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Determination of B-Vitamins in Infant Formula and Adult/Pediatric Formulas

ESI LC-MSMS

1

Applicable for the determination of B-Vitamins (Thiamine, Riboflavin, Nicotinic acid, Niacinamide,

Pantothenic acid, Pyridoxine, Biotin and Folic acid) in infant, adult, and/or pediatric formula (powders,

ready to feed liquids and liquid concentrates). The LOQs of Biotin and Folic acid are 0.05 mg/kg and for

remaining vitamins, 0.5 mg/kg.

Caution:

Refer to Material Safety Data Sheet (MSDS) of chemicals prior to use, and follow the

suggested personal protective equipment.

A. Principle:

Samples are extracted with water in presence of ascorbic acid and adjusted to pH 4.8 with

ammonia solution to precipitate proteins. Extract is filtered and subjected to LC-MSMS. Results are

quantified against certified reference materials by external standard method.

B. Apparatus:

a) Liquid Chromatograph:

Agilent UHPLC Infinity series with Binary Solvent Manager capable of

1200 bar pressure.

b) Column:

Zorbax RRHD, C18, 3.0 x 100 mm, 1.8 μm.

c) Detector:

Agilent 6460A triple quadrupole mass spectrometer.

d) Injector:

Agilent Infinity Series Auto sampler with column oven.

e) Data System:

Mass Hunter, Agilent.

f)

pH meter.

g) Syringe filters:

0.22 μm, PTFE.

C. Reagents:

a) Ascorbic acid

, AR grade.

b) Ammonium Formate

, MS grade.

c) Formic acid

, MS grade.

d) Ammonia

, Mass fraction of about 30%.

e) Acetic acid

, Glacial, AR grade.

f) Potassium Hydrogen Carbonate

, AR grade.

g) Methanol,

absolute, ULC/MS Grade

h) De-ionized laboratory water -

≥ 18 Mohm/cm

i) Volumetric flasks (Low actinic),

10ml, 25ml, 50ml, 100ml

D. Standards:

a) Thiamin Hydrochloride

,from Supelco, Cat. No. 47858

b) Riboflavin

, from Supelco, Cat. No. 4-7861

c) Niacinamide or Nicotinamide

, from Sigma, Cat. No. 72340

d) Nicotinic acid

, from Supelco, Cat. No. 47864

e) D-Pantothenic acid hemicalcium salt

, from Sigma, Cat. No. 21210

f) Pyridoxine Hydrochloride

, from Sigma-Aldrich, Cat. No. P9755

g) Biotin

, Sigma-Aldrich, Cat. No. B4501

h) Folic acid

, from Sigma, Cat. No. F7876

E.

Solutions Preparation:

a) Mobile Phase A:

Dissolve 0.63 g of ammonium formate in water, add 0.5ml formic acid and

dilute to 500ml with water. Mix and degas for 10 min in ultrasonic bath.

b) Mobile Phase B:

Dissolve 0.63 g of ammonium formate in methanol, add 0.5ml formic acid and

dilute to 500ml with methanol. Mix and degas for 10 min in ultrasonic bath.

BVit-03 (February 2016)

FOR ERP USE ONLY

DO NOT DISTRIBUTE