SPSFAM ERP

AOAC Candidate Method SUG-01

Submitter Name Submitter Email Organization Method Type Method Name

Jack Stevens

Jack.Stevens@genmills.com

General Mills

Sugar

Analysis of the Labeling Sugars in Feed, Food and Ingredients

Method Author(s) Method Applicability

Jack Stevens

This method determines the quantity of the nutritional leveling sugars fructose, glucose, galactose, sucrose, maltose, and lactose in feed, foods and ingredients by RI-HPLC utilizing normal phase chromatography.

Upload method here Analysis of labeling sugars in food.pdf Author Notes / Comments I’m attaching our method we use here for the analysis of the labeling sugars in feed and food for humans and pets for consideration.

Analysis of the Labeling Sugars in Feed, Food and Ingredients This method determines the quantity of the nutritional labeling sugars fructose, glucose, galactose, sucrose, maltose, and lactose in feed, foods and ingredients by RI-HPLC (Refractive Index-High Performance Liquid Chromatography) utilizing normal phase chromatography. This method is applicable for all feed, food, and ingredients and which contain a minimum of 0.1 % sugar. Principle: Sugars are extracted from the sample matrices by mixing with water. The aqueous solution is diluted with acetonitrile as a clean-up preparative step to remove high molecular weight soluble oligosaccharides, fats, and proteins. The samples are filtered through a 0.2 µm membrane and the sugars separated by their adsorption on an amino column. The concentration of sugar is determined from the linear regression of the sugars in the standards compared to the sugars response detected in the samples by refractive index along with the response of the internal standard in the sample matrix and standards. Matrices containing high salts (e.g cations) will appear as an extraneous peak between fructose and glucose and can be removed by a solid phase extraction cleanup step.

Chemicals (equivalent chemicals can be used): 1. Nanopure deionized water

Barnstead Corporation

18 MΩ

www.barnsteadthermolyne.com

2.

Acetonitrile, HPLC grade Burdick & Jackson

VWR International LLC Cat. No. AH015-4PC www.vwr.com

3.

D (-) Fructose

Sigma Aldrich Cat. No. F0127 www.sigma.com Sigma Aldrich Cat. No. G7528 Sigma Aldrich Cat. No. S7903 Sigma Aldrich Cat. No. M9171 Sigma Aldrich Cat. No. 61339

4.

D (+) Glucose

5.

Sucrose

6.

D (+) Maltose monohydrate

7.

D (-) Lactose monohydrate

8. JT Baker Cat. No. 3624-19 9. D (+) Galactose Sigma Aldrich Cat. N. G0750 10. D(-) Ribose Sodium Chloride

Sigma Aldrich W379301-1kg

Apparatus (equivalent equipment can be used):

1. 2 L heavy wall filter flask

VWR International LLC Cat. No. 29415-143 VWR International LLC Cat. No. 24710-252

2. 2 L graduated cylinder

3. 500 mL graduated cylinder

VWR International LLC Cat. No. 24710-500 VWR International LLC Cat. No. 29619-267 VWR International LLC Cat. No. 5642-250 VWR International LLC VWR International LLC Cat. No. 7100-100 VWR International LLC Cat. No. 7100-50 VWR International LLC Cat. No. 7100-20 VWR International LLC Cat. No. 48888-708 VWR International LLC Cat. No. 89039-670 VWR International LLC Cat. No. 13910-143 Fisher Scientific Cat. No. 11-471-134 VWR International LLC Cat. No. 301073 VWR International LLC Cat. No. 28145-487

4. 500 mL class A volumetric flask

5. 250 mL class A volumetric flask

6. 100 mL class A volumetric flask

1. Cat. No. 5642-100

7. 100 mL class A pipet

8. 50 mL class A pipet

9. 20 mL Class A pipet

10. Eppendorf Research Pro Pipettor

11. 15 mL disposable centrifuge tubes

12. 50 mL beaker

13. Shaker, Glas-Col Benchtop

14. Syringe, disposable B-D 3 cc

15. Filter, Whatman 25 mm 0.20 µm nylon disc filter

16. Bottle, Nalgene screw top 250 mL

VWR International LLC Cat. No. 16129-028

15. Glass vial, 2 dram w Teflon-lined screw top

Scientific Specialties Service Cat. No. B69306

16. Polypropylene Membrane Disc Filter

VWR International LLC Cat. No. 28140-549 VWR International LLC Cat. No. 66020-953 VWR International LLC Cat. No. C4011-1A

17. Clear Crimptop Vials

18. 11MM Crimp Seals Teflon

19. Analytical balance (4 decimal places)

Mettler Toledo Model AB204

20. Acquity UPLC system

Waters Corporation www.waters.com Sigma Aldrich Part # 58989C30

21. Supelcosil™ LC-NH2 column

(3 x 150 mm, 3 µm particle size)

22. Analog Vortex Mixer

VWR International LLC Catalog # 58816-121 VWR International LLC Catalog # 72888-012 Catalog # 47746-472 Bischoff www.bischoff-chrom.de

23. Bottle top dispensers

0-10 mL 10-100 mL

24. Refractive Index Detector Model 8120

25. Samco Transfer disposable pipets

Samco Scientific www.samcosci.com Catalog # 262

9” length 5 mL volume

26. Pipettor Tips

VWR International LLC Catalog # 82018-840 Catalog # 83007-378

5000 µL 1000 µL

27. Microwave

Panasonic Model NN-4471A

28. Clinical 200 Centrifuge

VWR International LLC Catalog #82017-654

29. Supelco standalone guard column holder

Sigma Aldrich Part # 21150AST

2 cm x 4 cm

30. Supelguard™ LC-NH2 guard column

Sigma Aldrich Part # 59568-c30

2 cm x 3 mm

31. Gilson Microman M250

Gilson Inc.

Positive displacment pipette Replacement capillary pistons

Catalog #F148505 Catalog #F148114

32. 0.1 µm 25 mm Cameo syringe filters

VWR International LLC Catalog #Z741925 VWR International LLC Cat. No. 6870-2504

33. Filter, Whatman 25 mm

0.45 µm nylon disc filter

34.

0.5 um precolumn filter HPLC

Sigma Aldrich 55214-4

Reagents: 1.

