487 / 696
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