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H
ostetler
:
J
ournal of
AOAC I
nternational
V
ol
.
100, N
o
.
3, 2017
1
Determination of Lutein and β-Carotene in Infant Formula
and Adult Nutritionals by Ultra-High-Performance Liquid
Chromatography: Single-Laboratory Validation, First
Action 2016.13
G
regory
L. H
ostetler
Perrigo Nutritionals, Analytical Research and Development, 147 Industrial Park Rd, Georgia, VT 05468
An ultra-HPLC method for the determination of
lutein and β-carotene in infant formula and adult
nutritionals was validated using both unfortified and
fortified samples provided by the AOAC Stakeholder
Panel on Infant Formula and Adult Nutritionals
(SPIFAN). All experiments showed separation of
all-
trans
-lutein and β-carotene from their major
cis
isomers, apocarotenal, α-carotene, lycopene,
and zeaxanthin. Samples spiked with all-
trans
-
lutein and β-carotene showed no isomerization
during sample preparation. Linearity of the
calibration solutions correlated to approximately
0.8–45 μg/100 g (reconstituted basis) for samples
prepared for the lowest sample concentrations. With
dilutions specified in the method, the range can be
extended to approximately 2250 μg/100 g. The LOD
for both lutein and β-carotene was 0.08 μg/100 g,
and the LOQ for both was 0.27 μg/100 g. For all
measurements in the range of 1–100 μg/100 g,
repeatability RSD was ≤5.8% for lutein and ≤5.1%
for β-carotene. For measurements >100 μg/100 g,
repeatability RSD was ≤1.1% for lutein and ≤1.7%
for β-carotene. Accuracy was determined by
recovery from spiked samples and ranged from
92.3 to 105.5% for lutein and from 100.1 to 107.5%
for β-carotene. The data provided show that the
method meets the criteria specified in the
Standard
Method Performance Requirements
for carotenoids
(SMPR 2014.014).
T
he carotenoids present in human milk include α-carotene,
β-carotene, β-cryptoxanthin, lutein, zeaxanthin, and
lycopene (1–3). Of these, lutein and β-carotene are
most commonly added to infant formula and adult nutritionals.
Whereas β-carotene has provitamin A activity (4), lutein may
play a role in vision and cognitive function (5). Both lutein and
β-carotene can occur as all-
trans
and
cis
isomers, and there is
interest in separating these because of differences in absorption
and biological activity. In addition to having twice the vitamin
A activity of
cis
isomers, all-
trans
-β-carotene is preferentially
absorbed over 9-
cis
-β-carotene (6). All-
trans
-lutein is the most
common isomer found in human retinas (7) and infant brains (8).
Because there were no official methods for the determination
of lutein or β-carotene in infant formula and adult nutritionals,
the current method was developed based on existing extraction
and chromatographic procedures from various carotenoid
methods. The saponification procedure was adapted from
Granado et al. (9), the extraction solvents from Craft (10),
the use of apocarotenal as an internal standard from Marx et
al. (11), and the use of 10 mM α-tocopherol as an antioxidant
from Scita (12). Chromatographic separation of lutein and
β-carotene isomers with C30 columns and a methanol–methyl
tert-butyl ether (MTBE) mobile phase has been demonstrated
in several reports (11, 13–16), and the current method adapted
these procedures for optimum resolution, sensitivity, and
run time. Calculations for standard concentrations were
based on purity from Müller et al. (17) using extinction
coefficients from Craft and Soares (18). Response factors for
cis
isomers of β-carotene relative to the all-
trans
form were
taken from Schierle et al. (13) and align with AOAC
Official
Method
SM
2005.07
(19) and the United States Pharmacopeia
(USP) monograph for β-carotene (20).
AOAC Official Method 2016.13
Lutein and β-Carotene in Infant Formula and
Adult Nutritionals
Reversed-Phase Ultra-High-Performance Liquid
Chromatography
First Action 2016
(Applicable to the determination of all-
trans
-lutein,
cis
isomers of lutein, all-
trans
-β-carotene, and
cis
isomers of
β-carotene in infant formula and adult nutritionals from 1 to
1300 μg/100 g reconstituted basis. Materials tested must not
contain measurable levels of β-apo-8′-carotenal.)
Caution
: Tetrahydrofuran (THF) can form peroxides, and only
THF stabilized with butylated hydroxytoluene (BHT)
should be used. Refer to Material Safety Data Sheets
when using any reagent, and use appropriate personal
protective equipment when performing analyses.
Note:
Throughout this method, estimated sample
concentrations for standard and sample preparations are stated
per 100 g on a reconstituted basis [as is for ready-to-feed (RTF)
INFANT FORMULA AND ADULT NUTRITIONALS
Received November 16, 2016. Accepted by SG January 20, 2017.
This method was approved by the AOAC Expert Review Panel for
SPIFAN Nutrient Methods as First Action.
The Expert Review Panel for SPIFAN Nutrient Methods 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.
Corresponding author’s e-mail:
gregory.hostetler@perrigo.comDOI: 10.5740/jaoacint.16-0386
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