Mobile Phase : Concentration varies to achieve good separation of analytes. Range will be from an initial 75 % Acetonitrile to 82 % Acetonitrile at the end of the column’s lifetime. The following recipe is for 75 % Acetonitrile/25 % water. a. Dispense 750 mL HPLC reagent grade acetonitrile into a glass container. b. Dispense 250 mL deionized water into the same glass container. c. Mix contents well and vacuum filter through a 0.45 µm nylon filter membrane. d. Stable for six months stored at room temperature. 2. Strong Needle Wash/Strong Seal Wash: 100 % water a. Dispense 1000 mL deionized water and filter through a 0.45 µm nylon filter membrane. b. Stable for six months stored at room temperature. 3. Weak Wash/Seal Wash/Syringe Wash Solutions: 75 % Acetonitrile/25 % water (or current mobile phase can be used) a. Dispense 750 mL HPLC reagent grade acetonitrile into a glass container. b. Dispense 250 mL deionized water into the same glass container. c. Mix contents well and vacuum filter through a 0.45 µm nylon filter membrane. d. Stable for six months stored at room temperature. 4. Sugar Standard Solutions a. Weigh the sugars as outlined in the chart below into a 250 mL volumetric flask to generate a three tier regression range for fructose and glucose at one linear range, galactose, maltose and lactose at another linear range, and sucrose at a higher linear range to assay samples containing a maximum of 35 %, 14 % and 43 % sugar, respectively (assuming minimum 7.5 g target weight). Sugar Std # mg/mL Glucose & Fructose, g Galactose 1 g Maltose & Lactose 2 g Sucrose g IS Ribose g 1 0 0 0 0 0 0.5 2 0.5/0.5/0.5 0.125 0.125 0.131 0.125 0.5 3 2.5/1/3 0.625 0.625 0.263 0.75 0.5 4 5/2/6 1.25 1.25 0.525 1.50 0.5 5 10/4/12 2.5 0 1.050 3.0 0.5 b. Add 100 mL of deionized water using a class A glass pipette and swirl/sonicate to dissolve the sugars. c. Add 150 mL of acetonitrile using a class A glass pipette (3 x 50 mL). d. Stopper, invert and shake to mix well. Do not dilute to volume. The 10/4/12 standard will need to be at room temperature to completely dissolve, since the addition of acetonitrile lowers the temperature of the solution. 3 1 Due to solubility, the maximum concentration of galactose was achieved with less overall standards. 2 Maltose and lactose are 95% pure because they are monohydrate salts, thus the target weight has been adjusted to account for this.

e. Dispense as needed into auto sampler vials, do not pre-vial and store on the counter 4 . f. Stable for six months stored at room temperature with a parafilm seal. 5. 5 mg/mL Sodium Chloride Standard Solution a. Weigh 0.5 g sodium chloride and dispense into a 100 mL volumetric flask. b. Add 40 mL of deionized water using a 20 mL class A glass pipette (2 x 20 mL) and swirl to dissolve the salt. c. Add 60 mL of acetonitrile using a 20 mL class A glass pipette (3 x 20 mL). d. Stopper, invert and shake to mix well. Do not dilute to volume. e. Dispense as needed into autosampler vials, do not pre-vial and store on the counter. f. Stable for six months stored at room temperature with a parafilm seal. 6. 60 % Acetonitrile:40 % Water Diluent Solution a. Dispense 150 mL HPLC reagent grade acetonitrile into a 250 mL volumetric flasks using a 50 mL class A glass pipette (3 x 50 mL). b. Dispense 100 mL of deionized water using a class A glass pipette. c. Mix contents well. d. Stable for six months stored at room temperature 6. Diluant - 5 mg/mL of ribose (Internal Standard) in water

a. Weigh 5 g grams of ribose and dispense into a 1 L volumetric flask. b. Add 500 mL of deionized water and dissolve by hand mixing. c. Dilute to volume with deionized water. d. Stable for 2 weeks stored at room temperature.

Sample Preparation: 1.

Grind samples as homogeneous as possible in air-tight, dark containers to a fine powder. Upon receipt of the delivered samples to the storage area, the handling and storage of the samples should be in accordance with good laboratory practices. Allow refrigerated or frozen samples to equilibrate to room temperature before weighing. Sample Size: The method was optimized to achieve a LOQ of 0.1% for the labeling sugars in various matrices with consideration to moisture and sample weight as denoted in the yellow region as a guideline.

3 Accelerate temperature acclimation to RT by ~30 seconds immersion in hot water from a sink spigot. 4 Over time, acetonitrile will evaporate out of vials crimp sealed.

Procedure: 1. Place a 250 mL Nalgene polypropylene bottle on the balance and tare it to zero. 2. Dispense a minimum of 7.5 g sample 5 into the container and record the weight. 3. Add 100 mL of diluant 5 mg/mL IS to the container using an automatic pipet or calibrated bottle top dispenser and securely fasten the bottle with a screw cap lid. 4. Perform a manual shake of the bottle and inspect if the contents are visually dissolving in solution, and place these samples on the shaker for a minimum of 15 minutes at a rate of 110-130 shakes per minute. 5. For samples not visually dissolving, microwave each individual bottle for 25 seconds and then place on the shaker for a minimum of 15 minutes at a rate of 110-130 shakes per minute. Representative Food Matrices requiring the Microwave Step (not an exclusive list): Gelatin, pectin, guar gum etc. Peanut butter, ground nuts Cottage or cream cheese Potato or corn chips Date or almond paste Dried fruit Butter, margarine mayonnaise, oil Frosting Chocolate bar Cheese 6. Post shaking, let solids settle out of solution to the bottom and fat rise to the surface for not less than 10 minutes. 7. If after 10 minutes the samples do not have a clear region to acquire an aliquot, centrifuge sample in a disposable centrifuge tube for 5 minutes at 3000 RPM 6 . 8. Pipette 2 mL of aqueous extract into a 2 dram vial. 9. Dispense 3 mL of acetonitrile into each 2 dram vial and briefly vortex mix. 10. Let mixture sit for a minimum of 15 minutes to allow the precipitation of proteins to the bottom of the tube and oleaginous materials rise to the surface 7 . 5 For samples containing >50% estimated sugar content (i.e. marbits), weigh samples accordingly, (eg. less). For samples containing >70% estimated water (i.e. yogurt and beverages), weigh 20 g of sample. For all other matrices, inclusive of maltodextrin or soluble corn fiber samples, weigh a minimum of 7.5g of sample. 6 Some RTE cereals, feed samples, fiber ingredients, and infant formula will require centrifugation. Infant formula and milk were found to require a minimum of 10 min centrifugation. 7 If dilution is required for quantitation of any individual sugar, an aliquot from this solution may be diluted with 60:40 acetonitrile: water using positive displacement pipettes.

