC Calibration Report Date Printed: Deviation2 Sugar Std cal inDej 1uviatL ion 3514 Maltose Level 1 133.000 889.048 143.482 7.88 No -5.11 1 No No 0.53 15 No No Report Method ID: 2927 2/4/2015 -6.19 31 SugaNo r Std Nocal in0.j 541uL 15 No3516 NoMaltose Level 1 133.000 983.771 149.205 12.18 No 7.88 2 No No 12:38:27 PM US/Eastern 8.53 42 SugaNo r Std Nocal inj 2uL 3518 Maltose Level 2 267.000 2963.948 266.023 -0.37 No 12.18 3 No No 11.65 53 SugaNo r Std Nocal inj 2uL 3520 Maltose Level 2 267.000 2844.989 259.151 -2.94 No -0.37 4 No No 0.74 64 SugaNo r Std Nocal inj 2uL 3522 Maltose Level 2 267.000 2724.392 252.166 -5.56 No -2.94 5 No No -4.12 75 SugaNo r Std Nocal inj 5uL 3524 Maltose Level 3 667.000 10845.862 687.319 3.05 No -5.56 6 No No Peak: Maltose -5.26 86 SugaNo r Std Nocal inj 5uL 3526 Maltose Level 3 667.000 9908.221 640.276 -4.01 No 7 3.05 No No Calc. % Sample Name Result Id Peak Name Level X Value Response Manual 2.22 97 SugaNo r Std Nocal inj 5uL 3528 Maltose Level 3 667.000 10421.724 666.129 -0.13 No -4.01 8 No No Value Deviation -3.19 108 SugaNo r Std Nocal inj 7uL 3530 Maltose Level 4 933.000 16482.005 956.178 2.48 No -0.13 19 SugaNo r Std Nocal inj 1uL 3507 Maltose Level 1 133.000 604.361 126.202 -5.11 No 0.38 119 SugaNo r Std Nocal inj 7uL 3532 Maltose Level 4 933.000 15595.366 915.334 -1.89 No 2.48 102 SugaNo r Std Nocal inj 1uL 3514 Maltose Level 1 133.000 889.048 143.482 7.88 No 2.33 1210 SugaNo r Std Nocal inj 7uL 3534 Maltose Level 4 933.000 16195.141 943.018 1.07 No -1.89 113 SugaNo r Std Nocal inj 1uL 3516 Maltose Level 1 133.000 983.771 149.205 12.18 No -1.91 1311 SugaNo r Std Nocal inj 10uL 3536 Maltose Level 5 1333.000 25146.237 1331.767 -0.09 No 1.07 124 SugaNo r Std Nocal inj 2uL 3518 Maltose Level 2 267.000 2963.948 266.023 -0.37 No 0.88 1412 SugaNo r Std Nocal inj 10uL 3538 Maltose Level 5 1333.000 24914.904 1322.233 -0.81 No -0.09 135 SugaNo r Std Nocal inj 2uL 3520 Maltose Level 2 267.000 2844.989 259.151 -2.94 No -0.23 1513 SugaNo r Std Nocal inj 10uL 3540 Maltose Level 5 1333.000 25349.509 1340.124 0.53 No -0.81 146 SugaNo r Std Nocal inj 2uL 3522 Maltose Level 2 267.000 2724.392 252.166 -5.56 No -0.62 14 No No Peak: Maltose 7 Sugar Std cal inj 5uL 3524 Maltose Level 3 667.000 10845.862 687.319 3.05 No Reported by User: System Project Name: Sugars in Honey with RI % % 8 Sugar Std cal inj 5uL 3526 Maltose Level 3 667.000 9908.221 640.276 -4.01 No ReportedManual by UsIgernore: System Manual Ignore Project Name: Sugars in Honey with RI Report Method: LC Calibration Report Date Printed: Deviation Deviation 9 Report Method: LC Calibration Report Date Printed: ReportSuga Mer thStodd ca IDl :in 2927j 5uL 3528 Maltose Level 3 667.000 10421.724 666.129 -0.13 2/4/2015No -5.11 Re1 portNo MethodNo ID: 29270.53 15 No No 2/4/2015 10 Sugar Std cal inj 7uL 3530 Maltose Level 4 933.000 16482.005 956.12:3178:827 PM2.48 US/EasteNorn 12:38:27 PM US/Eastern 7.88Figure2 No 2 showsNo the overlay of 12 replicate 667-µg/mL sugar standard injections,11 Sugar Std ca demonstratingl inj 7uL 3532 Ma ltexceptionalose Level 4 933. reproducibility.000 15595.366 915. Retention334 -1.89 No 12time.18 3 %No RSDsNo were also quite exceptional, exemplified by 0.026% RSD for12 Suga fructose.r Std cal inj 7uL 3534 Maltose Level 4 933.000 16195.141 943.018 1.07 No -0.37 4 No No 13 Sugar Std cal inj 10uL 3536 Maltose Level 5 1333.000 25146.237 1331.767 -0.09 No -2.94 5 No No 14 Sugar Std cal inj 10uL 3538 Maltose Level 5 1333.000 24914.904 1322.233 -0.81 No

