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7

ascorbic acid and 0.1 % (v/v) 2,3ͲdimercaptoͲ1Ͳporpanol. Flush tubes with N

2

gas in

order to minimise folate degradation then cap the tubes.

(h) Concentrate eluents by evaporation using a Savant SpeedVac to achieve 500 μL

volume.

(i) Aliquot concentrated extracts into microcon with 3 kDa molecular weight cutͲoff and

centrifuge at 4 °C for 30 minutes at 14,900 g

F. Determination

Chromatography

(a) Mobile phase system: 0.1 % (v/v) formic acid in MilliͲQ water (eluent A) and

acetonitrile (eluent B)

(b) The flow rate: 200

P

L/min

(c) Gradient elution: the initial conditions are 99.5 % A and 0.5 % B, maintain for 0.2

minutes following injection. Raise the proportion of B to 35 % at 2.2 minutes, then

to 70 % at 3.0 minutes and hold at that composition until 3.2 minutes. Return to

initial conditions at 3.5 minutes and reͲequilibrate until 6 minutes

(d) Injection volume 25 μL

Mass spectrometry

(a) Positive ion electrospray ionisation (ESI) mode

(b) Fix spray voltage of the mass spectrometer at 3,500 V

(c) Spray current approximately 10 μA.

(d) Maintain the entrance capillary at 350

o

C.

(e) Selected reaction monitoring mode. For quantitation purpose, monitor the following

transitions:

m/z

442ї295 (folic acid), 447ї295 (

13

C

5

folic acid), 460ї313 95Ͳ

methyltetrahydrofolate ) and 465ї313 (

13

C

5

5Ͳmethyltetrahydrofolate)

H. Calculations

Calculate analyte response as the ratio of the chromatographic peak area of each analyte

relative to that of the corresponding internal standard. Plot calibration curves by linear

regression. Obtain FA level in the reconstituted milk sample (μg/g).

Fol-20 (February 2016)

FOR ERP USE ONLY

DO NOT DISTRIBUTE