11. After 15 minutes, centrifuge samples that do not have a clear zone present for five minutes at 3000 RPM 8 . 12. Transfer a sample aliquot, avoiding precipitate and fat, into a 3 cc disposable syringe equipped with a 0.20 µm 25 mm syringe filter. 13. Dispense filtered sample into a 2 mL auto sampler vial, and enclose with a Teflon coated crimp seal 9 . Occasionally, samples may need to be prefiltered through a 0.45 um filter first, and subsequently through a 0.2 um filter. Salt Removal For soy sauce and fermented condiments, meat snacks, premixes and fortified grains an additional cleanup step is required to remove the cations. 14. At step 6, insert a 10 ml disposable syringe into the aqueous central layer and collect 6 mL of sample 15. Attach a 25 mm 0.45 mm syringe filter, and filter the sample into a clean vessel for each sample (e.g test tube). 16. Dispense 4 mL of the filtered sample from step 15 onto a dry strata ABW SPE cartridge to elute a minimum of 3 mL into a clean test tube by gravity (or light pipet bulb encouragement) 17. Continue with step 8 as outlined above 18. Analyze by RI-HPLC according to the following parameters on the chromatography system that is equipped with a 0.5 um column prefilter, then a guard column, followed by the analytical amino column. Injection volume: 10 µL Flow Rate: 0.5 mL min, isocratic Column temperature: ambient Sample temperature: ambient Mobile Phase startup~75 % acetonitrile/25 % water, up to 82 % acetonitrile/18 % water over time. Typical Chromatogram of the 0.5 mg/ml Sugar Standard

8 Centrifuge maltodextrin or soluble corn fiber samples to precipitate these out of solution. Milk protein isolate concentrates should be centrifuged at 6000 RPM for 20 minutes. 9 Alternatively, a 0.1 um 25 mm syringe filter membrane may be used at the discretion of the chemist

Relative elution time of various analytes on the amino column

Analyte Ribose Erythritol

Retention Time (min)

2.31 2.33

Xylose Xylitol

2.739 3.009 3.038 3.21 4.086 4.095 4.42 4.705 7.35 8.640 8.927 9.137 9.782 3.5

Arabinose

Sodium chloride

Fructose Mannose Sorbitol Glucose Galactose Sucrose Inositol Lactulose Maltitol Maltose Lactose Isomalt Lactitol

10.851 11.582

11.918 Samples with high salt content (cations), ie premixes, meat snacks, soy sauce will have a

large peak manifesting between fructose and glucose/galactose. Typical Chromatogram of 5 mg/mL Sodium Chloride Standard

Calculations: % Sugar Calculation

ℎ ℎ 10 ( / 100) ( ℎ ℎ ) /10

where: A = Analyte peak height of the sample

IS mg/mL = concentration of internal standard added IS peak height = peak height of internal standard added RF = response factor of analyte S w = Sample weight, g 100 = g of water in sample prep 10 = conversion of mg/ml to % sugar RF Calculation ℎ ℎ ℎ ℎ Purity Correction Calculation for standards × (1 − − ) × where: C a = Concentration of Standard Analyte W = measured water fraction as determined by Karl Fisher (%) S = salt form present, i.e. monohydrate H i = Vendor stated dry weight basis of analyte i as stated on the CoA (% HPLC purity)

10 Peak height was chosen due to two factors: Rs of galactose is < 1.5, and at LOQ easier to discern peak height vs peak area with respect to precision due to variability in analysts interpreting the baseline.

Validation Linear Regression

Limit of Detection and Quantitation The limit of detection and quantitation of the assay was calculated based on the standard deviation (SD) of the slope response and the slope (S) of the calibration curve according to the formulas: = 3.3 × × = 10 × × where : D f = Dilution Factor of sample prep C f = Correction factor, mg/mL to %

P e a k H e I g h t mg mL fructose glucose galactose sucrose maltose lactose 0 0 0 0 0 0 0 0.0125 202 269 125 222 109 104 0.025 428 528 334 450 224 252 0.05 931 811 427 938 391 348 0.125 1657 1731 903 2174 929 1016 0.25 3675 3151 1731 3894 1719 1640 0.5 7019 6290 3446 7798 3581 3929 slope 13976.77 12302.61 6741.92 15458.59 7067.38 7657.21 intercept 65.62 134.11 68.13 85.30 21.52 -11.58 cc 0.998 0.999 0.998 0.999 0.999 0.99

lod mg ml loq mg ml

0.006 0.019 0.028 0.020 0.018 0.019 0.017 0.059 0.051 0.061 0.055 0.056

LOD % 0.008 0.028 0.040 0.029 0.026 0.027 LOQ % 0.025 0.084 0.073 0.088 0.078 0.081

Based on these results, the limit of detection and quantitation for the sugars was calculated to be 0.04 % and 0.1 %, respectively. The limit of quantitation was verified by spiking in triplicate a sugar series into various matrices and calculating the % recovery obtained. All sample matrices were prepared at a 7.5 g target weight, with the exception of yogurt. Yogurt was found to achieve adequate recoveries at a 15 g sample weight for fructose, sucrose and maltose, but at 20 g was able to recover all sugars.

LOQ Verification :

% Recovery in Saltine Cracker Matrix (Sector 5) % fortified fructose glucose galactose sucrose maltose lactose 0.1 107 94 123 112 117 128 0.2 108 105 108 91 102 92 0.3 108 91 118 110 102 98 % Recovery in Oil Matrix (Sector 1) % fortified fructose glucose galactose sucrose maltose lactose 0.1 105 114 120 119 87 102 0.2 108 111 104 105 109 83 0.3 104 105 85 109 88 98 % Recovery in Gelatin Matrix (Sector 9) % fortified fructose glucose galactose sucrose maltose lactose 0.1 92 96 97 96 88 101 0.2 97 95 99 91 87 87 0.3 95 89 93 104 91 88 % Recovery in protein powder (Sector 9) % fortified fructose glucose galactose sucrose maltose lactose 0.1 93 109 115 93 86 103 0.2 97 97 115 87 88 82 0.3 106 89 82 95 90 97 % Recovery in yogurt (sector 6) % fortified fructose glucose galactose sucrose maltose lactose 0.1 117 120 109 99 108 128 0.2 112 109 121 99 99 101 0.3 101 110 104 99 106 109

Accuracy: Accuracy was performed on four matrices at four levels and analyzed according to the method and the % recovery calculated from the slope of the linear regression.

AOAC

% Recovery of the Sugars

Matrix water butter

Sector fructose glucose sucrose maltose lactose

---

97

98

99

103 106 103

103 104 102

1

102 101

105 102

101 100

corn starch 5 refried beans 9

91

96

94

94

93

The accuracy of galactose in the method was ascertained with passing a proficiency testing program. Precision: The precision of the method was evaluated by the analysis of different food matrices in triplicate over a five-day timeframe for the presence or absence of fructose, glucose, sucrose, maltose and lactose. Only the sugars that were found above the LOQ in the various food matrices and beverages evaluated are reported.