-5.56 6 No No Peak: Maltose 1580000.Suga0 r Std cal inj 10uL 3540 Maltose Level 5 1333.000 25349.509 1340.124 0.53 No 3.05 7 No No Calc. % Sample Name Result Id Peak Name Level X Value Response Manual Value Deviation 70000.0 Peak: Maltose -4.01 8 No No8.00 % % 1 Sugar Std cal inj 1uL 3507 Maltose Level 1 133.000 604.361 126.202 -5.11 No Manual Ignore Manual Ignore -0.13 9 No No Deviation60000.0 Deviation 2 Sugar Std cal inj 1uL 3514 Maltose Level 1 133.000 889.048 143.482 7.88 No 2.48 10 No No -5.11 150000.No0 No 0.53 15 No No 3 Sugar Std cal inj 1uL 3516 Maltose Level 1 133.000 983.771 149.205 12.18 No -1.89 11 No No7.00 7.88 ea 2 No No

4 Sugar Std cal inj 2uL 3518 Maltose Level 2 267.000 2963.948 266.023 -0.37 No Ar 40000.0 1.07 12 No No 12.18 3 No No 5 Sugar Std cal inj 2uL 3520 Maltose Level 2 267.000 2844.989 259.151 -2.94 No -0.09 13 No No -0.37 430000.No0 No 6 Sugar Std cal inj 2uL 3522 Maltose Level 2 267.000 2724.392 252.166 -5.56 No -0.81 14 No No6.00 -2.94 520000.No0 No 7 Sugar Std cal inj 5uL 3524 Maltose Level 3 667.000 10845.862 687.319 3.05 No -5.56 6 No No Re8 portedSuga rby St Usd caerl: in Syj 5ustLem 3526 Maltose Level 3 667.000 Proj9908ect Na.221me: 640.Sugars276 in Ho-4.0ne1y with RINo 10000.0 3.05 7 No No Report Method: LC Calibration Report Date Printed: 9 Sugar Std cal inj 5uL 3528 Maltose Level 3 667.000 10421.724 666.129 -0.13 No 0.0 Report Method 5.ID00: 2927 2/4/2015 -4.01 8 No No 10 Sugar Std cal inj 7uL 3530 Maltose Level 4 933.000 16482.005 956.178 2.48 No 12:38:27 PM US/Eastern -0.13 9 No 0.00 No 200.00 400.00 600.00 800.00 1000.00 1200.00 11 Sugar Std cal inj 7uL 3532 Maltose Level 4 933.000 15595.366 915.334 -1.89 No Concentration 2.48 10 NoPeak NaNome: Fructose; RT: 3.430; Fit Type: Quadratic (2nd Order); Cal Curve Id: 3503; R: 0.999954; 12 Sugar Std cal inj 7uL 3534 Maltose Level 4 933.000 16195.141 943.018 1.07 No 4.00 -1.89 11 NoR^2: 0.999908;No Weighting: None; Equation: Y = 8.88e-003 X^2 + 4.57e+001 X - 4.23e+003; 13 Sugar Std cal inj 10uL 3536 Maltose Level 5 1333.000 25146.237 1331.767 -0.09 No Normalized Intercept/Slope: ; RSD(E): 1.220112

