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

10.

Hydrolysis

10.1. 10.2. 10.3.

Warm up the waterbath to 60°C

Dissolve the dried residue in 1000 µL of the diluted internal standard solution. (5.6.1) Pipet 400 µL and add 25 µL Beta-galactosidase (5.7.) in a 2 mL safelock tube (hydrolyzed sample). Pipet 400 µL and add 25 µL of the diluted internal standard solution (5.6.1) in a 2 mL safelock tube (non-hydrolyzed sample for determination of lactose residue). Pipet 400 µL of the diluted internal standard solution (5.6.1) and add 25 µL Beta- galactosidase (5.7) in a 2 mL safelock tube (enzyme blank).

10.4.

10.5.

10.6. 10.7.

Homogenize the solutions and incubate for 30 min at 60°C.

Cool down the hydrolyzed samples and blanks to room temperature and centrifuge for 10 min at 13,000 rpm.

10.8.

Add 200 µL of the centrifuged samples in vials with micro insert.

11.

Quantification Analyze the samples with method GOS quantification (6.2) between standards (5.6.2) (approximately 10 sample injections).

12.

Calculation:

12.1.

Make a quadratic calibration curve for the ratio of the area of galactose / internal standard (x-axis) against the galactose concentration (y-axis; in µg/mL). This results in the formula y= ax 2 + bx + c. Calculate the calibration curve for lactose accordingly. The concentration galactose and lactose in the samples are calculated from these calibration curves (calculate between standards; bracketed 1 ). Correct the galactose concentration for the residual lactose and galactose from the non- hydrolyzed sample and the enzyme blank.

12.2.

12.3.

1 Calibration curve based on the preceding and succeeding standards

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