AOAC SPIFAN Review Team (August 2016)

- Sodium cyanoborohydride

- Formic acid, GR for analysis

- Acetic Acid anhydrous, GR for analysis

- Acetonitrile, Gradient grade for LC

- Ammonium hydroxide solution (25%), GR for analysis

- Di-potassium hydrogen phosphate (K

HPO

)

- Potassium dihydrogen phosphate (KH

)

- Hydrochloric acid solution (1 mol/L)

- Sodium hydroxide solution (1 mol/L)

- Bovine Serum Albumin (BSA)

- Laboratory water Type I

- Laminaritriose, >90 %

- Maltotriose min. 95 %

- Amyloglucosidase (AMG enzyme) from aspergilus niger (Roche #11202367001)

- High purity β-Galactosidase (4000 U/mL) from aspergilus niger (Megazyme E-BGLAN)

Maltotriose stock solution (4 µmol/mL):

Into a 50-mL volumetric flask, weigh 100 mg ± 5 mg of

maltotriose standard and dissolve with 30 mL water, then complete to the mark with water. (This

can be divided into 5 mL aliquots, and stored frozen for up to 1 year).

Laminaritriose stock solution (2 µmol/mL):

Weigh 50 mg of Laminaritriose into a 50-mL volumetric

flask, dissolve in 30 mL water, then complete to the mark with water. (This can be divided into

4 mL aliquots, and stored frozen for up to 1 year).

Laminaritriose working solution (0.3 µmol/mL):

Using a pipette transfer 3.8 mL of Laminaritriose

stock solution to a 20-mL volumetric flask and complete to the mark phosphate buffer. (This can be

divided into 5 mL aliquots, and stored frozen for up to 1 year).

Ammonium acetate buffer (0.1 mol/L, pH 5.5):

In a 100-mL beaker, dissolve 0.771 g ± 0.005 g (10

mmol) of anhydrous ammonium acetate with 80 mL ± 5 mL of water and adjust pH to 5.50 ± 0.05

with acetic acid. Transfer quantitatively to a 100-mL volumetric flask and complete to the mark

with water.

Potassium phosphate buffer (0.2 mol/L, pH 6.0):

Into a 1000-mL beaker dissolve 22.0 g potassium

dihydrogen phosphate KH

and 4.59 g di-potassium hydrogen phosphate K

HPO

in 600 mL of

water (using a magnetic stirrer). Adjust the pH to 6.0 with hydrochloric acid (1 mol/L) or sodium

hydroxide (1 mol/L). Transfer the solution to a 1000-mL volumetric flask and make up to the mark

with water.

BSA stock solution (15 mg/mL):

Weigh 30 mg of bovine serum albumin (BSA) in to a 5-ml plastic

tube. Add 2.0 mL of potassium phosphate buffer (0.2 mol/L, pH 6.0) and mix well.

BSA working solution (0.5 mg/mL):

Into a 5-mL plastic tube, pipette 50 µL of BSA stock solution.

Add 1450 µL of phosphate buffer pH 6.0 and mix well.

Β-Galactosidase solution (2000 U/mL with BSA (0.25 mg/mL)):

In a 1.5-mL microtube mix 300 µL of

β-galactosidase (4000 U/mL) with 300 µL of BSA working solution and mix well.

NOTE: This method has been validated with β-galactosidase from Megazyme (# E-BGLAN). Other sources of β-

galactosidase must be evaluated to ensure that there are no side activities on other oligosaccharides which may be

METHOD

FOR ERP USE ONLY

DO NOT DISTRIBUTE