Journal of Food Composition and Analysis 36 (2014) 6671

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Journal of Food Composition and Analysis

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Original Research Article

Selected vitamins, minerals and fatty acids in infant formulas in the

United States
Pamela R. Pehrsson *, Kristine Y. Patterson, Mona A. Khan
USDA Agricultural Research Service, Nutrient Data Laboratory, Building 005, BARC-West, 10300 Baltimore Avenue, Beltsville, MD 20705, USA

A R T I C L E I N F O A B S T R A C T

Article history: Infant formulas (IF) are consumed during a short but critical growth period and are the sole source of
Received 26 December 2012 nutrition for at least part of this time for non-breastfed infants. The 1980 Infant Formula Act (IFA) and
Received in revised form 3 June 2014 subsequent legislation mandate fortication of all IF at specic levels of vitamins and minerals;
Accepted 5 June 2014
manufacturers must assure these levels in their products. In order to determine the actual amounts of
Available online 23 August 2014
nutrients in IF and to determine how closely these conform to the IFA and labels amounts, fteen highly
consumed milk- or soy-based infant formulas were sampled nationally at 12 retail locations. These IF
Keywords:
were analyzed, and thiamin, vitamins C, A, D, E and K, choline, calcium, iron, and arachidonic,
Infant formula
Minerals
docosohexanoic and linoleic acids values examined. Within analytical uncertainty, all IFs except linoleic
Vitamins acid in one formula met label claim and were within allowed ranges. These results suggest IFs provide
National sampling nutrition at the level of the label or more. For some vitamins, there was substantially more present in the
Calcium formula than the label amount, e.g. vitamin C, 25  16 vs. 8 mg per 100 kcal (n = 20), but the variability
Iron among brands was high. These data help researchers and consumers understand the nutritional impact of
Choline formulas in the diet of infants.
Essential fatty acids 2014 Published by Elsevier Inc.
Food labeling standards
Food composition
Food analysis

1. Introduction
The primary responsibility of the Nutrient Data Laboratory
(NDL), Agricultural Research Service, USDA, is to provide authori-
tative food composition data on foods and dietary supplements
available in the United States. The USDA National Nutrient
Database for Standard Reference (SR) (USDA, 2012) includes
nutrient proles for over 8000 foods, for up to 146 food
components. These data are used by the Food Surveys Research
Group of the USDA along with data from the National Health and
Nutrition Examination Survey (CDC, 2010) to make estimates of
nutrient intake for the U.S. population presented in several ways
including by age.
In the past, data on infant formulas (IF) in SR have been based on
label claims. The purpose of this study was to provide analytical data
on IF for SR and thus to improve the accuracy of the estimates of
nutrient content as well as variance indicators in this important
* Corresponding author at: USDA Agricultural Research Service, Nutrient Data
Laboratory, Building 005, BARC-West, 10300 Baltimore Avenue, Beltsville, MD
20705, USA. Tel.: +1 301 504 0693; fax: +1 301 504 0692.
E-mail address: pamela.pehrsson@ars.usda.gov (P.R. Pehrsson).
food. Researchers and nutrition public policy makers can use these
data for a more accurate assessment of the nutrient content (means
and variability) of IF when evaluating the intake of very young
children. This study also provides information on how well label
claims conform to the actual nutrient content of the IF, and if these IF
conform to specic nutrient concentrations (as minimum allowed)
or ranges for a 100 kcal serving of IF mandated in the Infant Formula
Act (IFA) of 1980 (CFR, 2013) and related amendments.
The National Food and Nutrient Analysis Program (NFNAP) is a
research program initiated in 1997 in collaboration with the
National Institutes of Health to generate a body of nutrient data
with unprecedented analytical quality and which is nationally
representative of the U.S. food supply (Pehrsson et al., 2003).
Nutrient data presented, vitamins (A, D, E, C, K and thiamin),
minerals (calcium and iron), and choline, are among many
determined to be important to normal infant growth (e.g. bone,
brain and visual development; (IOM, 1998, 2001, 2002, 2010). Fatty
acids are also needed for development in the infant with one, linoleic
acid, being required by the U.S. Food and Drug Administration (FDA)
regulations (CFR, 2013). IF, therefore, must not only be in compliance
with the label but should meet recommended levels. Table 1 lists
recommendations for adequate intakes (AIs) for infants from the
Institute of Medicine (IOM) (IOM, 2011 and IOM, 2002), required

http://dx.doi.org/10.1016/j.jfca.2014.06.004
0889-1575/ 2014 Published by Elsevier Inc.
 P.R. Pehrsson et al. / Journal of Food Composition and Analysis 36 (2014) 6671 67

Table 1
Adequate intake (AI), and FDA requirements and ESPGHAN recommended amounts for select nutrients in infant formulas.

