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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 9 OF 13

377.00

198.00

6.54 Riboflavin_3

145

5

50

14

Analyte_1 is for Quantitation.Analyte_2 and 3 for confirmation.

Parameter

Period 1 Experiment 1

CUR

20.00

IS

5500.00

TEM

500.00

GS1

80.00

GS2

80.00

CAD

Medium

Valco value

Diverter

No.

Total Time (min

Position

1

0.0

A-Waste

2

2.0

B-MSMS

3

7.0

A-Waste

Typical LC Time

Program

Step

Total Time

(min)

Flow rate

(µL/min)

A (%)

B(%)

0

0.00

400

100

0

1

0.05

400

100

0

2

1.50

400

85

15

3

5.00

400

80

20

4

6.50

400

10

90

5

8.50

400

10

90

6

9.00

400

100

0

7

12.00

400

100

0

Stop Time

12 minutes

Retention Time

Nicotinic acid approximately 2.0-2.5 minutes

Nicotinamide approximately 2.0-2.5 minutes

Pyridoxal approximately 2.8-3.3 minutes

Pantothenic acid approximately 2.8-3.3 minutes

Pyridoxamine approximately 2.8-3.3 minutes

Pyridoxine approximately 3.1-3.6 minutes

Thiamin hydrochloride approximately 4.0-4.5 minutes

Biotin approximately 6.0-6.5 minutes

Riboflavin approximately 6.3-6.8 minutes

D. System Suitability Requirements

a. Plot calibration between Concentration (ng/ml) and Area ratio

≥ 6 points

. The correlation coefficient of

each calibration

should be not less than 0.995. % Error of concentration should be ≤ 10%

.

b. When stable baseline is obtained, inject a middle level of working standard three times in succession

and determine the %RSD, The %RSD should be not more than 3%.

c. Analyte a middle level of working standard between 10 to 12 samples. % Error od centration should be

≤ 10%

d. For the confirmation, ion ratio should be with in 20%

BVit-01

FOR ERP USE ONLY

DO NOT DISTRIBUTE