IU 1.07 12 No No 14 Sugar Std cal inj 10uL 3538 Maltose Level 5 1333.000 24914.904 1322.233 -0.81 No

µR -0.09 13 No No 15 Sugar Std cal inj 10uL 3540 Maltose Level 5 1333.000 25349.509 1340.124 0.53 No 50000.0 80000.0 3.00 -0.81 14 No No Peak: Maltose 70000.0 40000.0 % % Reported by User: System Project Name: Sugars in Honey with RI Manual Ignore Manual Ignore Deviation60000.0 Deviation Report Method: LC Calibration Report Date Printed: Report Method ID: 2927 2/4/2015 -5.11 1 No No2.000.53 15 No No 30000.0 50000.0 12:38:27 PM US/Eastern ea

7.88 ea 2 No No Ar

Ar 40000.0 12.18 3 No No 20000.0 -0.37 430000.No0 No1.00 -2.94 5 No No 20000.0 10000.0 -5.56 6 No No 10000.0 80000.0 3.05 7 No No 0.00 0.0 -4.01 8 No0.0 No 70000.0

-0.13 9 No 0.00 No 200.00 400.00 600.00 800.00 1000.00 1200.00 60000.0 0.00 200.00 400.00 600.00 800.00 1000.00 1200.00 Concentration Concentration 2.48 10 No No Peak Na-1me.0: Fr0uctose; RT: 3.430; Fit Type: Quadratic (2nd Order); Cal Curve Id: 3503; R: 0.999954; 50000.0Peak Name: Glucose; RT: 3.772; Fit Type: Quadratic (2nd Order); Cal Curve Id: 3504; R: 0.999959;

-1.89 11 NoR^2: 0.999908;No 0. 00Weighting: None0.; 50 Equation: Y = 1.8.8800e-003 X^2 + 4.1.57e+50001 X - 4.23e+003;2.00 2.50 3.ea 00 R^2: 0.999918;3.50 Weighting: None4.00; Equation: Y4. = 505.30e-003 X^2 +5. 2.0079e+001 X - 2.5.54e+003;50 6.00

Ar 40000.0Normalized Intercept/Slope: ; RSD(E): 1.148411 1.07 12 NoNormalizNoed Intercept/Slope: ; RSD(E): 1.220112 Minutes -0.09 13 No No 30000.0 50000.0 -0.81 14 No No Figure 2. Overlay of 12 replicates of the 667 µg/mL sugar standard. 20000.0 Reported by User: System Project Name: Sugars in Honey with RI Re40000.ported0 by User: System Project Name: Sugars in Honey with RI Re10000.port0 Method: LC Calibration Report Date Printed: Report Method: LC Calibration Report Date Printed: Report Method ID: 2927 2/4/2015 0.0 FigureReport 3Me thshowsod ID: 2927 the calibration results for all four sugars over a2/ 4/concentration2015 range of 133 to 1333 µg/mL. All four sugars12 :3followed8:27 PM US/Eas tearn 30000.0 12:38:27 PM US/Eastern nd 2 0.00 200.00 400.00 600.00 800.00 1000.00 1200.00 quadraticea (2 order) fit and had R coefficients > 0.999 (n = 3 at each level). Concentration Ar Peak Name: Fructose; RT: 3.430; Fit Type: Quadratic (2nd Order); Cal Curve Id: 3503; R: 0.999954; 20000.0 R^2: 0.999908; Weighting: None; Equation: Y = 8.88e-003 X^2 + 4.57e+001 X - 4.23e+003; Normalized Intercept/Slope: ; RSD(E): 1.220112 50000.0 10000.0 50000.0 80000.0 Fructose Glucose 40000.0 0.0 70000.0 Reported by User: System 40000.0 Project Name: Sugars in Honey with RI 60000.0 0.00 200.00 400.00 600.00 800.00 1000.00 1200.00 Report Method: OverConcenlatryatio nReport 30000.0 Date Printed: 30000.0 50000.0Peak Name: Glucose; RT: 3.772; Fit Type: Quadratic (2nd Order); Cal Curve Id: 3504; R: 0.999959; ea ea ea R^2: 0.999918; Weighting: None; Equation: Y = 5.30e-003 X^2 + 2.79e+001 X - 2.54e+003; Ar Report Method ID2232: 2232 20000.0 1/30/2015 Ar 40000.0Normalized Intercept/Slope: ; RSD(E): 1.148411 Ar 20000.0 30000.0 Page: 1 of 1 3:05:55 PM US/Eastern 10000.0 20000.0 10000.0 Reported by User: System Project Name: Sugars in Honey with RI 10000.0 Report Method: LC Calibration Report 2 Date Printed: 0.0 2 Report Method ID: 2927 R = 0.999954 2/4/2015 0.0 R = 0.999959 0.0 12:38:27 PM US/Eastern 0.00 200.00 400.00 600.00 800.00 1000.00 1200.00 0.00 200.00 400.00 600.00 800.00 1000.00 1200.00 0.00 200.00 400.00 600.Co00ncentration 800.00 1000.00 1200.00 Concentration Peak Name: Sucrose; RT: 4.464; Fit Type: QuadraCoticncen (2trndatio nOrder); Cal Curve Id: 3505; R: 0.999952; Peak Name: Fructose; RT: 3.430; Fit Type: Quadratic (2nd Order); Cal Curve Id: 3503; R: 0.999954; PeakR^2: 0.999903;Name: Gl uc Weose;ig ht RTing:: 3.None772;; FiEqt uaTytipeon: :Qu Y adra= 5.1ti4e-003c (2nd OrdeX^2 +r) ;2. 66e+Cal Curv001 eX Id - :2. 350448e+003;; R: 0. 999959; R^2: 0.999908; Weighting: None; Equation: Y = 8.88e-003 X^2 + 4.57e+001 X - 4.23e+003; RNorm^2: 0.999918;alized Inte rc Weept/Sightloinpeg:: ;None RSD(; E): Eq 1.252103uation: Y = 5.30e-003 X^2 + 2.79e+001 X - 2.54e+003; Normalized Intercept/Slope: ; RSD(E): 1.220112 Normalized Intercept/Slope: ; RSD(E): 1.148411 50000.0 30000.0

