AOAC SPIFAN Review Team Meeting Book-July 22, 2015

Abbott Nutrition Division

Simultaneous Determination of Total Vitamin B

and B

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.

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.

Compounds

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

ClNO

and its molecular weight is

205.64 g/mol.

The molecular formula for Pyridoxine is C

and its molecular weight is 169.18 g/mol.

Common synonyms for Pyridoxine include vitamin B

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:

BVIT-02

FOR ERP/WORKING GROUP USE ONLY

DO NOT DISTRIBUTE