Fructose AOAC Food Matrix Sectors

Glucose

AOAC Food Matrix Sectors

---

% 1

2

4

5

5

---

%

1

4

5

salad dressing

cream cheese

sweet potatoes V8 juice

salad dressing

potato chips

cream cheese rte cereal

sweet potatoes V8 juice

#

#

1 2 3 4 5 6 7 8 9

6.5 6.6 6.5 6.5 6.6 5.9 6.8 7.0 6.9 6.8 7.0 6.9 6.8 6.8 7.1 6.7 0.3

2.1 2.1 2.1 2.1 2.7 2.6 2.6 2.6 2.2 1.9 2.0 2.1 2.1 2.3 2.2 2.2 0.3

0.8 0.8 0.8 1.0 0.9 0.9 0.9 0.9 0.9 1.0 1.0 1.1 1.1 0.9 0.7 0.9 0.1

1.2 1.2 1.2 1.5 1.5 1.4 1.2 1.1 1.3 1.2 1.2 1.1 1.2 1.2 1.1 1.2 0.1

1 2 3 4 5 6 7 8 9

4.7 4.7 4.5 5.3 4.9 4.5 5.2 4.5 4.3 5.6 5.1 5.0 4.9 4.8 4.8 4.9 0.4

1.1 1.3 1.0 1.0 1.4 1.1 1.2 1.1 1.2 1.2 1.0 1.0 1.3 1.2 1.2 1.2 0.1

2.4 2.5 2.4 2.1 2.1 2.5 2.4 2.4 2.2 2.7 2.8 2.9 2.4 2.5 2.6 2.5 0.2 9.1

2.2 2.2 2.2 1.9 2.2 2.4 2.3 2.1 2.0 1.9 2.1 2.0 1.8 2.1 2.0 2.1 0.2 8.1

4.3 4.2 4.6 3.9 4.3 4.9 3.7 3.9 3.9 4.3 4.3 4.5 4.1 4.0 3.9 4.2 0.3 7.5

1.4 1.4 1.7 1.2 1.4 1.1 1.4 1.6 1.3 1.5 1.5 1.5 1.4 1.4 1.4 1.4 0.1

10 11 12 13 14 15

10 11 12 13 14 15

 σ

 σ

% RSD 4.4

11.5 10.0 9.9

% RSD 7.3

10.1

10.4

Sucrose

AOAC Food Matrix Sectors

% 1

1

2

2

3

4

5

5

7

8

9

salad dressing

cake frosting

chocolate bar

potato chips

peanut butter

cream cheese rte cereal

sweet potatoes

Cat Chow Bac'Os

Beef Jerky 17.7 16.7 16.6 16.2 15.8 16.5 16.4 17.1 17.0 16.6 16.6 16.4 16.5 16.5 16.4 16.6

#

1 2 3 4 5 6 7 8 9

6.4 6.4 6.2 6.0 5.8 6.0 6.5 6.8 6.3 6.4 6.6 6.5 6.2 6.4 6.5

48.8 49.8

2.0 10.8 8.4 20.4

1.6 1.5 1.6 1.2 1.7 1.3 1.5 1.4 1.6 1.5 1.4 1.6 1.7 1.6 1.8 1.5 0.2

1.4 1.5 1.5 1.3 1.3 1.4 1.5 1.5 1.4 1.4 1.4 1.5 1.7 1.4 1.3 1.4 0.1

7.8 7.7 7.8 7.1 7.2 7.2 7.3 7.5 7.4 7.6 7.9 7.6 7.7 7.7 7.8 7.6 0.3 3.4

46.1 50.1 2.0 10.7 8.4 20.3 46.9 50.5 2.0 10.7 8.3 20.2 45.5 47.6 3.1 10.8 8.2 19.0 46.3 47.5 3.2 11.3 8.1 19.4 44.8 46.1 3.1 11.1 8.4 19.3 46.6 49.2 2.0 10.1 8.4 19.6 46.4 49.3 2.1 10.3 7.6 19.6 45.1 49.3 2.0 10.6 8.1 20.1 45.4 47.8 2.0 10.5 8.0 19.9 44.8 48.5 2.0 10.4 8.4 19.2 46.7 51.0 3.2 10.4 8.6 19.1 49.0 49.3 3.0 10.8 8.7 19.7 49.1 48.8 2.9 10.9 8.5 19.8 44.7 47.2 3.1 9.9 8.0 19.0

10 11 12 13 14 15

6.3 46.4 48.8

2.5 10.6 8.3

19.6

 σ

0.2

1.5 3.2

1.4 2.8

0.6

0.4 3.4

0.3 3.4

0.5

0.4 2.6

% RSD 3.9

22.4

2.4 10.4 6.7

Maltose

AOAC Food Matrix Sectors

Lactose

AOAC Food Matrix Sectors

% 1

2

5

5

6

% 1

2

4

4

6

cake frosting

potato chips rte cereal

sweet potatoes

Infant Formula

salad dressing

chocolate bar

american cheese

cream cheese

Infant Formula

#

#

1 2 3 4 5 6 7 8 9

8.0 6.2 6.4 6.7 7.9 7.8 6.8 6.8 6.8 6.4 6.5 6.3 6.5 6.9 7.0 6.9 0.6

1.5 1.4 1.4 1.5 1.6 1.4 1.2 1.5 1.8 1.2 1.7 1.8 1.4 1.4 1.7 1.5 0.2

1.0 1.0 1.0 1.1 1.1 1.2 1.2 1.2 1.2 1.2 1.1 1.2 1.2 1.1 1.1 1.1 0.1 7.3

5.8 5.5 5.8 4.9 5.3 5.6 5.4 5.2 5.3 5.9 6.1 5.8 5.1 5.0 5.0 5.4 0.4 6.9

1.3 1.6 1.6 1.3 1.5 1.3 1.3 1.4 1.4 1.5 1.5 1.5 1.4 1.6 1.7 1.5 0.1 9.5

1 2 3 4 5 6 7 8 9

1.8 1.7 1.7 1.7 1.6 1.6 1.7 1.6 1.8 1.9 1.9 1.9 1.8 1.8 1.7 0.1

9.2 9.2 9.3 9.3 9.6 9.6 9.5 9.5 9.0 9.1 8.9 9.2 9.1 9.4 0.5 4.9

5.2 5.2 5.3 4.7 4.9 4.7 5.2 5.2 5.4 5.0 4.9 4.8 5.4 5.2 0.3 6.7

1.8 23.7 1.7 22.7 1.8 23.2 2.0 23.4 1.8 24.9 1.8 24.2 1.8 22.0 2.1 22.8 1.6 23.0 1.9 21.8 2.0 22.4 2.0 21.7 1.8 20.5 1.9 20.8 2.0 20.7 1.9 22.5

10.4 5.9

1.7 10.4 5.8

10 11 12 13 14 15

10 11 12 13 14 15

 σ

 σ

0.1 6.1

1.3 5.7

% RSD 8.4

12.6

% RSD 6.5

Galactose is a monosaccharide inherent to dairy products and was evaluated along with lactose in Yoplait original and greek style yogurt with Saco buttermilk dried powder 11 .

galactose

lactose

Day-Sample greek saco yoplait

Day-Sample greek yoplait

1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3

0.79 0.79 0.80 0.82 0.81 0.80 0.65 0.64 0.70 0.70 0.71 0.72 0.71 0.68 0.69 0.73 0.06 8.52

