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Abbott Nutrition Division
Simultaneous Determination of Total Vitamin B
6,
B
2,
B
3
and B
1
in Infant
Formula Products by LC-MS/MS Using Enzymatic Digestion
compound during sample preparation and chromatographic separation but be readily differentiated by
the mass spectrometer.
The quantitation of a compound is performed by the simultaneous analysis of the native and the
isotopically labeled compounds. The isotopically labeled compound is added to each standard
solution and sample preparation at a constant level. The calibration curve is generated from the
concentration of the native compound versus the response ratio, which is defined as the ratio of the
peak response of the native compound to the isotopically labeled compound.
3.
Solvent Purity
All components that enter the ESI source are continuously ionized. As a result any impurities in the
solvents or mobile phase additives can and often do result in a measurable loss of ionization efficiency
and loss of instrument sensitivity. This exact cause leading to a loss of sensitivity and impact on the
analytical data can be difficult to diagnose from review of the data. It is therefore critical that only the
best quality solvents be used. Specifically, Burdick and Jackson HPLC grade Methanol has been
identified as containing one or a number of components that, when present in ether the mobile phase
or sample preparation, results in a loss of up to 50% of the ion signal for selected compounds. The use
of LC-MS grade Methanol is required for successful completion of this method.
4.
Compounds
a)
Pyridoxine
Pyridoxine is added to Abbott Nutrition premixes as Pyridoxine Hydrochloride. Pyridoxine
Hydrochloride is readily soluble in water, sparingly soluble in ethanol and only slightly soluble in
diethyl ether and chloroform. The powder is reasonably stable to light and air and aqueous
solutions are stable to air and heat. The UV absorbance maxima of Pyridoxine Hydrochloride are
pH-dependent. At pH=4.5 the maxima are at 290 nm and 324 nm.
The chemical formula for Pyridoxine Hydrochloride is C
8
H
12
ClNO
3
and its molecular weight is
205.64 g/mol.
The molecular formula for Pyridoxine is C
8
H
11
NO
3
and its molecular weight is 169.18 g/mol.
Common synonyms for Pyridoxine include vitamin B
6
and 2-methyl-3-hydroxy-4,5-
dihydroxymethyl pyridine.
The CAS# for Pyridoxine is 65-23-6.
The chemical structure of Pyridoxine is shown below:
21
BVit-02
FOR ERP USE ONLY
DO NOT DISTRIBUTE