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Simultaneous Determination of Thiamine HCl, Riboflavin, Niacin (Nicotinic Acid and Nicotinamide),

Pantothenic acid, Vitamin B6 (Pyridoxine, Pyridoxal and Pyridoxamine) and Biotin in Infant

Formula and Adult Nutritionals

CONFIDENTIAL METHOD FOR ERP ASSESSMENT ONLY

PAGE 8 OF 13

Parameters

Column

Shishedo, CAPCELL PAK C18 column, MGIII-H, 100mm x 2.0mm x 3

μm or equivalent.

Mobile Phase A

0.04% HFBA-0.05% Formic acid-5mM Ammonium formate in water

Mobile Phase B

MeOH

Flow Rate

0.4mL/min

Injection Volume

20 µL

Wash location

Flush port

Wash time

5 seconds

Tray temperature

4±2 °C

Column Oven

35±2 °C

Run Time

12 minutes

MSMS condition

ESI Positive mode

MRM detection

window

70 seconds

Resolution tables

Quad 1 and Quad 3: Unit, Scan speed = 10 Da/s

Scheduled MRM

Q1

Mass

(Da)

Q3

Mass

(Da)

Time

(min)

Analyte

DP

(volts)

EP

(volts)

CE

(volts)

CXP

(volts)

128.00

84.00

2.27 IS Nicotinic acid

96

10

29

12

124.00

80.05

2.27 Nicotic acid_1

86

10

27

10

124.00

78.02

2.27 Nicotic acid_2

86

10

29

10

124.00

52.99

2.27 Nicotic acid_3

86

10

41

8

127.00

84.00

2.27 IS Nicotinamide

78

3

28

11

123.00

80.00

2.27 Nicotinamide_1

82

4

38

4

123.00

78.00

2.27 Nicotinamide_2

82

4

29

10

123.00

53.00

2.27 Nicotinamide_3

82

4

41

7

171.00

153.00

2.91 IS Pyridoxal

34

3

20

2

168.02

150.10

2.91 Pyridoxal_1

46

10

15

10

168.02

94.00

2.91 Pyridoxal_2

46

10

31

8

168.02

122.02

2.91 Pyridoxal_3

46

10

25

16

224.00

94.00

2.93 IS Patothenic acid

12

3

20

12

220.00

90.00

2.93 Pantothenic acid_1

35

5

20

11

220.00

202.00

2.93 Pantothenic acid_2

35

5

17

20

220.00

184.00

2.93 Pantothenic acid_3

35

5

19

21

172.00

155.00

3.03 IS Pyridoxamine

28

8

17

19

169.00

152.00

3.03 Pyridoxamine_1

25

13

19

10

169.00

134.00

3.03 Pyridoxamine_2

25

13

26

13

169.00

77.00

3.03 Pyridoxamine_3

25

13

45

8

174.00

156.00

3.30 IS Pyridoxine

25

4

19

3

170.00

152.00

3.30 Pyridoxine_1

47

10

29

20

170.00

134.00

3.30 Pyridoxine_2

47

10

38

16

170.00

77.00

3.30 Pyridoxine_3

47

10

47

11

268.00

122.00

4.25 IS Thiamine HCl

40

5

20

15

265.00

122.00

4.25 ThiamineHCl_1

22

4

21

14

265.00

144.00

4.25 ThiamineHCl_2

22

4

20

19

265.00

81.00

4.25 ThiamineHCl_3

22

4

43

9

247.00

229.00

6.24 IS Biotin

56

10

21

16

245.00

227.20

6.24 Biotin_1

30

10

21

15

245.00

123.00

6.24 Biotin_3

30

10

37

15

245.00

97.00

6.24 Biotin_2

30

10

39

9

383.00

249.00

6.54 IS Riboflavin

49

3

31

22

377.00

243.00

6.54 Riboflavin_1

145

5

33

20

377.00

172.00

6.54 Riboflavin_2

145

5

48

10

BVit-01

FOR ERP USE ONLY

DO NOT DISTRIBUTE