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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