AOAC SPIFAN Review Team Meeting Book-July 22, 2015

BVIT-02 FOR ERP/WORKING GROUP USE ONLY DO NOT DISTRIBUTE

Abbott Nutrition Division Simultaneous Determination of Total Vitamin B 6, B 2, B 3 in Infant Formula Products by LC-MS/MS Using Enzymatic Digestion and B 1

c) Mass Spectrometer Data Acquisition Parameters Table 3. Mass Spectrometer data collection parameters.

SRM Transition Typ. RT (min) Fragment Ion Molecular Ion

Mass Spec Conditions

End (min)

Start (min)

Compound

Cone Voltage

CE Voltage

Dwell Time (msec)

Pyridoxamine

0.3 0.3

3.0 3.0

169.1 172.1

151.8 154.6

0.70 0.70

20 20

15 15

40 40

2 H 3

-Pyridoxamine

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 0.3 0.3 0.8 0.8

3.0 3.0 3.0

128.0 170.2 174.2 265.3 268.3 122.9 127.0 377.2 383.2

84.0

1.25

44 25 25 20 20

18 13 13 15 15 16 16 20 20

40 40 40 40 40 40 40 40 40

Pyridoxine

151.7 155.7 122.0 122.0 100.0 242.8 248.6 96.0

1.5 1.5

13 C 4

-Pyridoxine

Thiamine

1.75 1.75

1.56 1.56 2.67 2.67 3.67 3.67

13 C 3

-Thiamine

Niacinamide

1.75 1.75 1.75 1.75

3.5 3.5 3.5 3.5

8 8

2 H 4

-Niacinamide

Riboflavin

30 30

13 C 4

, 15 N 2

-Riboflavin

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.

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