AOAC SPIFAN Review Team Meeting Book-July 22, 2015

ABBOTT LABORATORIES

INTEROFFICE CORRESPONDENCE

Date:

2/13/12

From:

Louis Salvati

Subject: Validation of LC/MS Determination of Total B

, B

, and B

Using an

Enzymatic Preparation.

Background

Although internal methodologies are available for the determination of the

fortified forms of pyridoxine (PYR), thiamin (THI), and riboflavin (RIB); Abbott

Nutrition does not currently have a method for the determining the total content of these

three nutrients. While the overwhelming majority of each vitamin in Abbott Nutrition’s

products comes from the fortified form, some comes from the natural ingredients used in

the products. Natural ingredients such as milk contain two forms or pyridoxine:

pyridoxal (PAL) and pyridoxamine (PAM) as well as phosphorylated versions of these

two forms. Milk also contains the fortified form of riboflavin with two phosphorylated

analogs and the fortified form of thiamin with two phosphorylated analogs. Government

regulations in certain countries require the total content to be determined. This method

would also be a useful internal research tool.

In order to assay the inherent nutrient content that is bound in the sample, some

form of matrix digestion is needed; and additionally, since those forms are

phosphorylated, a dephosphorylating agent is also necessary. The matrix digestion can be

carried out by strong acid and heat, or enzymatically. A potential procedure was proposed

by C. Hasselmann

which used papain, and alpha-amylase to break down the sample

matrix to free bound nutrients. Acid phosphotase is also added to dephosphorylate the

nutrients. The separation developed is based loosely on work done by Hong Zhang

using an Acquity BEH C18 column (2.1 mm x 50 mm, 1.7 um) and a 20 mM ammonium

formate and methanol gradient. The separation is done in less than 7 minutes and is

illustrated in Figures 1 and 2.

A limited sample group consisting only of milk-based infant formulas was chosen

for this validation because milk based ingredients are the dominant source of the inherent

vitamins. The goal was to first establish a solid basis using the most affected sample

type. Two standard reference materials were analyzed along with a commercial infant

formula and a placebo infant formula not fortified with these nutrients. Samples spiked

with the phosphorylated forms of each nutrient were tested; however, the spiking levels

BVIT-02

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