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S

alvati

et al

.:

J

ournal of

AOAC I

nternational

V

ol

.

99, N

o

.

3, 2016 

1

Simultaneous Determination of Total Vitamins B

1

, B

2

, B

3

, and

B

6

in Infant Formula and Related Nutritionals by Enzymatic

Digestion and LC-MS/MS: Single-Laboratory Validation, First

Action 2015.14

L

ouis

M. S

alvati

, S

ean

C. M

c

C

lure

, T

odime

M. R

eddy

,

and

N

icholas

A. C

ellar

1

Abbott Nutrition, 3300 Stelzer Rd, Columbus, OH 43219

This method provides simultaneous determination

of total vitamins B

1

, B

2

, B

3

, and B

6

in infant formula

and related nutritionals (adult and infant). The method

was given First Action for vitamins B

1

, B

2

, and B

6

,

but not B

3

, during the AOAC Annual Meeting in

September 2015. The method uses acid phosphatase

to dephosphorylate the phosphorylated vitamin

forms. It then measures thiamine (vitamin B

1

);

riboflavin (vitamin B

2

); nicotinamide and nicotinic

acid (vitamin B

3

); and pyridoxine, pyridoxal, and

pyridoxamine (vitamin B

6

) from digested sample

extract by liquid chromatography-tandem mass

spectrometry. A single-laboratory validation was

performed on 14 matrixes provided by the AOAC

Stakeholder Panel on Infant Formula and Adult

Nutritionals (SPIFAN) to demonstrate method

effectiveness. The method met requirements of

the AOAC SPIFAN

Standard Method Performance

Requirement

for each of the three vitamins, including

average over-spike recovery of 99.6 ± 3.5%, average

repeatability of 1.5 ± 0.8% relative standard deviation,

and average intermediate precision of 3.9 ± 1.3%

relative standard deviation.

A

OAC Stakeholder Panel on Infant Formula and Adult

Nutritionals (SPIFAN) released a call for methods for total

vitamins B

1

, B

2

, B

3

, and B

6

in infant formula and related

nutritionals. In the European Union and other countries, label

claim is regulated based upon total vitamin content and not just

the fortified form. Historically, microbiological methods were used

to estimate total vitamin. However, these methods are challenged

with newer, more diverse nutritional products and are no longer

considered the gold standard. Newer, chromatographic methods,

especially with mass spectral detection, are quickly becoming

the new standard because their specificity enables accurate

quantitation across more complex and diverse matrixes. However,

that specificity then requires explicit definition of the vitamin forms

necessary for a “total” vitamin determination. SPIFAN gathered

experts in industry, government, and academia to provide these

definitions. Total vitamin B

1

is defined as the sum of thiamine,

thiamine monophosphate, thiamine pyrophosphate, and thiamine

triphosphate in the

Standard Method Performance Requirement

(SMPR

®

; 1). Total B

2

is defined as riboflavin, riboflavin-5′-

phosphate, and flavin adenine dinucleotide (2). Total B

3

is defined

as the sum of nicotinic acid and nicotinamide (3). Finally, total B

6

includes five forms: pyridoxine, pyridoxal, pyridoxal-5′-phosphate,

pyridoxamine, and pyridoxamine-5′-phosphate (4).

SPIFAN defined forms of vitamins B

1

, B

2

, B

3

, and B

6

that

represent the major contributors to total vitamin concentration in

formulas and enter the formulation through both fortification and

from ingredient sources. For example, depending on the protein

fraction of milk and the degree of processing, the contribution of

inherent (unfortified) vitamin can be virtually 0 to >45% of the

total value (5). But, regardless of source, the concentration of

each vitamin must be verified to meet label claim. Remarkably,

the necessary sample treatments and separation as described

later are similar for the intended vitamins and thus lend

themselves to simultaneous determination, saving both time

and cost. Further, the availability of modern mass spectrometry

(MS) instrumentation with electrospray ionization (ESI)

facilitates simultaneous determination by removing remaining

hurdles associated with detection. Suppression of ionization is

problematic for quantitation with ESI, but is overcome with the

use of stable-isotope labeled internal standards. The associated

cost of isotopically labeled standards, although perceived as

great, only adds a few cents to the cost of a sample because

of the small amount necessary. By contrast, the syringe filter

required to prevent clogging the liquid chromatography (LC)

column adds about $1 (USD) to the cost of a sample.

The combined method was developed to measure thiamine,

riboflavin, pyridoxamine, pyridoxal, pyridoxamine, nicotinic acid,

and nicotinamide directly. Separation was achieved with 20 mM

ammonium formate mobile phase without ion pairing agent.

Thiamine is not well retained in reverse phase at low pH without

an ion pairing agent (6). However, ion pairing agents bring

additional challenges to LC-tandem MS (MS/MS) determination.

Improved retention of thiamine has been previously demonstrated

by increasing the mobile phase pH (6). In fact, there is a striking

improvement in retention for many water-soluble vitamin under

reverse-phase conditions at moderate pH (5–7). This improvement

in retention was harnessed to achieve good method performance

for a subset of the targeted vitamin forms; however, elution

of phosphorylated compounds is notoriously difficult (8). The

phosphate moiety complexes with Fe

3+

and thus phosphate

INFANT FORMULA AND ADULT NUTRITIONALS

Submitted for publication February 18, 2016.

The method was approved by the AOAC Expert Review Panel for

SPIFAN Nutrient Methods as First Action.

The expert review panel invites method users to provide feedback

on the First Action methods.

Feedback from method users will help verify that the methods

are fit-for-purpose and are critical for gaining global recognition and

acceptance of the methods. Comments can be sent directly to the

corresponding author or

methodfeedback@aoac.org

.

1

Corresponding author’s e-mail:

nick.cellar@abbott.com

DOI: 10.5740/jaoacint.15-0315

105