0.43 0.47 0.35 0.42 0.43 0.45 0.38 0.44 0.40 0.40 0.47 0.40 0.36 0.38 0.36 0.41 0.04 9.54

1.05 1.12 1.05 1.15 1.00 1.07 0.91 0.98 1.00 1.04 1.00 0.99 1.02 1.01 1.00 1.03 0.06 5.72

1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3

2.63 2.64 2.64 2.44 2.45 2.43 2.37 2.34 2.38 2.49 2.52 2.48 2.48 2.43 2.45

4.83 4.77 4.81 4.16 4.14 4.39 4.16 4.24 4.30 4.52 4.49 4.43 4.43 4.42 4.35

mean

mean

2.48

4.43

std dev

std dev

0.093 0.226

% rsd

% rsd

3.77

5.10

11 Low levels of fructose and glucose were detected in the yogurt, but not reported since the emphasis was on galactose and these sugars were already covered previously. Saco buttermilk contained lactose at >50% and was not reanalyzed by dilution of the samples to be within linear range of the calibration curve.

AOAC Candidate Method SUG-002 Submitter Name

Barry McCleary

Submitter Email Organization Method Type Method Name

barry@megazyme.com

Megazyme

Fructans

Single Lab Validation of Megazyme’s Fructan Assay Kit (K-FRUC) Method for the Determination of Fructan (Inulin, FOS, Levan and Branched Fructan) in Animal Food (Animal Feed, Pet Food, and Ingredients) Barry V. McCleary, Lucie M. J. Charmier, Vincent A. McKie, Ciara McLoughlin and Artur Rogowski.

Method Author(s)

Method Applicability Upload method here

-

1. Fructan SMPR McCleary.pdf

Single Lab Validation of Megazyme’s Fructan Assay Kit (K-FRUC) Method for the Determination of Fructan (Inulin, FOS, Levan and Branched Fructan) in Animal Food (Animal Feed, Pet Food, and Ingredients)

Objective

Evaluation of the Megazyme Fructan Assay Kit method (K-FRUC; updated with inclusion of endo -levanase) for the measurement of total dietary fructan, such as inulin, levan, branched fructans including agave fructans) and fructo-oligosaccharides (FOS), in animal feed, pet food, and the corresponding ingredients. The method can distinguish dietary fructans from interfering compounds such as free glucose, fructose, sucrose, starch and other polysaccharides.

Authors

Barry V. McCleary, Lucie M. J. Charmier, Vincent A. McKie, Ciara McLoughlin and Artur Rogowski.

Status

Unpublished but commercially available.

Conclusion The method developed meets the standard method performance requirements that can be tested in a single lab. This method is believed to be, currently, the only method that can specifically measure inulin, levan and Agave (branched) fructan in foods, feeds and pasture grasses.

Date of Issue 29 th June, 2018.

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1. Abstract The Fructan Assay Kit is an enzymatic test kit developed by Megazyme for the determination of fructan [inulin, FOS, agave (branched) fructan and levan] in a variety of animal feed and food samples and in ingredients. For the purpose of this submission, the kit has been validated in a single laboratory for analysis of pure inulin, FOS (non-reducing), agave fructan and levan and a range of fructan containing samples. The commercially available Fructan Assay Kit (Megazyme cat no. K- FRUC) contains all components required for the analysis. Quantification is based on complete hydrolysis of fructan to fructose and glucose by a mixture of exo -inulinase, endo -inulinase and endo -levanase and measurement of these sugars using the PAHBAH reducing sugar method, which gives the same color response with fructose and glucose. Before hydrolysis of fructan, interfering sucrose and starch in the sample are hydrolyzed to glucose and fructose and these sugars and any other reducing sugars in the sample extract are “removed” by borohydride reduction. The Single Lab Validation (SLV) outlined in this document was performed on commercially available inulin (Raftiline) and agave fructan (Frutafit ® agave fructan), levan purified from Timothy grass, two grass samples, a sample of legume hay, two animal feeds and two barley flours, one of which (Barley MAX ® ) was genetically enriched in fructan through plant breeding. Parameters examined during the validation included Working range, Selectivity, Recovery, Limit of Detection (LOD), Limit of Quantification (LOQ), Trueness (bias), Precision (reproducibility and repeatability), Robustness and Stability. The method outlined within allows accurate enzymic measurement of fructan in a wide range of animal feeds, pet foods and ingredients. It is a robust, quick and easy method for analysis of fructan, based on the commercial Fructan Assay Kit (K- FRUC) as developed by Megazyme.

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The results of the SLV are summarized in Table 1 .

Table 1. Results of the SLV for the Fructan Assay Kit.

AOAC SMPR ® 2018.002

Megazyme (K-FRUC)

0.21 to 98.4 a

Operating range (% w/w)

0.2 to 100

Limit of quantitation (LOQ) (% w/w)

0.119 b

0.20

RSD r RSD r RSD r RSD R RSD R Fructan) Fructan)

, % (0.2 to 1% w/w Fructan) , % (>1 to 10% w/w Fructan)

7 5 3

4.74 3.59 2.96 8.47 6.36

, % (>10 to 100% w/w

, % (0.2 to 1% w/w Fructan)

14 10

, % (>1 to 10% w/w

RSD R

, % (>10 to 100% w/w

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5.77

Fructan)

a Precise range dictated by fructan content in samples tested. b Based on replicate measurements for a sample with ~ 1% (w/w) fructan.

2. Introduction Fructans are defined as any compound where one or more fructosyl-fructose linkage constitutes a majority of the linkages. This refers to polymeric material as well as oligomers as small as the trisaccharide, kestose. Material included in this definition may or may not contain D-glucosyl substituents. Fructans are widely distributed in the plant kingdom. They are present in monocotyledons, dicotyledons and in green algae. Fructans differ in molecular structure and in molecular weight. They may be classified into three main types: the inulin group, the levan group and the branched (agave-type) group. The inulin group consists of material that has mostly or exclusively the (2→ 1) fructosyl-fructose linkage. Levan is material that contains mostly or exclusively the (2→ 6) fructosyl-fructose linkage. The branched group has both (2→ 1) and (2→ 6) fructosyl-fructose linkages in significant amounts (e.g. graminan from Gramineae and agave fructan).

