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

Sample preparation/fractionation:

7.

7.1.

Ready to feed samples can be used as it is. 25 g of powders need to be reconstituted in 200 g of water. Heat the solutions for 30 min at 60°C and cool down to room temperature. Liquid concentrates need to be diluted 1 on 1 by weight.

Depending on the amount of GOS in the ready to consume product, the product weight can be adjusted.

Table 5.5: Required product weight for sample preparation GOS concentration (%)

Amount of product solution (mg) to be weight

0.2 2.5 5.0

7500

600 300

7.2. 7.3. 7.4. 7.5. 7.6. 7.7. 7.8.

Weigh accurately according Table 5.5 an amount of product in a 50 mL volumetric flask.

Add Milli-Q until ¾ of the flask is filled.

Add 750 µL Carrez reagent 1 (5.2.) and homogenize thoroughly. Add 750 µL Carrez reagent 2 (5.3.) and homogenize thoroughly.

Adjust the volume to 50 mL and homogenize.

Fill a safelock tube with sample and centrifuge for 10 min at 13,000 rpm.

After centrifugation filtrate the supernatant through a 0.2 µm microfilter and transfer to an HPLC vial.

7.9.

Run a sequence with method “GOS fractionation” (6.1) . Perform at least 3 injections to stabilize the system.

7.10. 7.11. 7.12. 7.13.

Run lactose standard (5.8) and a sample to check the fractionation window.

Run the samples (bypass or switch of detector).

Monitor the time on screen and collect the fraction 1.0 min up to 2.8 min (begin lactose peak) in a 15 mL tube. Discard the lactose peak from 2.8 min up to 3.2 min (waste). Collect again from 3.2 min up to 7.2 min, in the same 15 mL tube. Collect fractions in duplicate. Solid Phase Extraction (SPE) Prepare a Carbograph column by vacuum elution. Make sure that the flow of the eluents is constant and at a rate of 1 to 2 drops per second. Do not let the column bed run dry. Close the tap exactly when the eluent level reaches the top of the column bed.

8.

8.1. 8.2. 8.3. 8.4. 8.5.

1.5 mL 80% ACN + 0.1% TFA (5.4.) » waste

1.5 mL Milli-Q

» waste » waste » waste

Total fraction (7.1.13)

1.5 mL Milli-Q

Remove the SPE column from the vac-elute. Add 1.5 mL 40% ACN + 0.05% TFA (5.5.) and collect the fraction in a 2 mL safelock tube by using air pressure from a pipet to get a flow rate of 1 or 2 drops per second. Carefully (watch out for bubbles) press all liquid into the tube until the Carbograph column is dry. Speedvac Dry the collected samples overnight in the Eppendorf concentrator under vacuum at room temperature.

9.

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