332 / 435
C
ampos
-G
iménez
et al
.:
J
ournal of
AOAC I
nternational
V
ol
.
96, N
o
. 5, 2013
1065
Vitamin C in Infant Formula andAdult/Pediatric Nutritional
Formula by Ultra-Performance Liquid Chromatography with
Ultraviolet Detection: First Action 2012.22
E
sther
C
ampos
-G
iménez
,
P
atric
F
ontannaz
, K
arine
R
edeuil
, and
T
amara
K
ilinc
Nestlé Research Center, Nestec Ltd, Department of Quality Assurance, Vers-Chez-les-Blanc, 1000 Lausanne 26, Switzerland
D
awn
D
owell
AOAC INTERNATIONAL, 481 North Frederick Ave, Gaithersburg, MD 20877-7089
Submitted for publication April 22, 2013.
The method was approved by the Expert Review Panel on Infant
Formula and Adult Nutritionals as First Action.
The AOAC Stakeholder Panel on Infant Formula and Adult
Nutritionals (SPIFAN) 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 to gaining global
recognition and acceptance of the methods. Comments can be sent
directly to the corresponding author or
methodfeedback@aoac.org.Corresponding author’s e-mail: esther.campos-gimenez@rdls.
nestle.comDOI: 10.5740/jaoacint.13-141
INFANT FORMULA AND ADULT NUTRITIONALS
During the AOAC Annual Meeting held in Las
Vegas, NV from September 30 to October 3, 2012,
the Stakeholder Panel on Infant Formula and Adult
Nutritionals convened to review single-laboratory
validation data submitted for the method, Vitamin C
in Adult/Pediatric Formula by Ultra-Performance
Liquid Chromatography with Ultraviolet Detection.
This method is a modified version of the method
“HPLC-UV Determination of Total Vitamin C in a
Wide Range of Fortified Food Products” previously
published in
Food Chem.,
94, 626–631 (2006). The
SLV data from the modified method were reviewed
and compared to the standard method performance
requirements (SMPR 2012.012), and it was concluded
that the method meets the requirements. The
method was approved as AOAC Official First Action.
The method is based on the acidic extraction of
ascorbic acid in the presence of Tris[2-carboxyethyl]
phosphine (TCEP) as a reducing agent. Separation
was achieved on a C
18
column with a sodium
acetate eluent (pH 5.4) combined with TCEP and
decylamine as an ion-pairing agent. Accuracy rates
were between 90 and 100%. Repeatability RSD
(RSD
r
) ranged from 1.4 to 2.5%, and intermediate
reproducibility RSD (RSD
iR
) ranged from 1.3 to 7.5%.
V
itamin C, L-ascorbic acid, is a water-soluble vitamin
that plays an important role in oxidative stress reactions
and takes part in a number of metabolic functions (1).
This makes its incorporation into the human diet essential; its
deficiency results in scurvy. It is found naturally in fresh fruits
and green vegetables and is largely used in food fortification to
compensate for losses during industrial processes. The method
described here, a modified version of a previously published
method (2), has been evaluated and determined to meet the
Standard Method Performance Requirements (SMPRs; 3) for the
determination of vitamin C in infant formula and adult/pediatric
nutritional formula.
AOAC Official Method 2012.22
Vitamin C in Infant Formula and Adult/Pediatric
Nutritional Formula
Ultra-Performance Liquid Chromatography
with Ultraviolet Detection
First Action 2012
[Applicable to the determination of vitamin C (L-ascorbic
acid) in infant formula and adult/pediatric nutritional formula
by ultra-performance LC (UPLC
®
)-UV.]
Caution
: Refer to Material Safety Data Sheets prior to use of
chemicals. Use appropriate personal protective equipment when
performing testing.
A. Principle
Ascorbicacidisextractedfromthesampleusingtrichloroacetic
acid (TCA) in the presence of Tris[2-carboxyethyl]phosphine
(TCEP) as a reducing agent. Ascorbic acid is then determined
by UPLC
®
-UV at 265 nm. Separation takes place on a C
18
column with sodium acetate (pH 5.4) as the eluent, combined
with TCEP and decylamine as an ion-pairing agent.
B. Apparatus
(
a
)
Balances.—
With readability of 0.1 mg and 0.01 g.
(
b
)
pH meter.—
Metrohm 691 (Herisau, Switzerland) or
equivalent.
(
c
)
LC column.—
Waters (Milford, MA) Acquity UPLC
®
ethylene bridged hybrid (BEH) C
18
column, 1.75 µm,
2.1×100 mm or equivalent.
(
d
)
UPLC
®
system.—
Waters
Acquity UPLC
®
system
equipped with a Waters Acquity UPLC
®
photodiode array
detector or equivalent.
C. Reagents and Standards
(
a
)
Acetonitrile.—
HPLC grade, Merck (Geneva, Switzerland)
or equivalent.
(
b
)
Ascorbic acid.—
Fluka (Buchs, Switzerland) or equivalent.
(
c
)
Decylamine.—
Fluka or equivalent.
(
d
)
Phosphoric acid.—
85%, Merck or equivalent.
AOAC OMB Meeting Book
307