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3. Definitions a) Animal and pet foods. —Material consumed or intended to be consumed by animals other than humans that contributes nutrition, taste, or aroma or has a technical effect on the consumed material. This includes raw materials, ingredients, and finished product. (AAFCO) b) Fructans. —Any DP ≥ 3 carbohydrate with one or more fructosyl-fructose linear or branched linkages which constitute a majority of the linkages in which fructose is the major constituent and glucose content is 33% or less. Examples include inulin, levan, branched fructans (e.g., agave fructans), and fructooligosaccharides (FOS). 4. General Information This method is based on the Fructan Assay Kit (Megazyme cat. no. K-FRUC) which is available for purchase from Megazyme directly at www.megazyme.com Technical support can be provided to the user via email (contact scientist@megazyme.com ). 5.1 Scope of the Method i. Target analyte – Fructan, including inulin, FOS (non-reducing), levan and branched fructans (e.g. agave fructan). ii. Matrices – For this validation, the Fructan Assay Kit (K-FRUC) was tested with the following matrices: 1. Animal feed and pet food samples Brett Brothers swine feed (purchased from Glanbia, Ireland). Purina GoCat dry cat food (purchased from Tesco Ireland, Greystones, Ireland). Top Spec TopChop alfalfa (purchased from TopSpec Equine Ltd, Middle Park Farm, Pickhill, Thirsk, North Yorkshire, YO7 4JN, UK). 5. Method c) Ingredients. —The base materials used in the formulation of animal and pet foods.

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Timothy grass hay (Lot no. 171201)(supplied by Alison Muirhead, Grass Seed Direct, Menstrie Mains, Menstrie, Clackmannanshire, Scotland). Rye grass hay (supplied by Dr Joe Panozzo, Seed Phenomics and Quality Traits Agriculture Victoria Research, Horsham, Victoria, Australia). 2. Barley grain/flour Conventional barley flour (Lot no. 60301a) (from Megazyme, Bray, Ireland). Barley MAX ® (from Paul Sims, The Healthy Grain Pty. Ltd., South Yarra, Victoria, Australia). 3. Pure, or high purity commercial of purified fructans: Raftiline ® (Native Chicory Inulin – supplied by BENEO Tienen , Aandorenstraat 1, 3300 Tienen, Belgium). Frutafit ® agave fructan (supplied by Sensus, Borchwerf 3, Roosendaal, The Netherlands). Levan (cat. no. P-LEVAN; Lot: 171114 purified from Timothy grass - from Megazyme, Bray, Ireland).

ii. Certified reference materials (CRMs) Certified reference materials are not currently available for fructan.

iii. Expression of results – The concentration of fructan is expressed as % w/w of the sample analyzed.

5.2 Method Principle Products to be analyzed are suspended in water in tubes that are heated in a boiling water bath to dissolve the fructan. Sucrose in the sample is hydrolyzed at 30 o C by a specific sucrase enzyme (1), which has no action on lower degree of polymerization (DP) FOS, such as 1-kestose and 1,1-kestotetraose.

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Starch and maltodextrins are concurrently hydrolyzed to maltose and maltotriose by pullulanase and β -amylase at 30 o C, and these oligosaccharides are then hydrolyzed to D-glucose by maltase (2).

D-Glucose and D-fructose are reduced at 40 o C by sodium borohydride to the corresponding sugar alcohols, D-sorbitol and D-mannitol (3). Native fructans and non-reducing FOS such as Neosugars ® are not affected by this reaction.

Fructans and FOS are specifically hydrolyzed by exo - and endo -inulinase and endo - levanase at 40 o C to D-glucose and D-fructose (4).

D-Fructose and D-glucose derived from fructan are measured using the PAHBAH reducing sugar method (5). This method is simple to use and the color response for D- fructose and D-glucose is the same (Figure 1).

5.3 Equipment Required

( a ) Grinding mill .- Centrifugal, with 12-tooth rotor and 0.5 mm sieve, or similar device. Alternatively, cyclone mill may be used for small laboratory samples.

(b) Microfuge.- (required speed 13,000 rpm).

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( c ) Disposable 2.0 mL polypropylene microfuge tubes .- e.g. Sarstedt cat. no. 72.691 (www.sarstedt.com).

(d ) Glass test tubes .- 16 120 mm, round bottom, 17 mL.

(e) Pyrex screw cap culture tubes (25 x 150 mm).- with PTFE lined phenolic caps (e.g. Fisher cat. no. 14-933D).

( f ) Volumetric flasks .-50 and 100 mL.

(g) Micro-pipettors.- e.g. Gilson Pipetman® (100 µ L and 200 µ L).

(h) Positive displacement pipettor .- e.g. Eppendorf Multipette® with 5.0 mL Combitip® (to dispense 0.2 mL aliquots of sucrase mixture and 0.1 mL aliquots of fructanase, and other solutions and buffers); with 50 mL Combitip ® (to dispense 5.0 mL aliquots of PAHBAH Working Reagent). (i) Bottle-top dispenser.- e.g. Brand ® S Digital 2.5 - 25 mL cat. no. 4600351 to dispense 25 mL of 200 mM sodium acetate buffer (pH 5.0).

(j) Water bath .-Maintaining 30± 0.1°C and 40 ± 0.1°C.

(k) Boiling water bath .- Deep-fat fryer filled with water at 95-100°C.

(l) Analytical balance.

(k) Spectrophotometer .- Operating at 410 nm.

( e ) Vortex mixer .

( f ) pH Meter .

( g ) Stop-clock timer .

5.4 Chemicals and Reagents

All reagents should be of analytical purity grade.

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( a ) Sodium maleate buffer .- 100 mM, pH 6.5. Dissolve 11.6 g maleic acid in 900 mL distilled water and adjust pH to 6.5 with 2 M NaOH (8.0 g NaOH/100 mL) and dilute to volume in 1 L volumetric flask with water. Store at 4°C. ( b ) Sodium acetate buffer .- 100 mM, pH 4.5. Pipet 5.8 mL glacial acetic acid (1.05 g/mL) into 900 mL distilled water. Adjust to pH 4.5 using 1M NaOH and dilute to 1 L with water. Store at 4°C. ( c ) p-Hydroxybenzoic acid hydrazide (PAHBAH) reducing sugar assay reagent .- ( 1 ) Solution A .- Add 10 g PAHBAH (e.g., Sigma cat. no. H-9882, Sigma Chemical Co., PO Box 14508, St. Louis, MO 63178, USA) to 60 mL water in 250 mL beaker on magnetic stirrer. Stir slurry and add 10 mL concentrated HCl. Adjust to 200 mL with distilled water and store at room temperature (ca 22°C). Solution is stable for at least 2 years. ( 2 ) Solution B .-Add 24.9 g trisodium citrate dihydrate to 500 mL distilled water and stir to dissolve. Add 2.20 g CaCl 2 ·2H 2 O and dissolve by stirring. Then add 40.0 g NaOH and dissolve with stirring. (Solution may be milky, but will clarify on dilution.) Adjust volume to 2 L. The solution is stable for at least 2 years at room temperature (ca 22°C). ( 3 ) PAHBAH working reagent .-Immediately before use, add 20 mL Solution A to 180 mL Solution B and mix thoroughly. This solution should be stored on ice and is stable for ca 4 h.