50000.0 Sucrose Maltose 25000.0 40000.0 Reported by User: System Project Name: Sugars in Honey with RI 40000.0 Re20000.port0 Method: LC Calibration Report Date Printed:

30000.0 Report Method ID: 2927 2/4/2015 12:38:27 PM US/Eastern 30000.0 15000.0 ea ea ea Ar Ar 20000.0 Ar 10000.0 20000.0

10000.0 5000.0 50000.0 10000.0

2 0.0 2 0.0 R = 0.999952 R = 0.999940 0.0 40000.0 -5000.0 0.00 200.00 400.00 600.00 800.00 1000.00 1200.00 0.00 200.00 400.00 600.00 800.00 1000.00 1200.00 0.00 200.00 400.00 600.Co00ncentration 800.00 1000.00 1200.00 Concentration Concentration 30000.0 Peak Name: Sucrose; RT: 4.464; Fit Type: Quadratic (2nd Order); Cal Curve Id: 3505; R: 0.999952; Peak Name: Maltose; RT: 5.155; Fit Type: Quadratic (2nd Order); Cal Curve Id: 3506; R: 0.999940;

PeakR^2: 0.999903;Name: Gl uc Weose;ig ht RTing:: 3.None772;; FiEqt uaTytipeon: :Qu Y adra= 5.1ti4e-003c (2nd OrdeX^2 +r) ;2. 66e+Cal Curv001 eX Id- :2. 350448e+003;; R: 0.999959; ea R^2: 0.999881; Weighting: None; Equation: Y = 3.27e-003 X^2 + 1.56e+001 X - 1.42e+003;

Figure 3. RNormResults^2: 0.999918;alized of Inte 5-level rc Weept/Sightloin pecalibrationg:: ;None RSD(; E): Eq 1.252103ua setstion: Yfor = 5.30 fructose,e-003 X^ 2glucose, + 2.79e+001 maltose X - 2.54e+003; and sucrose. Ar Normalized Intercept/Slope: ; RSD(E): 1.389827 Normalized Intercept/Slope: ; RSD(E): 1.148411 20000.0 30000.0

