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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