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Determining Total Sugar Content in Maple Syrup to Meet FDA Nutrition Labeling Requirements

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Determining Total Sugar Content in Maple Syrup to Meet FDA Nutrition Labeling Requirements Agilent Application Solution Determining total sugar content in maple syrup to meet FDA nutrition labeling requirements Application Note Food Safety e s o r c u S Agilent 1260 Infinity LC 3 7 . 0 Author nRIU 1 35000 Syed Salman Lateef 30000 e e s s Agilent Technologies, Inc o o t c 25000 c u u l r F Bangalore, India G 5 20000 7 8 0 . 2 6 1 1 15000 10000 5000 0 0 5 10 15 20 25 min Abstract Nutrition labels on food items provide valuable information to consumers. To ensure authenticity, the Food and Drug Administration (FDA) checks the reported values on the nutrition labels. This Application Note shows a total sugar analysis method for commer - cially available maple syrup using the Agilent 1260 Infinity Binary LC system coupled with the Agilent 1260 Infinity Refractive Index Detector. The method involved the use of Agilent HiPlex Ca column with 100% water as mobile phase. The method was validated for area and retention time (RT) precision, linearity, and accuracy. Robustness of the method was tested by varying pump flow, column temperature, injection amount, and refractive index optical unit temperature. The total sugar value reported on the maple syrup’s nutrition label was verified. This method can be used by food manufacturers to report total sugar content to meet nutrition labeling requirements. Introduction Experimental The nutrition Labeling and Education Instrument/Experimental parameters Details Act (NLEA) requires vendors to report Column: Agilent Hi-Plex Ca Column, 7.7 mm × 300 mm, ≥ 1 g per serving size of total sugar. 8.0 µm (p/n Pl1170-6810), operated at 85 °C The FDA monitors food items for com - Mobile phase: 100% water (Milli-Q), isocratic run pliance with the labeling requirement 1. Flow: 0.5 mL/min Total sugars or sugar alcohols are reported separately from carbohydrates. Injection volume: 3 µL, samples maintained at room temperature Total carbohydrates are measured 1 by Run time: 30 min subtracting the sum of crude protein, Agilent 1260 Infinity Binary Pump G1312C total fat, moisture and ash from the Agilent 1260 Infinity High Performance Autosampler G1367E total weight of the food. The total sugars are measured as the sum of dis - Agilent 1290 Infinity Thermostatted Column G1316C Compartment accharides (sucrose, lactose, mannose) Agilent 1260 Infinity Refractive Index Detector G1362A and monosaccharides (glucose, galactose and fructose). The choice of Agilent 1260 Infinity Standard Degasser G1322A method in sugar analysis is based on Agilent ChemStation for Liquid Chromatography B.04.02[SP1] the prior knowledge of composition of Agilent 1260 Infinity Refractive Index Detector parameters sugars in food. For example, sucrose, a Optical Unit Temperature: 30 °C disaccharide, is the major constituent present in maple syrup which also may Attenuation: 500000 nRIU contain trace amount of glucose and Material and instrumentation Recovery studies fructose. An acceptable method to ana - lyze total sugars in maple syrup would Maple syrup samples were obtained The recovery experiments were per - require analysis of sucrose. from local stores in the Unites States of formed by spiking commercially avail - America. Sucrose, glucose and fructose able maple syrup with sucrose, at two UV absorbance at ~200 nm shows poor standards were obtained from Sigma- different sucrose concentrations. At a response for sugars due to low extinc - Aldrich and dried for 12 hrs at 50 °C high concentration spike, sucrose was tion coefficients. The AOAC method, under vacuum. spiked to 40% higher sucrose than 920.189 2, describes the analysis of reported on the nutrition label. At a low sucrose in maple products using Extraction procedure concentration spike, where sucrose polarimeter before, and after inversion 0.75 mL of maple syrup was added into was spiked at 20% higher sucrose than using Invertase enzyme. Due to pre-weighed 50 mL centrifuge tube. The reported on the nutrition label. The advancements in chromatography and difference in weight was recorded. recovery is measured as a difference universal analysis of sugars by refrac - Twenty-five mL of water was added and between high concentration spike and tive index detectors (RI) 3, RI is the pre - vortexed. The centrifuge tubes were low concentration spike where the area ferred method for sugar analysis. The placed in sonicator for 25 min and the obtained was back calculated to con - AOAC methods, 977.20 and 980.13, for solution was filtered using glass centration using the linearity curve. The analysis of total sugars in honey, and microfiber and cellulose nitrate 2 in 1 density of maple syrup used in the cal - chocolates 4,5 involve carbohydrate syringe filter 0.45 µm (p/n 5042-1391). culation was empirically determined as column and refractive index detector. The filtered solution was used for 1.3 g/mL. Due to the viscous nature of Here we report a total sugar analysis analysis. maple syrup, the weight was used to method for commercially available maple obtain accurate volume and also to nor - syrup using Agilent HiPlex Ca Column, a malize samples (mg of sucrose per cation exchange column and Agilent gram of maple syrup). The variation 1260 Infinity Refractive Index Detector. from the added value was reported in percentage as recovery. 