( d ) Sodium hydroxide .- 50 mM. Dissolve 2.0 g NaOH in 900 mL distilled water. Adjust volume to 1 L. Store at room temperature (ca 22°C).

( e ) Alkaline borohydride .- Accurately weigh approximately 50 mg sodium borohydride (Sigma cat. no. S-9125) into polypropylene containers (10 mL volume with screw cap). Record weight on tubes (ca 10 for convenience); seal tubes and store in a desiccator for future use. Immediately before use, dissolve sodium borohydride (at 10 mg/mL) in 50 mM NaOH solution, 5.4 ( d ). This solution is stable for 4-5 h at room temperature.

( f ) Acetic acid .- 200 mM. Add 11.5 mL glacial acetic acid to distilled water and adjust volume to 1 L. Store at room temperature (ca 22°C).

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( g ) Sucrase/amylase mixture (Sucrase/ β -amylase/ pullulanase/maltase).- 12 units (U) sucrase/mL. Dissolve contents of 1 vial containing sucrase (270 U) plus β -amylase ( Bacillus cereus , 320 U), pullulanase ( Bacillus licheniformis , 64 U), and maltase (yeast, 1600 U) (as a freeze-dried powder) in 22 mL sodium maleate buffer, 5.4 ( a ). Divide enzyme solution into 5 mL aliquots, and store frozen in polypropylene containers to prevent microbial contamination. If not diluted in buffer, the freeze- dried enzyme is stable 5 years when stored below -10°C. One unit (U) of sucrase activity is the amount of enzyme required to release 1 µmole glucose/min from sucrose at pH 6.5 and 30°C. ( h ) Fructanase solution .- 360 U/mL exo -inulinase, 11 U/mL endo -inulinase and 7.3 U/mL endo -levanase. Dissolve contents of 1 vial containing 8,000 U exo -inulinase, 240 U endo -inulinase and 160 U endo -levanase in 22 mL sodium acetate buffer, 5.4 ( b ). Divide enzyme solution into 5 mL aliquots and store frozen in polypropylene containers to prevent microbial contamination. If not diluted in buffer, the freeze- dried enzyme is stable 5 years when stored below -10°C. One unit (U) of exo - inulinase activity is the amount of enzyme required to release 1 µmole of reducing- sugar equivalents (as fructose)/min from kestose (10 mg/mL) at pH 4.5 and 30°C. ( i ) Inulin control powder .- Containing a known amount of fructan (inulin) from dahlia tubers freeze-dried in the presence of α -cellulose. Stable when stored dry at room temperature. ( j ) Levan control powder .- Containing a known amount of levan from Timothy grass freeze-dried in the presence of α -cellulose. Stable when stored dry at room temperature.

( k ) Sucrose control powder .- Sucrose freeze-dried in the presence of -cellulose. Stable when stored dry at room temperature (ca 22°C).

( l ) D-Fructose standard stock solution .- 1.5 mg/mL in 0.2% benzoic acid solution. Before preparing solution, dry powdered crystalline fructose (purity > 97%) 16 h at 60°C under vacuum.

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Items ( g )-( l ) are supplied in the Fructan Assay Kit (cat. no. K-FRUC) available from Megazyme, Bray Business Park, Bray, County Wicklow, Ireland, but preparations of enzymes and standards which meet these criteria may also be used.

5.5 Safety Considerations

The general safety measures that apply to all chemical substances should be adhered to. For more information regarding the safe usage and handling of this product please refer to the associated SDS that is available from the Megazyme website; www.megazyme.com.

5.6 Preparation of Test Materials

All products should be equilibrated to room temperature (22°C) before they are weighed.

( a ) Test samples .- For dry food, animal feed or grass samples, grind ca 50 g of sample in a grinding mill to pass 0.5 mm sieve. Transfer all material into wide-mouthed plastic jars and mix well by shaking and inversion. For wet products such as canned pet foods, homogenise samples with a Waring blender and freeze-dry approximately 100 g. Analyse a representative sample of the dry product. Record sample wet weight and dry weight. (b) Fructose standard working solution .- Add 0.2 mL fructose standard stock solution [1.5 mg/mL, 5.4(l)] to 0.9 mL acetate buffer, 5.4(b), and mix thoroughly. Dispense 0.2 mL aliquots of this solution (containing 54.5 µg fructose) in quadruplicate to the bottoms of 4 glass test tubes, 5.3(d). Add 0.1 mL acetate buffer, 5.4(b), to each tube. Immediately before incubation in boiling water bath, add 5.0 mL PAHBAH working reagent, 5.4(c).

(c) Reagent blank .-Transfer 0.3 mL acetate buffer, 5.4(b), into test tubes and proceed with the standard assay procedure from 5.7(c)( 3 ).

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(d) Sucrose control powder. - Containing ca 10% w/w sucrose. Extract and analyse 400 mg of this powder as per 5.7(a)( 1 ). This product contains no fructan and is used to check the effectiveness of the sucrase and borohydride treatments. The calculated fructan content should be less than 0.3% w/w.

(e) Inulin and levan control powders. - Extract and analyse 100 mg of these control powders as per 5.7(a)( 2 ).

5.7 Assay Procedure

(a) Extraction of fructan .-

( 1 ) Products containing 0-10% w/w fructan .- Run D-fructose working standard solution (in quadruplicate), reagent blank (in duplicate), fructan control flour, and sucrose control flour with each set of tests. Use reagent blank to zero the spectrophotometer. (i) Accurately weigh approximately 400 mg test portion of sample into a dry pyrex screw cap culture tube (25 x 150 mm) and add 25 mL of distilled water. Loosely cap the tube. Place the tube into a boiling water bath and heat for a total of 10 min. After 5 min, tighten the tube cap and vigorously mix the contents on a vortex mixer. Return the tube to the boiling water bath. After a further 5 min, remove the tube from the boiling water and mix the contents by inversion and shaking.

(ii) Cool the solution to room temperature and mix thoroughly. Transfer 2.0 mL into a disposable 2.0 mL polypropylene microfuge tube 5.3 ( c ).

(iii) Centrifuge the solution at 13,000 rpm for 5 min in a microfuge 5.3 ( b ), and analyze within 1 h after centrifugation. The supernatant may be slightly turbid. This is not a problem. (If the solution is stored for several hours at low temperature before analysis, the fructan may precipitate from solution. In such cases, reheat solution to ~80°C and let cool to room temperature before removing solutions for analysis.) Use this solution for determination of fructan as per 5.7(b)( 1 ).