Nutrient AI units per day FDA required ESPGHAN recommended

amount per 100 kcalc amount per 100 kcald

Vitamin A 400500 mg (RAE) 75225 mg 60180 mg (REe)

Vitamin D 10 mg 12.5 mg (as D3) 12.5 mg (as D3)
400 IU 40100 IU

Vitamin K 22.5 mg 4 mg 425 mg

Vitamin E 45 mg 0.315 mg (natural)
0.469 mg (synthetic) 0.55 mg (a-TEf)
0.7 IU

Vitamin C 4050 mg 8 mg 830 mg

Thiamin 0.20.3 mg 0.04 mg 0.060.3 mg
Choline 125150 mg 7 mg 750 mg
Calcium 200260 mg 60 mg 50140 mg

Iron 0.27 mga 0.153.0 mg 0.31.3 mgg

6.9 mg (EAR)b 0.452.0 mgh

Linoleic acid 4.44.6 g 0.3 g 0.31.2 g

a
Iron value for ages 06 months.
b
Iron value for ages 712 months.
c
Single values are the minimum with no maximum mandated; ranges give the minimum and maximum (CFR, 2013).
d
Koletzko et al. (2005).
e
1 mg RE (Retinol equivalent) is equal to 1 mg of retinol; RAE = Retinol activity equivalent = 1 mg retinol.
f
1 mg a-TE (a-tocopherol equivalent) = 1 mg D-a-tocopherol.
g
Iron value for formula based on cows milk protein and protein hydrolysate.
h
Iron value for formula based on soy protein isolate.

formula concentrations/ranges from ofcial FDA regulations in (FDA, 2006). These fatty acids are added to support brain
amounts per 100 kcal (CFR, 2013) and, also, recommendations from development and visual acuity in young formula-fed infants (CFR,
an expert panel convened by the European Society for Pediatric 2013; IOM, 2004); addition of these fatty acids to IF is also supported
Gastroenterology, Hepatology and Nutrition (ESPGHAN) at the by the International Formula Council (IFC, 2010). If fortied, FDA has
request of the Codex Committee on Nutrition and Foods for Special permitted the following levels: DHA must be 0.20.5% by of weight of
Dietary Uses (Koletzko et al., 2005). total fatty acids; the amount of ARA must be DHA (FDA, 2006).
Two of the vitamins in this study are thiamin and vitamin C.
Thiamin, or vitamin B1, was the rst of the essential B vitamins 2. Methods
identied and is important for converting carbohydrates into
energy. Vitamin C has a high reducing capacity, which helps it to 2.1. Sampling
act as a cofactor for several enzymes (Englard and Seifter, 1986).
Within limits, excesses of these water-soluble vitamins are A nationally representative sampling frame shown was
excreted and no maximum level is set (Table 1). developed for collection of IF under the USDA National Food and
Vitamins D, K, E and A are fat-soluble. Vitamin D3 has been Nutrient Analysis Program (NFNAP) (Pehrsson et al., 2003). The
incorporated into cows milk since the early part of the 20th stratied, probability proportional-to-size (PPS) statistical design
century for the prevention of rickets. Vitamin K, phylloquinone, is was based on the 2000 U.S. Census data (U.S. Bureau of the Census)
necessary for normal blood clotting and synthesis of proteins. It is (Perry et al., 2003). Fifteen high-consumption IF were obtained
also helpful for cell growth and maintains their healthy structure from grocery stores in 12 statistically selected locations in the U.S.
(IOM, 2001). Vitamin E functions as an antioxidant preventing (Fig. 1). Three major manufacturers and several types of IF (both
propagation of lipid peroxidation with the a-tocopherol form soy- and cow milk-based) were represented in the sampling; two
being the most active (IOM, 2000). Vitamin A, as retinol in IF, is toddler formulas were included for analysis because they
important for normal vision, gene expression and for the immune presented similar label claims but were analyzed solely for fatty
system (IOM, 2001). Maximum levels for both vitamins A and D in acids and vitamin D, and store-brand formulas, milk and soy were
IF are set by the FDA (Table 1). obtained. IF were combined, composited, a priori into six random
Choline is a nutrient essential for the production of acetylcho-
line, a neurotransmitter that plays a role in the proper functioning
of many organs including the brain and liver (Zeisel et al., 1986). In
1998, the Institute of Medicine (IOM, 1998) set intake require-
ments for infants and the FDA has established fortication levels
for IF (Table 1).
The two minerals in this study, calcium and iron, are essential
for healthy growth in infants: iron in the body is incorporated into
the red blood cells and calcium is an element essential for bone
growth and other functions.
The FDA has set a minimum amount of linoleic acid for IF at
300 mg/day (CFR, 2013). Voluntary fortication with the long chain
polyunsaturated fatty acids docosahexaenoic (DHA) and arachidonic
(ARA) began in 2002 after the FDA recognized DHA and ARA derived
from various sources as being Generally Recognized As Safe (GRAS)
at the amounts and ratios specied by the formula manufacturers Fig. 1. Sampling plan locations for procurement of infant formulas.
68 P.R. Pehrsson et al. / Journal of Food Composition and Analysis 36 (2014) 6671

