AOAC SPIFAN Review Team (August 2016)

Single Lab Validation of a Method for the Determination of β‐

Galactooligosaccharides in Infant Formula & Adult Nutritionals / Sean Austin (NRC,

Lausanne)

25 Jul 2016

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Page 6 / 35

‐ 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.

VALIDATION REPORT

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