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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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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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