Table 2
The vitamin content of 15 infant formulas giving the number of composites, mean, standard deviation (where n > 2) and the (%) percent difference from label. Not all formulas
were analyzed for all nutrients.

Formulaa Vitamin C Thiamin Vitamin D Vitamin K Vitamin A Vitamin E

(mg/100 kcal) (mg/100 kcal) (IU/100 kcal) (mg/100 kcal) (mg/100 kcal) (IU/100 kcal)

n mean %b n mean SD % n mean SD % n mean % n mean SD % n mean SD %

A-1 2 12 100 6 0.07 0.01 90 6 75 2 120 2 10 120 6 141 2 160 6 4.2 0.9 210
A-2 1 20 160 6 0.11 0.02 140 7 90 12 150 1 12 150 6 112 2 120 4 3.9 0.5 190
A-3 2 85 140 2 3.7 180
A-4 2 16 130 4 0.14 0.02 170 4 70 4 140 2 10 120 4 130 6 140 4 3.8 0.7 190
B-5 2 19 220 4 0.16 0.03 160 4 72 5 120 2 10 130 4 131 14 150 4 4.5 0.5 230
B-6 2 0.17 170 2 74 120 2 132 150 2 4.5 230
C-7 1 81 140
C-8 1 38 420 5 0.17 0.02 170 5 72 7 120 1 20 260 5 180 9 200 5 3.7 0.3 250
C-9 1 47 520 5 0.19 0.01 320 5 81 6 180 1 21 140 5 174 5 190 5 5.5 0.1 360
C-10 2 54 360 4 0.34 0.05 160 4 94 4 130 2 21 190 4 214 5 150 4 8.0 0.9 220
SB-11 2 14 120 2 0.19 240 6 80 7 130 2 11 140 2 130 140 2 3.6 180
A-12 2 17 140 5 0.10 0.02 120 5 75 10 120 2 13 160 5 129 4 140 5 3.7 0.4 190
B-13 2 23 190 2 0.15 260 2 73 120 2 10 110 2 127 140 2 4.8 240
C-14 1 51 560 5 0.19 0.03 230 6 77 5 130 1 18 170 5 187 5 210 5 4.2 0.2 280
SB-15 2 16 130 2 0.22 270 2 81 130 2 10 120 2 123 140 2 4.6 230
a
The letter indicates the manufacturer or SB for store brand, and the number indicates the formula. Formulas 111 are milk based; formulas 1215 are soy based.
b
Percent of label value.

