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Contribution of minor folates to the total folate content of infant formula and

adult/pediatric nutritionals / F. Martin (NRC, AS)

03 Feb 2016

CONFIDENTIAL

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Page 3 / 13

M E T H O D

Sample reconstitution

Powder samples were reconstituted by dissolving 25 g of powder sample in 200 g warm water (40 °C).

The SRM 1849a was reconstituted by dissolving 10 g of powder in 90 g warm water (40 °C).

Extraction

An aliquot of 15 g of reconstituted sample or 15 g of readyͲtoͲfeed (RTF) sample were weighed into a

250 mL flatͲbottomed amberͲglass flask.

50 mg of ɲͲamylase were added and sample was kept at 40 °C for 15 min. 40 mL of buffer (100 mmol/L

phosphate buffer, 2 % ascorbic acid, 0.1 % dithiothreitol (DTT), pH=4.5) were added and the flask was

then heated at 90 °C for 30 min, while stirring. After cooling to room temperature, 2 mL of a protease

solution (4 mg/mL) were added and incubation was carried out in water bath at 37 °C for 30 min. After

cooling to room temperature, the sample was transferred in a 100ͲmL volumetric flask and the volume

made up to the mark with water. After filtration through folded paper filter, 50 μL of a 5 μg/mL

internal standard solution were added to 10 mL of filtrate. From this solution, 3 mL were loaded on a

strong anion exchange (SAX) cartridge (previously washed and equilibrated). After loading, the

cartridge was washed with 6 mL of washing solvent and then analytes were eluted with 4 mL of eluting

solution. Eluate was then evaporated under controlled temperature at 50 °C and nitrogen flow.

Extracts were then reconstituted in 1.5 mL of reconstitution solution (H

2

O, 1 % ascorbic acid, 0.5 %

DTT) and filtered through 0.22 μm membrane into an amber LC vial.

To assess the effect of deconjugation, 1 mL of rat serum was added after the incubation with the

protease. Prior of this addition, samples were heated at 90 °C for 5 min to deactivate the protease.

Incubation was carried out in water bath at 37 °C for 2 hours. After cooling to room temperature the

extraction procedure was carried on as described above.

Standard solutions

Stock standard solutions (100 μg/mL of free form) were diluted in reconstituted solution to obtain a

5 μg/mL “mix 1” solution. It was further diluted to obtain a 75 ng/mL “mix 2” solution.

In 5 mL volumetric flasks, aliquots of either mix 1 or 2 solution were pipetted and made up to volume

(5 mL) with reconstituted solution to yield working standard solutions (1.00, 4.00, 10.0, 40.0, 100,

200, 300 and 400 ng/mL). To evaluate the linearity following SMPR, 3 additional standards were

prepared to yield 0.3, 700 and 900 ng/mL.

UHPLC conditions

2013.13 (Fol-21) / (February 2016) - NOTES

FOR ERP USE ONLY

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