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Abbott Nutrition Division

Simultaneous Determination of Total Vitamin B

6,

B

2,

B

3

and B

1

in Infant

Formula Products by LC-MS/MS Using Enzymatic Digestion

f)

Data Processing: Waters MassLynx 4.1 (or later version) with TargetLynx data processing

package.

g)

Nitrogen Generator: Peak Scientific Nitrogen Generator; Model # NM30LA, or equivalent.

3.

Instrument Conditions

a)

UPLC Instrument Configuration and Operating Parameters

Table 1. UPLC instrument configuration details (Injection 1).

Mobile Phase A1

20 mM Ammonium Formate

Mobile Phase B1

Methanol

Flow Rate

250 µL/min

Flow Rate into MS

250 µL/min

Degasser

On

Column

Waters Acquity UPLC BEH C-18 1.7 µm 2.1 x 50 mm with a Waters BEH C18 1.7 µm

2.1 x 5mm Vanguard Column

Column Temperature

40

°

C

Injection Type

10 µL full loop injection with 10 µL sample loop installed

Loop Volume Overfill

Automatic

Sample Temperature

25 ºC

Injector Wash Settings

Strong Wash - Solvent

Methanol

Strong Wash – Volume

600 µL

Weak Wash – Solvent

Water

Weak Wash - Volume

1000 µL

Gradient Program

Time

% A1

% B1

Curve

0.0

99.0

1.0

6

1.5

99.0

1.0

6

1.6

75.0

25.0

6

3.5

75.0

25.0

6

3.6

1.0

99.0

6

5.0

1.0

99.0

6

5.1

99.0

1.0

6

7.0

99.0

1.0

6

27

BVit-02

FOR ERP USE ONLY

DO NOT DISTRIBUTE