Analysis of Sugars in Honey

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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. 2 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.
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