SPIFAN Nutrients ERP Book_9-29-15

GOS-02 SLV FOR ERP USE ONLY DO NOT DISTRIBUTE

Single Lab Validation Report for GOS in Infant Formula and Adult Nutritionals

Table 5.1: Preparation of sugar stock solutions Internal standard

MW (mg/mMol) Conc (mg/1000 mL) Purity (%)

L(+)-arabinose

150.14

15

99

Sugar

MW (mg/mMol) Conc (mg/100 mL)

Purity (%)

D(+)-galactose

180.17

100 100

99 99

Lactose monohydrate 360.31

5.6.1.

Internal standard solution (15 µg/mL in 40 mM phosphate buffer pH 6.0): Prepare the internal standard by weighing 15 ± 2 mg arabinose in a 200 mL beaker. Dissolve the arabinose in about 100 mL Milli-Q water. Transfer analytically the arabinose stock solution to a 1 L volumetric flask. Add 200 mL of the 0.2 M potassium phosphate buffer pH 6.0 (5.1) and adjust to 1 L with Milli-Q water. Standard series: Prepare the sugar stock solutions according to Table 5.1 by weighing 100 ± 5 mg lactose and galactose to separate 100 mL volumetric flasks and adjust to 100 mL with the internal standard solution (5.6.1) . The concentration of the sugar stock solution is 1000 µg/mL (galactose or lactose) plus 15 µg/mL internal standard. Prepare dilutions from the standard sugar stock solutions in 50 mL volumetric flasks according to Table 5.2.

5.6.2.

Table 5.2: Preparation of standards

Added volume stock (mL)

Concentration galactose + lactose (µg/mL)

Standard

galactose

lactose

1 2 3 4 5

1.000 0.750 0.500 0.125 0.000

1.000 0.750 0.500 0.125 0.000

20 15 10

2.5 0.0

For each standard, add galactose and lactose stock to one volumetric flask. Adjust to 50 mL with the diluted internal standard solution (5.6.1) . Due to addition of enzyme solution in the sample (including arabinose) the standards need to be corrected by adding 25 µL buffer solution to 400 µL standard (or by correcting for this in the calculation). Beta-galactosidase solution (400 U/mL): • Weigh 40 mg enzyme and dissolve in 1 mL with the diluted internal standard solution (5.6.1) . • Prepare fresh on daily basis. Carrez treated lactose standard 60 µg/mL: Pipet 3 mL lactose stock (1 mg/mL) (5.6.2.) in a 50 mL volumetric flask. Add Milli-Q water until ¾ of the flask is filled. Pipet 750 µL of Carrez 1 (5.2.) and 750 µL of Carrez 2 (5.3.) to the flask and mix. Adjust the volume until 50 mL with Milli-Q water, homogenize and centrifuge for 10 min at 13,000 rpm. Filtrate the supernatant through a 0.2 µm microfilter and transfer to an HPLC vial. This standard is used to determine the fractionation time.

5.7.

5.8.

16