Review Team (February 2016).pdf

D. Preparation of Standard Solutions

(a) Stock standard solutions (100 μg/mL). Accurately weigh 10.00 mg folic acid and 5Ͳ

methyltetrahysdrofolate under subdued light into a separate 100 mL volumetric

flask. Add 100 mL 0.1 M phosphate buffer (pH 6.1) with antiͲoxidants containing 1%

(w/v) ascorbic acid and 0.1% (v/v) 2,3 dimercaptoͲ1Ͳpropanol.

(b) Similarly, dissolve 1.00 mg stable isotope labelled

folic acid and

5Ͳ

methyltetrahydrofolate which serve as internal standards in 10 mL phosphate buffer

and antiͲoxidants

months.

(d) Prepare working internal standard solutions (10 μg/mL) on day of extraction.

(e) Calibration standard solutions. Dilute stock analyte solutions using phosphate buffer

containing antioxidants and internal standards. Prepare these solutions fresh and

analyse them at the beginning of every analytical run.

E. Sample and Test Portion Preparation

(a) Reconstitute (in duplicate) formula powder according to the manufacturer’s

recommendation (100 mL).

(b) Dissolve 25.0 g of SPIFAN samples (n=2) with 200.0 g of lukewarm water.

into centrifuge tubes.

(d) Pipette 2 mL of phosphate buffer containing both internal standards (0.7μg) per 1.00

g sample, and then vortex.

(e) Place centrifuge tubes in a boiling water bath for 15 minutes, cool down on ice and

then centrifuge at 10,000 g at 4 °C for 15 minutes.

(f) Filter supernatants using Whatman 41 ashͲless filter paper

(g) Solid phase extraction: Activate styreneͲdivinylbenzene (SDBͲL) sorbents with

acetonitrile (5 mL) and then equilibrate with 15 mL of 0.2 % (v/v) formic acid in MilliͲ

Q water. Load samples under gravity and wash the sorbents with 0.2 % formic acid

(15 mL). Elute analytes with 2.25 mL of 40 % (v/v) acetonitrile in MilliͲQ water under

gravity into glass tubes, which have 250 μL antiͲoxidants consisting of 2 % (w/v)

Fol-20 (February 2016)

FOR ERP USE ONLY

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