pairs based on location. Because not all IF were found in each retail
location, the number of composites ranged from two to six by
brand/type. When the ready-to-feed (RTF) form of the IF was not
found, the powder or liquid concentrate form was selected.
The IF types for milk-based included: regular formula, lactose-
free formula, formula for neonates (premature infants), hypoaller-
genic formula and formula for toddlers. One soy-based IF was
obtained from each of the three major manufacturers as well as
store-brand.
2.2. Sample preparation
All the individual samples of IF (samples units) were shipped to
the Food Analysis Laboratory Control Center (FALCC) at Virginia
Tech and none of them were beyond their sell-by date. The
sample units were pooled into composites as described above.
Sample units were combined and homogenized in acid cleaned
bowls, stirred thoroughly and aliquots were dispensed into 1-oz.
jars, sealed under nitrogen with Teon lined caps, and stored
frozen at  60 8C until analyzed. The aliquots of the composites
and quality control (QC) materials were shipped to the laboratories
overnight on dry ice. The QC materials were identied as controls
but no other information was given.
2.3. Analysis of vitamins, mineral and choline
The composite aliquots were analyzed by laboratories that had
been prequalied for capability in making specic nutrient
measurements using valid methods. Most of the vitamin, mineral
and fatty acid methods were AOAC (AOAC, 2000), thiamin AOAC
942.23, vitamin C 967.22, iron and calcium 985.01 and 984.27,
and fatty acids 996.06 with some modications (e.g. 100 m
column). Vitamin D analysis methodology used high performance
liquid chromatography (HPLC) with ultraviolet detection (Patter-
son et al., 2010) and choline analyses were done by isotope dilution
mass spectrometry (Koc et al., 2002). Phylloquinone (vitamin K)
was determined using HPLC with uorescence detection (Booth
and Sadowski, 1997). Vitamin A, retinol, was determined by HPCL,
using a modied carotenoid method (Craft, 2001) and a-
tocopherol, vitamin E, using HPLC with UV detection, external
calibration and internal standard recovery. Reference materials
from the National Institute for Standards and Technology (NIST)
were included in all analyses for quality control purposes, and
strict quality control (QC) measures were employed. The NIST SRM
1846 Infant Formula was used as the reference material for all
nutrients except vitamins A and E, and fatty acids. For vitamins A
and E, NIST SRM 2383 Baby Food was used, and for fatty acids, NIST

Table 3
The mineral and choline content of 15 infant formulas giving the number of composites, mean, standard deviation (where n > 2) and the (%) percent difference from label. Not
all formulas were analyzed for all nutrients.

Formulaa Calcium (mg/100 kcal) Iron (mg/100 kcal) Linoleic acid (mg/100 kcal) Choline (mg/100 kcal)

n Mean SD %b n Mean SD % n Mean SD % n Mean %

A-1 6 85 6 100 6 1.8 0.0 100 6 901 36 100 2 30 120

A-2 6 79 5 100 6 2.0 0.0 110 6 902 20 100 1 53 220
A-3 2 854 100
A-4 4 99 3 110 4 2.0 0.1 110 4 634 15 70 2 31 130
B-5 4 71 12 110 4 1.7 0.1 110 4 973 10 110 2 38 160
B-6 2 220 120 2 2.2 110 2 952 110
C-7 1 1170 120
C-8 5 87 1 110 5 2.1 0.0 120 5 1180 24 120 1 18 110
C-9 5 119 6 110 5 1.9 0.1 100 5 2320 37 120 1 16 130
C-10 4 122 2 120 4 2.0 0.1 110 4 1110 65 150 2 22 140
SB-11 2 76 100 2 2.0 110 7 980 65 110 2 29 120
A-12 5 112 3 110 5 2.0 0.0 110 5 953 27 110 2 29 120
B-13 2 105 100 2 1.9 100 2 991 110 2 30 130
C-14 5 124 2 120 5 2.0 0.0 110 5 1170 18 120 1 12 100
SB-15 2 115 110 2 1.9 100 2 927 120 2 26 110
a
The letter indicates the manufacturer or SB for store brand, and the number indicates the formula. Formulas 111 are milk based; formulas 1215 are soy based.
b
Percent of label value.
 P.R. Pehrsson et al. / Journal of Food Composition and Analysis 36 (2014) 6671 69