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( 2 ) Products containing 10-40% w/w fructan. - Run D-fructose working standard solution (in quadruplicate), reagent blank (in duplicate), fructan control flour, and sucrose control flour with each set of tests. Use reagent blank to zero the spectrophotometer. (i) Accurately weigh approximately 100 mg of the sample into a dry pyrex screw cap culture tube (25 x 150 mm) and add 25 mL of distilled water. Loosely cap the tube. Place the tube into a boiling water bath and heat for a total of 10 min. After 5 min, tighten the tube cap and vigorously mix the contents on a vortex mixer. Return the tube to the boiling water bath. After a further 5 min, remove the tube from the boiling water bath and mix the contents by inversion and shaking. (iii) Centrifuge the solution at 13,000 rpm for 5 min in a microfuge, 5.3 ( b ), and analyze within 1 h after centrifugation. Supernatant may be slightly turbid. This is not a problem. (If the solution is stored for several hours at low temperature before analysis, the fructan may precipitate from solution. In such cases, reheat solution to ~80°C and let cool to room temperature before removing solutions for analysis.) Use this solution for determination of fructan as per 5.7(b)( 1 ). ( 3 ) Products containing 40-100% w/w fructan .- Add 1 mL of solution 5.7(a)(2)(ii) to two mL of water and mix thoroughly. Use this solution for determination of fructan as per 5.7(b)( 1 ). (ii) Cool solution to room temperature and mix thoroughly. Transfer 2.0 mL into a disposable 2.0 mL polypropylene microfuge tube 5.3 ( c ).

( b) Removal of sucrose, starch, and reducing sugars .-

( 1 ) Accurately transfer a 0.2 mL aliquot of filtrate to be analyzed (containing ca 0.1- 1.0 mg/mL fructan, or controls) to bottom of a glass test tube, 5.3 ( d ).

( 2 ) Add 0.2 mL diluted sucrase/amylase solution, 5.4 ( g ), to each tube and incubate at 30°C for 30 min.

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( 3 ) Add 0.2 mL alkaline borohydride solution, 5.4 ( e ), to each tube. Stir the tube vigorously, cover them with Parafilm R and incubate at 40°C for 30 min for complete reduction of reducing sugars to sugar alcohols. ( 4 ) Add 0.5 mL acetic acid, 5.4 ( f ), to each tube with vigorous stirring on Vortex mixer. If borohydride is fresh, a vigorous effervescence should be observed. If not, there is a problem with the borohydride; repeat the analysis with fresh borohydride. (This treatment removes excess borohydride and adjusts pH to ca 4.5.) This is Solution S .

(c) Hydrolysis and measurement of fructan .-

( 1 ) Transfer 0.2 mL aliquots of Solution S (in triplicate) to the bottoms of glass test tubes, 5.3 ( d ).

( 2 ) Add 0.1 mL fructanase solution, 5.4(h ), to two of these tubes, stir contents on Vortex mixer, and incubate at 40°C for 30 min for complete hydrolysis of fructan to fructose and glucose. To the third tube (the sample blank) add 0.1 mL of 100 mM sodium acetate buffer, 5.4(b) . ( 3 ) Add 5.0 mL PAHBAH working reagent to all tubes, including the fructose standard working solution, 5.6 ( b ), reagent blank, 5.6 ( c ), extracts of fructan control powder 5.6 ( e ), and sucrose control powder, 5.6 ( d ), and incubate in boiling water bath for exactly 6 min.

( 4 ) Remove all tubes from the boiling water bath and immediately place in cold water (18-20°C) for ca 5 min.

( 5 ) Measure the absorbances of all solutions at 410 nm against reagent blank as soon as possible after cooling. The PAHBAH color complex will fade with time. At room temperature, little change (< 5%) is seen over 10-15 min. The same change will be seen in the standard solutions.

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5.8 Calculations

Calculate total fructan content (% w/w, on “as-is” basis) in test samples as follows:

where: Δ A = absorbance of 0.2 mL reacted sample solutions minus the absorbance for the sample blank read against reagent blank; F = factor to convert absorbance values to µg fructose (= 54.5 µg fructose/absorbance value for 54.5 µg fructose); 5 = factor to convert from 0.2 mL as assayed to 1.0 mL; 25 = volume (mL) of extractant used; 1.1/0.2 = 0.2 mL was taken from 1.1 mL enzyme digest for analysis; W = weight (mg) of test portion extracted; 100/W = factor to express fructan as percentage of flour weight; 1/1000 = factor to convert from µg to mg; 162/180 = factor to convert from free fructose, as determined, to anhydrofructose (and anhydroglucose), as occurs in fructan; D = further dilution of the sample extract. Note: These calculations can be simplified by using the Megazyme Mega-Calc TM Excel-based calculator downloadable from the product page where this appears on the Megazyme website (www.megazyme.com).

5.9 Indicative Controls

Indicative controls are used as a check on assay conditions. The final absorbance of the sucrose/ α -cellulose control powder should be very low (< 0.08), which demonstrates the effectiveness of both the sucrase treatment step and the borohydride reduction step. If sucrose is not completely hydrolyzed by the sucrase/amylase treatment, it will then be hydrolyzed by the fructanase mixture and give erroneously high fructan values. Other indicative controls can be used, such as soluble starch, β - glucan and α -cellulose. The absorbance from α -cellulose should be negligible, i.e., < 0.02; for β -glucan and soluble starch, the absorbance should be very low, i.e. < 0.05.

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This result for starch demonstrates the effectiveness of the borohydride reduction step.

5.10 References McCleary, B.V., Murphy, A. and Mugford, D.C., J. AOAC Int. 83, 356-364 (2000).

McCleary, B.V. and Rossiter, P., J.AOAC Int ., 707-717 (2004)

McCleary, B. V., Charmier, L. M. H. McKie, V. A. and Rogowski, A. (unpublished).

6. Validation 6.1 Planning

The purpose of this report is to verify and validate a recently updated fructan assay procedure developed and supplied by Megazyme as detailed in the Fructan Assay Kit booklet (K-FRUC). This method is an extension of AOAC Method 999.03 (Measurement of total fructans in foods; enzymatic/spectrophotometric method). endo -Levanase has been added to the fructanase mixture (which also contains exo - inulinase and endo -inulinase). The presence of endo -levanase facilitates quantitative measurement of levan as well as inulin and branched fructans. 6.2 Performance characteristics Performance characteristics that are investigated within this study are working range, target selectivity (inulin, levan, branched fructans and FOS), specificity [removal of interfering sugars including sucrose, galactosyl-sucrose oligosaccharides (e.g. raffinose and stachyose) starch, maltodextrins and reducing sugars], Limit of Detection (LOD), Limit of Quantitation (LOQ), Trueness ( bias ) and Precision (repeatability and reproducibility). 6.2.1 Working Range The assay follows the Megazyme Fructan Assay Kit (cat. no. K-FRUC) and has a working range of 0.1 to 100% w/w fructan in the sample. For samples containing 0- 10% w/w fructan, 400 mg of sample is extracted with 25 mL of hot water to give a fructan content of 0.1-1.6 mg/mL; equivalent to approximately 3.6-58.2 µ g of sugar in the PAHBAH assay mixture. For samples containing 10-40% w/w fructan, 100 mg of

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