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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