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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

c)

Mass Spectrometer Data Acquisition Parameters

Table 3. Mass Spectrometer data collection parameters.

Compound

Start

(min)

End

(min)

SRM Transition Typ. RT

(min)

Mass Spec Conditions

Molecular

Ion

Fragment

Ion

Cone

Voltage

CE

Voltage

Dwell Time

(msec)

Pyridoxamine

0.3

3.0

169.1

151.8

0.70

20

15

40

2

H

3

-Pyridoxamine

0.3

3.0

172.1

154.6

0.70

20

15

40

Pyridoxal

0.3

3.0

168.1

149.8

1.2

15

15

40

2

C

3

-Pyridoxal

0.3

3.0

171.1

153.1

1.2

15

15

40

Nicotinic Acid

0.3

3.0

124.0

80.4

1.25

50

16

40

2

H

4

- Nicotinic Acid

0.3

3.0

128.0

84.0

1.25

44

18

40

Pyridoxine

0.3

3.0

170.2

151.7

1.5

25

13

40

13

C

4

-Pyridoxine

0.3

3.0

174.2

155.7

1.5

25

13

40

Thiamine

0.8

1.75

265.3

122.0

1.56

20

15

40

13

C

3

-Thiamine

0.8

1.75

268.3

122.0

1.56

20

15

40

Niacinamide

1.75

3.5

122.9

96.0

2.67

8

16

40

2

H

4

-Niacinamide

1.75

3.5

127.0

100.0

2.67

8

16

40

Riboflavin

1.75

3.5

377.2

242.8

3.67

30

20

40

13

C

4

,

15

N

2

-Riboflavin

1.75

3.5

383.2

248.6

3.67

30

20

40

4.

UPLC-MS System Equilibration

a)

Place freshly prepared mobile phases onto the system and purge old solvents from the solvent

lines and needle washes using the UPLC Control Panel.

b)

The sample cone must be cleaned prior to each sample set. Cleaning can only be performed by

trained personnel and requires less than 10 minutes.

c)

Start gas flow into the mass spectrometer. Once gas flow has stabilized load the mass

spectrometer Tune Page and allow the source temperatures to equilibrate.

NOTE: It is very important to complete this step before proceeding. Failure

to do so could result in the premature introduction of UPLC eluate

into the Mass Spectrometer Ionization source resulting in potential

damage to the Ionization source.

d)

After confirming equilibration of the Mass Spectrometer source, start LC flow to the column.

Monitor the column backpressure and allow it to stabilize too less than 50-psi deviation for a

minimum of one minute. Once equilibration has been established, allow the system to equilibrate

for 10 minutes with flow into the source before beginning the sample set.

29

BVit-02

FOR ERP USE ONLY

DO NOT DISTRIBUTE