10000.0 25000.0 Reported by User: System Project Name: Sugars in Honey with RI Reported by User: System Project Name: Sugars in Honey with RI Report Method: LC Calibration Report Date Printed: 3 Re20000.port0 Method: LC Calibration Report Date Printed: Report0.0 Method ID: 2927 2/4/2015 Report Method ID: 2927 2/4/2015 12:38:27 PM US/Eastern 12:38:27 PM US/Eastern 15000.0 0.00 200.00 400.00 600.00 800.00 1000.00 1200.00 ea Concentration Ar Peak Name: Sucrose; RT: 4.464; Fit Type: Quadratic (2nd Order); Cal Curve Id: 3505; R: 0.999952; 10000.0 R^2: 0.999903; Weighting: None; Equation: Y = 5.14e-003 X^2 + 2.66e+001 X - 2.48e+003; Normalized Intercept/Slope: ; RSD(E): 1.252103 5000.0 50000.0 30000.0

0.0 25000.0 40000.0 -5000.0 0.00 200.00 400.00 600.00 800.00 1000.00 1200.00 20000.0 Concentration 30000.0 Peak Name: Maltose; RT: 5.155; Fit Type: Quadratic (2nd Order); Cal Curve Id: 3506; R: 0.999940; 15000.0 ea R^2: 0.999881; Weighting: None; Equation: Y = 3.27e-003 X^2 + 1.56e+001 X - 1.42e+003; ea Ar Normalized Intercept/Slope: ; RSD(E): 1.389827 Ar 20000.0 10000.0

10000.0 5000.0 Reported by User: System Project Name: Sugars in Honey with RI Report Method: LC Calibration Report Date Printed: 0.0 Report0.0 Method ID: 2927 2/4/2015

12:38:27 PM US/Eastern -5000.0 0.00 200.00 400.00 600.00 800.00 1000.00 1200.00 0.00 200.00 400.00 600.00 800.00 1000.00 1200.00 Concentration Concentration Peak Name: Sucrose; RT: 4.464; Fit Type: Quadratic (2nd Order); Cal Curve Id: 3505; R: 0.999952; Peak Name: Maltose; RT: 5.155; Fit Type: Quadratic (2nd Order); Cal Curve Id: 3506; R: 0.999940; R^2: 0.999903; Weighting: None; Equation: Y = 5.14e-003 X^2 + 2.66e+001 X - 2.48e+003; R^2: 0.999881; Weighting: None; Equation: Y = 3.27e-003 X^2 + 1.56e+001 X - 1.42e+003; Normalized Intercept/Slope: ; RSD(E): 1.252103 Normalized Intercept/Slope: ; RSD(E): 1.389827

30000.0

25000.0 Reported by User: System Project Name: Sugars in Honey with RI Report Method: LC Calibration Report Date Printed: 20000.0 Report Method ID: 2927 2/4/2015 12:38:27 PM US/Eastern 15000.0 ea Ar

10000.0

5000.0

0.0

-5000.0 0.00 200.00 400.00 600.00 800.00 1000.00 1200.00 Concentration Peak Name: Maltose; RT: 5.155; Fit Type: Quadratic (2nd Order); Cal Curve Id: 3506; R: 0.999940; R^2: 0.999881; Weighting: None; Equation: Y = 3.27e-003 X^2 + 1.56e+001 X - 1.42e+003; Normalized Intercept/Slope: ; RSD(E): 1.389827

Reported by User: System Project Name: Sugars in Honey with RI Report Method: LC Calibration Report Date Printed: Report Method ID: 2927 2/4/2015 12:38:27 PM US/Eastern Overlay Report

45.00

40.00

35.00

30.00

25.00 IU

µR 20.00

15.00

10.00

Overlay Report 5.00 Using the same chromatographic conditions, four honey samples chromatograms of these honey samples with the sugar standards, were analyzed. The chromatographic results for Honey X, it can be observed that all three honey samples contain the same Honey Y and Honey Z are shown in Figure 4. Comparing the three sugars: fructose, glucose and small amounts of sucrose.

45.00

40.00

35.00

30.00

25.00 IU

µR Sucrose 20.00

15.00 Fructose

10.00 0.00 Glucose 5.00

0.00

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 Minutes

Figure 4. Overlaid chromatograms of Honey X (green), Honey Y (black) and Honey Z (blue).

