6. AOACSPIFANMethods-2018Awards

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8 H aselberger & Jacobs : J ournal of AOAC I nternati nal Vol. 99, No. 6, 2016

H aselberger & J acobs : J ournal of AOAC I nternational V ol . 99, N o . 6, 2016  1583

is WW. Allow to stir for 30 min, or until dissolved/uniformly suspended. (b) Sample dilution.— As with the qualitative analyses, samples require different dilutions according to their individual fructan content (Table 2016.06D ). Record all weights to 4 decimal places. This is SW 1 . (c) Removal of inherent glucose, fructose, and sucrose.— Transfer 0.2 g ± 10%of the diluted sample from step E(b) or K(b) above, as appropriate (as per guidelines in Table 2016.06D ) to a glass screw cap scintillation vial and record weight to 4 decimal places. This is SW 2 . Add 200 µL of sucrase solution, J(h) , to vial. Cap, swirl gently, and incubate at 40°C for 2 h (do not use foil lined caps). After the sucrase incubation, add 700 µL laboratory water to the scintillation vial. Then add 200 µL sodium borohydride solution, J(f) . Cap, swirl, and incubate at 40°C for 1 h. After the borohydride reduction is complete, neutralize the excess reagent with 500 µL of 0.2 M acetic acid, J(d) . Swirl gently (leave uncapped to allow gas generated to vent safely) and allow samples to sit at room temperature for 15 min. Note : Gas bubble formation after the addition of NaBH 4 , but prior to addition of acetic acid, may be a sign of improper sample pH and will negatively impact final results. If this is observed, further investigation of sucrase buffer and/or 50 mM NaOH solution is recommended to ensure the borohydride reagent is sufficiently basic to remain stable. (d) Fructan hydrolysis.— Add 100 µL glucoheptose IS solution, J(b) , and 100 µL fructanase solution, J(j) . Cap, swirl, and incubate at 40°C for 30 min. After the incubation is completed, swirl gently to ensure a homogeneous sample and filter through a 0.45 µm nylon syringe filter into autosampler vials (prepared samples can be stored in vials at 2–10°C for 5 days). L. Instrumental Analysis (a) Gradient.— Fructose and glucoheptose are eluted isocratically using 50 mM NaOH at 1.0 mL/min for 20 min. The column is then washed for 10 min with 400 mM NaOH and 60 mM sodium acetate. Following the wash step, the column is re-equilibrated with 50 mMNaOH for 15 min (Table 2016.06G ). Column and detector compartment are maintained at 20°C. It is recommended to clean the column and trap approximately

every five quantitative analytical sequences, with three complete cycles of the conditions outlined in Table 2016.06H . [Five sequences would roughly equate to ~130 sample and/or control injections. Failure to regularly clean the column/trap set may result in breakthrough of borate to the analytical column degrading method performance (primarily observed in the peak asymmetry of the glucoheptose).] (b) Injection.— Make a single 4 µL injection of each sample test solution. At the start of each sequence, make at least six equilibration injections to ensure that system is stable (note that an observed calibration error in excess of 5% on average for a level of standard may be indicative of insufficient equilibration time), followed by the four levels of WS. Bracket samples with WS after every 13 injections. Maintain autosampler at 10°C. (c) Electrochemical detector parameters.— This method utilizes the carbohydrate triple waveform per Table 2016.06F . Note : This waveform is not appropriate for disposable gold electrodes. The reference electrode is set to AgCl mode. (d) Retention times.— Typically fructose elutes around 10–11 min and glucoheptose around 15–18 min ( see Figures 2016.06D and E ). M. Calculations (a) Fructose stock standard .—Calculate the fructose stock concentration according to: Fructose concn, µg/mL = fructose wt. (g) × 1000000 µg/g × purity 500 mL where purity = %purity [from the Certificate of Analysis (CoA)/100%]. (b) WS .—Calculate WS concentrations (µg/mL) as follows: WS4 = Fructose stock × 50/100

WS3 = Fructose stock × 25/100

WS2 = Fructose stock × 5/100

WS1 = Fructose stock × 5/200 (c) Calibration .—Obtain the peak areas for fructose and

ED_1

WS4

75.0 060216-FRUCTAN #10 [modifiedbyHASELPA]

nC

70.0

60.0

50.0

40.0

30.0

20.0

2 -Glucoheptose - 15.834

1 - Fructose - 9.700

10.0

min

Figure 2016.06D Working standard (WS4) chromatogram (Part II). 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0 42.0 45.0 -2.0