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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
ͼ This document may not be reproduced or disclosed to third parties without prior authorization
Page 4 / 13
The 0.22 μmͲsyringeͲfiltered final extract (5 μL) and standards solutions (5 μL) were injected onto a
UHPLC system (Agilent 1290 Infinity) equipped with a Waters UPLC HSS T
3
, 1.8 μm; 2.1 x 150 mm
column. Mobile phase A consisted of H
2
O, 0.5 % acetic acid. Mobile phase B was acetonitrile.
Following injection, isocratic conditions of 0% of solvent B were initially employed for 0.5 min, then a
step direct to 10% of solvent B was achieved in 0.1 min. Isocratic conditions of 10% solvent B were
hold for 1.4 min and followed by a linear gradient to 25% solvent B for 3.5 min. Then, a step directly
at 99% B was achieved in 0.1 min and hold for 1.9 min before going back to start conditions (0% of
solvent B) in 0.1 min. Start conditions were kept for 2.4 min.
Tandem Mass Spectrometry conditions
Mass spectrometry was performed on an Agilent 6460 MS in positive electrospray ionization (ESI+)
mode operating at unit resolution.
ESI capillary voltage was set at 3.5 kV; nozzle voltage, 600 V; gas temperature, 300 °C; sheath gas
temperature, 350 °C; gas flow 10 L/min; sheath gas flow, 12 L/min; nebulizer pressure 30 psi.
MultipleͲreaction monitoring mode was applied for quantification and compound identification
conformation. The following transitions were followed:
Time
range
[min]
Analyte
Q1
Q3
Fragmento
r Voltage
[V]
Collision
Energy
[eV]
2.0 Ͳ 5.0
5ͲMe THF (Q)
460.2 313.1
108
14
2.0 Ͳ 5.0
5ͲMe THF (q)
460.2 180.0
108
42
2.0 Ͳ 5.0 5ͲMe THF ISTD 465.2 313.1
120
15
2.0 Ͳ 5.0 5Ͳ10MylͲTHF (Q)
456.2 412.1
110
20
2.0 Ͳ 5.0 5Ͳ10MylͲTHF (q)
456.2 416.1
110
40
2.0 Ͳ 5.0 5Ͳ10MylͲTHF ISTD 461.1 416.1
120
30
2.0 Ͳ 5.0
THF (Q)
446.2 299.1
106
14
2.0 Ͳ 5.0
THF (q)
446.2 166.0
106
46
2.0 Ͳ 5.0
THF ISTD
451.2 299.1
115
15
5.0 Ͳ 8.0
Folic Acid (Q)
442.2 295.1
90
10
5.0 Ͳ 8.0
Folic Acid (q)
442.2 176.0
90
40
5.0 Ͳ 8.0 Folic Acid ISTD 447.1 295.0
92
10
5.0 Ͳ 8.0 5CHOͲTHF (Q)
474.2 327.1
110
15
2013.13 (Fol-21) / (February 2016) - NOTES
FOR ERP USE ONLY
DO NOT DISTRIBUTE