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C
ampos
G
iménez
&
M
artin
:
J
ournal of
AOAC I
nternational
V
ol
.
100, N
o
.
1, 2017
139
Vitamin C in Infant Formula and Adult/Pediatric Nutritional
Formula by Liquid Chromatography with UV Detection:
Collaborative Study, Final Action 2012.22
E
sther
C
ampos
G
iménez
and
F
rédéric
M
artin
Nestlé Research Center, Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland
Collaborators: K. Schimpf; L. Butler Thompson; D. Aoude-Werner; J. Dalmas-Le Grandois; J. Wong; J. Chia; I. Malaviole; M. Mapar; T. Nguyen;
M. Hoard; G. Joseph; L. Ma; B. Wu; T. Rogers; J. Austad; B.M. Jensen; I. Pedersen; S. Meng Jensen; Y. Zhang; W. Jobgen; C. Weihong; L. Xiuying;
H. Chanyuan; D. Woollard; E. Kneeteman; O. Hernández Hernández; E. Beltrán; G. Weerasekera; J. Frueh; P. González; E. Román; M. Carreño;
G. Jaudzems; R. Nijman; T. Noorloos; L.K. Yap; S.H. Chooi; G.J. Lautenschlager; S. Christiansen; D. Piot; M. Barba Le Brun
To determine the repeatability and reproducibility
values of the AOAC INTERNATIONAL First Action
Method 2012.22,
Vitamin C in Infant Formula and
Adult/Pediatric Nutritional Formula by Liquid
Chromatography with UV Detection
, a collaborative
study was organized. The study was divided into two
parts: method setup and qualification of participants
(part 1) and collaborative study participation (part 2).
During part 1, each laboratory was asked to analyze
two practice samples using the aforementioned
method. Laboratories that provided results within
a range of expected levels were qualified for part 2,
where they analyzed 10 samples in blind duplicates.
Two of the samples were suspected of spoilage
during the test and new cans of the same type of
product were analyzed by a subset of laboratories
in part 3. The results were compared with
Standard
Method Performance Requirement
(SMPR
®
) 2012.012
established for vitamin C. The precision results were
within the requirements stated in the SMPR: 1.4–7.3%
and 3.2–11.4% respectively, for repeatability and
reproducibility. Finally, Horwitz ratio values were all
<2 (0.5–1.7). The Expert Review Panel for Stakeholder
Panel for Infant Formula and Adult Nutritionals
Nutrient Methods determined that the data presented
met the SMPR and therefore recommended the
method be granted Final Action status.
V
itamin C (
l
-ascorbic acid) plays an important role in
oxidative stress reactions and is involved in a number of
metabolic functions (1). Because humans are unable to
synthetize vitamin C, its supply must be ensured through adequate
dietary intake. Daily requirements can vary from 75 mg in adult
women to 90 mg in adult men (2), and Codex has established
a lower limit of 10 mg/100 kcal and a guidance upper level of
70 mg/100 kcal in infant formula and foods for special medical
purposes (3). Several official AOAC INTERNATIONAL
Methods exist for the analysis of vitamin C in foods, and in
particular, in infant formula, as reviewed elsewhere (4). Fontannaz
et al. (5) published a method for the quantification of ascorbic and
isoascorbic acid in fortified foods, including infant formula and
nutritional products.Amodification of thismethodwas proposed to
the Expert Review Panel for Stakeholder Panel for Infant Formula
and Adult Nutritionals Nutrient Methods (SPIFAN) and was
approved asAOAC FirstAction Method
2012.22
in 2012 (6), with
a recommendation to advance to a multilaboratory collaborative
study. This paper presents the results of the collaborative study.
Samples
The study took place using SPIFANmatrixes, which represent
most of the products in the scope of the project (infant formula
and adult nutritionals made from any combination of milk,
soy, rice, whey, hydrolyzed protein, starch, and amino acids,
with and without intact protein). All samples were blinded and
codified before being sent to participating laboratories.
AOAC Official Method 2012.22
Vitamin C in Infant Formula and Adult/Pediatric
Nutritional Formula
Liquid Chromatography with UV Detection
First Action 2012
Final Action 2016
[Applicable to the determination of vitamin C (
l
-ascorbic
acid) in infant formula and adult/pediatric nutritional formula
by LC–UV.]
Caution
: Refer to Material Safety Data Sheets prior to use
of chemicals. Use appropriate personal protective
equipment when performing tests.
A. Principle
l
-Ascorbic acid is extracted from the sample using
trichloroacetic acid (TCA) in the presence of tris [2-carboxyethyl]
phosphine (TCEP) as a reducing agent. Ascorbic acid is then
determined by ultra-HPLC (UHPLC) or HPLC with UV
detection at 265 nm. Separation takes place in a C
18
column
INFANT FORMULA AND ADULT NUTRITIONALS
Received July 21, 2016. Accepted by SG August 17, 2016.
The method was approved by the AOAC Official Methods Board
as Final Action.
See
“Standards News,” (2016)
Inside Laboratory
Management
, July/August issue.
The AOAC Stakeholder Panel on Infant Formula and Adult
Nutritionals (SPIFAN) invites method users to provide feedback on the
Final 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.
Corresponding author’s e-mail: esther.campos-gimenez@rdls.
nestle.comDOI: 10.5740/jaoacint.16-0232
222