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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
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I. SUMMARY
Water-soluble vitamins in infant formula or adult nutritional samples are extracted with a mixture of mild
acid-Methanol. Quantitation of the extracted solution is carried out via LC-MS/MS using ESI in the positive
mode.
a. Intended use of method: The method is applicable for the concurrent quantitative analysis
of
Thiamine HCl, Riboflavin, Niacin (Nicotinic Acid and Nicotinamide), Pantothenic acid,
Vitamin B6 (Pyridoxine, Pyridoxal and Pyridoxamine) and Biotin present in milk- and soy-
based infant formula and adult nutritionals and formulas containing hydrolyzed protein.
b. Critical components: Correct personal and environmental safety standards shall be used
while performing this analytical method. Laboratory personnel handling solvents, acids
and reagents should be knowledgeable of their potential hazards. Consult the Material
Safty Data Sheets for information on the hazards and take proper precautions.Transfer
solvents and acids inside efficient fume hoods and extractors. Ensure all glassware is free
from chipping and hairline cracks.
II. EQUIPMENT & APPARATUS
Equivalents may be used unless otherwise specified.
Equipment
Description (Brand, make, model, etc.)
LC-MS/MS
Agilent UHPLC 1290 series- ABSCIEX QTRAP 6500
ESI Positive mode or equivalent.
HPLC Column
Shishedo, CAPCELL PAK C18 column, MGIII-H, 100mm x 2.0mm x 3
μm
or equivalent.
Vortex mixer
Labnet VX100 or equivalent.
Ultrasonic bath
Ultrasonik 208H or equivalent.
High speed centrifuge
Hettich Rotina 420R, with rotor for 2mL centrifuge tubes and capable of
15000 rpm at 4
C or equivalent.
Hot-Air oven (105
C)
Memmert or equivalent.
Analytical balance
Mettler Teledo XP204, capable of weighing to four decimal places or
equivalent.
Mechanical Shaker
EYELA Multishaker MMS or equivalent
HPLC vials
Agilent Technologies, amber, 2mL capacity, with crimp caps and PTFE
seals.
Centrifuge tube
50 ml PP, Amber Falcon tube with screw cap or equivalent.
Microcentrifuge tube
2 ml PP, Amber, Axygen or equivalent.
Auto pipette with tip
Eppendorf, 10-100 µl, 100-1000 µl and 1-5 ml or equivalent.
Volumetric pipet
Witeg, class A: 2, 3, 4, 5, 10, 15, 25 ml or equivalent.
Volumetric flask
Witeg, class A, amber: 1 (clear), 10, 100, 200, 1000 ml or equivalent.
Syringe Filter Nylon
membrane
Filtrex, 0.2 µm, 13 mm or equivalent and MSF filter 0.2 µm, 47mm or
equivalent.
B-VIT-01
FOR ERP/WORKING GROUP USE ONLY
DO NOT DISTRIBUTE
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