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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 1 OF 13

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.

BVit-01

FOR ERP USE ONLY

DO NOT DISTRIBUTE