Based on standard calibration, the quantitative results for each Table 2. Quantitative Results. honey sample are shown in Table 2. Combining the fructose and Honey X: glucose percentages for each honey sample, the overall fructose Component Amount (mg) Percent Sugar (w/w) 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 and glucose content4. for Honey00 X, Y, and Z was determined to Fructose 556.054.5022.24 5.00 5.50 6.00 be 50.90%,Re 57.13%,ported by and Us er53.60%,: System respectively. These results Glucose Project Name716.48: Sugars in Hone28.66y with RI Report Method: Overlay Report Date Printed: are consistent with the accepted overall content of fructose and Sucrose 79.875 3.20 glucose in Rehoney,port expectedMethod IDto2232: be2232 somewhere around 60%.1 The 1/30/2015 Page: 1 of 1 4:03:09 PM US/Eastern sucrose content for each honey sample was determined to be Honey Y: 3.20%, 3.26% and 3.90%, respectively. These values are all Component Amount (mg) Percent Sugar (w/w) below the 5% mass ratio limit for sucrose that is allowed in Fructose 610.23 24.41 unadultered honey. Based on the data presented, the three Glucose 817.95 32.72 store-bought honey samples do not appear to be adultered Sucrose 81.525 3.26 Minutes with cheaper sweeteners. Honey Z: Upon closer examination of the chromatogram of Honey W, a smaller but significant peak was observed at about 5.10 minutes Component Amount (mg) Percent Sugar (w/w) (Figure 5). This matched the elution time for maltose in the Fructose 602.30 24.09 standard mix. The amount of maltose was calculated to be Glucose 737.78 29.51 43.85 mg, and the percent sugar was calculated to be 1.75% Sucrose 97.525 3.90 (w/w). Considering the 5% (by weight) limit that is allowed in commericially available “pure”-labeled honeys, the resulting maltose level found in Honey W suggests it was not adultered.

Reported by User: System Project Name: Sugars in Honey with RI Report Method: Overlay Report Date Printed: Report Method ID2232: 2232 1/30/2015 Page: 1 of 1 4:03:09 PM US/Eastern Overlay Report

30.00

25.00

Overlay Report 20.00

IU 15.00 µR

10.00

30.00

25.00 5.00

20.00 Fructose

IU 15.00 0.00 µR

10.00 Sucrose 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 Minutes Glucose 5.00 Maltose

0.00

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 Minutes

Figure 5. Overlay chromatograms of Honey W (red) and the 133 ppm sugar standard (black), zooming in on last eluting peak.

Conclusion References This work has demonstrated the effective chromatographic 1. W. Guo, Y. Liu, X. Zhu and S. Wang, "Dielectric properties of separation of four sugars using a PerkinElmer Altus HPLC System honey adulterated with surcrose syrup", Journal of Food with RI detection. The results exhibited very good retention time Engineering, pp. 1-7, 2011. Reported by User: System Project Name: Sugars in Honey with RI repeatability as well as excellent linearity over the tested 2. A. Moussa, D. Noureddine, A. Saad and S. Douichene, "The concentrationRe poranges.rt Method: Overlay Report Date Printed: Report Method ID2232: 2232 Relationship between Fructose, Glucose and Maltose1/30 /2Content015 Reported by User: System From a foodPage: quality 1 of perspective, 1 there is an ever growing emphasis with DiastaseProj Numberect andNa Anti-Pseudomonalme3:28: :5 0 PMSu ActivityUSgar/Eas oftes Naturalrn in Honey with RI on food monitoring. This is especially the case pertaining to the Honey Combined with Potato Starch", Organic Chemistry Report Method: Overlay Report adulteration of honey. With this in mind, this work focused on the Current Research, vol. 1, no. 5, pp. 1-5, 2012. Date Printed: sugar analysis of four store bought honeys, identifying the particular 3. Codex Alimentarius Commission, 2001; GB18796-2005, 2005 Report Method ID2232: 2232 analytes contained in each of the honey samples, as well as 1/30/2015 comparing the sugar profiles, both chromatographically and Page: 1 of 1 quantitatively. 3:28:50 PM US/Eastern

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