SRM 2387 Peanut Butter was used. A QC committee from the NDL
reviewed the analytical data and the QC reference material results.
Results met the acceptance criteria at NDL.
3. Results and discussion
3.1. Quality control results for nutrient analyses
iron 5.86  0.06 mg/100 g (certicate 6.31  0.40), vitamin C
103.3  4.3 mg/100 g (certicate 114.6  6.6), choline 153  7 mg/
100 g (certicate 125  12), vitamin K 84.5  4.5 mg/100 g (certicate
94.4  4.1), vitamin E 2.61  0.24 mg/100 g (certicate 2.5  0.33),
vitamin D 448  6 IU/100 g (certicate 468  44). Where values did
not fall within the certicate range, they did fall within an extended
range that allows for additional uncertainty during routine analyses.

The quality control results for the SRM materials were vitamin 3.2. Data handling
A 78  4 mg/100 g (certicate 80  15), thiamin 0.88  0.02 mg/100 g
(certicate 1.09  0.15), linoleic acid 13.43  0.02 g/100 g (certicate The laboratory data were received on a weight basis, which is
13.15  0.41), calcium 356  3 mg/100 g (certicate 367  20), suitable for SR, but for purposes of this report, were recalculated to

Fig. 2. Comparison of analytical values (mean, SD) in milk- and soy-based infant formula to FDA nutrient requirements for (a) vitamins and choline, and (b) minerals and
linoleic. Shaded areas are FDA ranges and dotted lines are FDA minimums.
70 P.R. Pehrsson et al. / Journal of Food Composition and Analysis 36 (2014) 6671
give them as per 100 kcal. For those composites that were not RTF
(i.e. concentrates or powder), it was necessary to adjust the data to
a RTF basis. The calculations were based on the reconstituting
directions on the labels. The data from the composites were
averaged to give a mean value by brand/type; each of these values
was based on the analysis of two to six composites. In addition, the
label values of vitamins A, D and E were given in IU, therefore the
analytical results were adjusted from mass units to IU for
comparison vitamin A (retinol) 0.3 mg per IU, vitamin D
0.025 mg per IU, and vitamin E (synthetic source) 0.667 mg per
IU or (natural source) 0.45 mg per IU (IOM, 2000, 2001, 2010).
Some of the formula labels listed rst mixed tocopherols and then
natural a-tocopherol in the ingredient list. The conversion of the
analytical data in mg to IU assumed that the vitamin E was half
synthetic and half natural for these formulas.
3.3. Vitamins and choline
The vitamin and choline content of all of the IF met the label
amounts with one exception where the value was very close
(Tables 2 and 3). The actual amounts, in some cases, exceeded the
label by a substantial amount especially for vitamin C. Gao et al.
(2008) also observed in their study that water-soluble vitamins in
IF exceeded label values. Since the shelf life of many IF is 23 years,
this may be done to ensure that the levels do not drop below the
label from deterioration over time. The label amounts for the
nutrients varied among products, e.g. the IF designated C-10 in
Table 2 is for premature infants, and many of the nutrient levels are
higher than for the other IF.
The products from manufacturer C for vitamin C exceeded the
label by 3.55.5 times which was far more than any of the other
vitamins or choline (Table 2). MacLean et al. (2010), in a Codex
Alimentarius study of infant formulas, noted that vitamin C is a
difcult nutrient for manufacturers due to degradation under
certain conditions. They stated that it would not be unusual to see
losses during shelf life of 3050% or higher. The addition of vitamin
C in excess of the label ensures that the product meet the label
claim through the entire shelf life.
Of all the vitamins, vitamin D results were the closest to the
label, ranging from 100 to 180%. These data are similar to early
research by Holick et al. (1992), who found that of 10 popular IF
Milk-based Formulas
120
Standard / Toddler
100
Vitamin D IU / 100 kcal
80
60
40
A B C D
Fig. 3. Vitamin D3 values by brand/type of infant formula showing individual sample values; shading shows FDA acceptable range and  the label value.
analyzed, all met or exceeded the amount on the label for vitamin
D; three IF analyzed contained 200% of the label declaration. The
vitamin D values for the individual composites are shown in
Fig. 3 separated by the types of IF: standard, standard toddler,
neonate and hypoallergenic as well as standard soy-based. The
different symbols within type are by manufacturer. Except for