2 Method validation e s o The sucrose method for maple syrup r c u was validated using aqueous standards S 3 of sucrose, glucose and fructose. 7 . 0 Precision, linearity, accuracy and carry - nRIU 1 over were obtained for sucrose. The 35000 validation for glucose and fructose was performed separately and the limit of 30000 detection (LOD) and the limit of quanti - e e s s tation (LOQ) were determined. The o o t c 25000 c u u l analyte concentration that provides a r F signal to noise ratio (S/N) of >3 was G 5 20000 7 8 0 . considered as LOD and analyte concen - . 2 6 1 tration with S/N > 10 was considered 1 as LOQ. A linearity curve for each com - 15000 pound was constructed from the LOQ level to a maximum concentration. Each 10000 linearity solution was injected six times. Accuracy was determined as back cal - 5000 culated concentration from the linearity equation. To determine the sensitive 0 parameters in the method, method 0 5 10 15 20 25 min robustness was performed by varying pump flow (± 2%), column temperature Figure 1 (± 5%), Injection volume (± 5%), and RI Chromatogram showing the separation of 10 mg/mL each of sucrose, glucose and fructose using an optical unit temperature (± 5%) 6. Each Agilent HiPlex Ca column. experimental variation run was per - formed for six replicates. Robustness testing was done using a mixture of 27 mg/mL of sucrose, 0.5 mg/mL each of glucose and fructose. Results and discussion Method development Individual standards of monosaccharide and disaccharides were injected on a Hi-Plex Ca column showing good reso - lution for sucrose, glucose and fructose as shown in Figure 1. Hi-Plex Ca is a ligand exchange column which requires low flow rate and high column tempera - ture for analysis. Water was used as mobile phase which displayed a flat baseline with the refractive index detector. 3 Filter test e Filters were tested using a standard s o r preparation by measuring the peak area c u with and without the filter. The results S 9 6 . show that nylon, PTFE and regenerated 0 nRIU 1 cellulose filters showed an area recov - 100000 ery of 60% while glass microfiber showed area recovery of 100%. Glass 80000 microfiber and cellulose nitrate 2 in 1 syringe filters showed an area recovery 60000 s s k of 104%. This filter was selected for the k a a e e p p analysis because of their pore size of x 40000 x i i r r t 0.45 u, as small pore size helps to t a a Blank M remove particulates from samples. For M 20000 3 chromatogram 6 6 8 . sample analysis, the area recovery from . 2 5 1 syringe filters are incorporated in calcu - 1 0 lations of total sucrose. 0 5 10 15 20 25 min Extraction procedure To obtain an extraction method opti - Figure 2 mized for maximum recovery, maple Agilent HiPlex Ca column separation of maple syrup extract spiked with high concentration of syrup was tested by dissolving it in sucrose, glucose and fructose peaks. Glucose and fructose peaks contribute 2% of the sucrose peak either water or 50% acetonitrile-water area. Matrix peaks occurs at 12.63 min and 15.86 min which overlap with the glucose and fructose followed by either heating at 85 °C or peaks (see Figure 1). The blank chromatogram showed flat baseline and low background interference to analyte peak. sonication for 25 minutes. In all cases, the extracted solvent was filtered with cellulose nitrate and glass microfiber 2 in 1 syringe filter. Dissolving in water followed by sonication yielded better chromatography. Comparable extraction was achieved using 50% acetonitrile- water solution followed by heating. Water as dissolution solvent followed by sonication was used in recovery and sample analysis. Recovery studies The recovery experiments were per - formed by spiking maple syrup with sucrose. Since sucrose free maple syrup is not available, the recovery is measured as a difference between high concentration spike and low concentra - tion spike. In food analysis, if a blank matrix is available, standards are spiked at two or three different concen - trations and compared with the blank 7. For this study, since a blank matrix is not available, sucrose is spiked at two 4 level concentrations to account for Validation protocol Experimental runs Results compounds already present in the matrix or account for matrix interfering Sucrose peaks.
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