one composite that was slightly high, all the results were within
the range required under the IFA designated by the shaded area
on the graph. The one sample that exceeded the acceptable
range was only 4% higher than the maximum amount and easily
within analytical variability. In earlier research conducted
under NFNAP, vitamin D was highly variable among fortied
milks of different brands and fat levels; this prompted the closer
scrutiny of IF.
The vitamin E (a-tocopherol) values in the IF were consistently
higher than label. For the most part, these were about twice label
however one was about 3.5 times label. This high IF was also one
that was very high in vitamin C (Fig. 2).
For the other vitamins and choline, none of the results were as
far over label as was the vitamin C. There were a few values that
exceeded 200% of label (Table 2), primarily for thiamin, the other
water soluble vitamin, in the soy formulas, and in a few cases for
the other vitamins and choline. When compared to FDA nutrient
requirements per 100 kcal, all vitamins and choline met or
exceeded the minimum, and, where the FDA gives a range of
acceptable content, fell within that range (Fig. 3).
3.4. Minerals
The minerals, in general, were much closer to label values
than the vitamins; this was expected since these nutrients do not
degrade over time. The minerals (calcium and iron) ranged from
an average of 100 to 120% above label; consistency within
manufacturer and across manufacturers was evident (Table 3).
The actual label value for calcium differed widely from a value of
67 for a standard IF to 190 mg per 100 kcal for a IF for an older
infant. The label values for iron were much closer, ranging from
1.5 to 2.0 mg per 100 kcal. As shown in Fig. 3, the mineral values
exceeded the minimum amounts set by FDA and for iron, where
there is a maximum given, none of the IF were above that
amount.
Soy-based Formulas
Hypo-allergenic/ Neo Standard
Lactose free
E F G H I J K L
Infant Formulas
 P.R. Pehrsson et al. / Journal of Food Composition and Analysis 36 (2014) 6671
0.045
0.040
0.035
0.030
g / 100 kcal
0.025
0.020 ARA
0.015
DHA
0.010
0.005
0.000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
A B C D
Manufacturer
Fig. 4. Amount of docosahexaenoic acid (DHA) and arachidonic acid (ARA) in
different types of infant formula from four sources.
3.5. Fatty acids
Linoleic acid results were very close to the label values (Table 3).
Except for one, the IF were all between 100 and 120% of label. One
IF fell signicantly below the label amount. Results for four
composites, representing several locations and lots, with a follow-
up repeat analysis of one at a later date conrmed the results. This
suggests manufacturing or labeling error and not analytical error.
Regardless, all of the linoleic acid results exceeded the FDA
minimum (Fig. 3).
The IF were not labeled with the content of the fatty acids ARA
or DHA but these are often added. DHA and ARA results were
variable among IF and averaged 0.24% and 0.49%, respectively, of
total fatty acids with all were within the requirement of 0.20.5% of
total fatty acids (FDA 2001, FDA, 2006); the ARA content always
exceeded the DHA content (range 1.62.8 times higher) (Fig. 4).
When compared to global averages for human breast milk, 0.32%
(DHA) and 0.47% (ARA) as % of total fatty acids (Brenna et al., 2007),
average DHA in these IF was lower and ARA about the same. Values
were consistent with concentrations found specically in ranges
for breast milk of U.S. and Canadian women (IOM, 2002): 0.10.4%
(DHA) and 0.10.6% (ARA). These fortied U.S. IF were within the
approved ranges for added DHA and ARA.
4. Conclusions
Nearly all analytical results for the nutrients reported here
met or exceeded the minimum nutrient content for infant
formula or fell within range as given in the Code of Federal
Regulations. For choline and vitamins, the label claim may
understate the actual amount in the product. For minerals, the
analytical results more closely match the label claims than for the
vitamins. The linoleic acid values were above the FDA minimum,
although one product failed to meet the label claim, and the other
fatty acids, arachidonic, docosohexanoic were found in the
correct proportions.
Acknowledgements
This project was supported by the Agricultural Research
Service, USDA. Partial support was received from the National
Institutes of Health, Agreement # Y1-HV-8116-15.
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