A Method for the Measurement of Lutein in Infant

Food and Nutrition Sciences, 2011, 2, 145-149
doi:10.4236/fns.2011.22020 Published Online April 2011 (http://www.scirp.org/journal/fns)
Copyright © 2011 SciRes. FNS
A Method for the Measurement of Lutein in Infant
Formula
Rebecca Yuhas1, Megan McCormick1, Stephen Yachetti1, Anita M. Burgher1, Kadeline Kong2,
John Walsh3
1Department of Nutrition Research, Pfizer Nutrition, Pennsylvania, USA;
2Department of Quality, Wyeth Nutrition (S) Pte Lt, Singapore;
3Department of Quality, Wyeth Nutritionals Ltd., Askeaton, County Limerick, Ireland.
E-mail: rebecca.yuhas@pfizer.com
Received October 21st, 2010; revised January 14th, 2011; accepted February 24th, 2011.
ABSTRACT
Lutein is an antioxidant that is deposited in the macular region of the eye and is crucial to macular function. Lutein is
present in human milk and recently has been added to infant and toddler formulas. A method for the extraction and
quantification of lutein from lutein-fortified infant formula and toddler milk products was developed. The lipids and
carotenoids were extracted from the samples with ethanol and hexane: tetrahydrofuran. Polar xanthophylls were extracted
from the organic mixture with ethanol: water, leaving behind lipids and less polar carotenoids. The ethanol:
water extracts were dried, reconstituted in mobile phase, and the lutein was quantified by high performance liquid
chromatography with UV detection. A C30 carotenoid column and a mobile phase gradient of methyl-tert-butyl ether
and methanol were used for the chromatographic separation. Validation data showed repeatability with relative standard
deviations < 4% and intermediate precision with relative standard deviations < 20% at a low fortification level, 25
mcg lutein/L, and < 12% at a high fortification level, 200 mcg lutein/L. Overall recoveries from sample matrix ranged
from 88% to 106%. This liquid:liquid extraction method for the quantification of lutein in infant formulas is precise and
accurate.
Keywords: Lutein, HPLC, Infant Formula, Carotenoids
1. Introduction
The nutritional intake of the neonatal infant must support
a period of rapid maturation and organ growth. Human
milk is ideally suited to provide the nutritional needs of
the infant and contains many bioactive components including
carotenoids [1,2]. The xanthophyllic carotenoids,
lutein and zeaxanthin must be obtained from dietary
sources as they cannot be synthesized by the human body
[3]. They occur naturally in human milk [4], but not in
infant formulas. Lutein and zeaxanthin are antioxidants
that are deposited in the macular region of the eye and
are crucial to macular function. These yellow pigmented
macular carotenoids are responsible for the yellow pigment
of the macula [5]. They have the ability to absorb
high-energy blue light and to act as part of the antioxidant
system of the retina [6].
Exposure to blue light at any age may cause eye damage
and infants and children are at the highest risk. The
lens of the neonate is transparent thus providing little
protection against this high energy light [6]. As the lens
gradually and naturally yellows, the level of blue light
reaching the retina is progressively reduced [6,7]. Lutein
has been shown to help protect the retina from blue light
damage in animal models by filtering out this high energy
light [6,8]. In addition, there is evidence that lutein
acts as an antioxidant in the eye [6,9,10] and as macular
lutein is located in close proximity to lipid targets susceptible
to oxidation by singlet oxygen, it is well suited
to act as a biological antioxidant [11].
Lutein levels from a multinational study of carotenoids
in human milk found a range of 14.8 - 43.8 mcg lutein/L
with an overall mean of 25 mcg/L [4]. Infant formula,
follow on formula and milks for toddlers and young
children were formulated to provide a source of lutein
and a validated method was necessary to confirm fortification
levels.
Due to the high fat content of the matrices, current
methods for the measurement of carotenoids in human
milk and formulas often use a saponification or enzyA
Method for the Measurement of Lutein in Infant Formula
Copyright © 2011 SciRes. FNS
146
matic hydrolysis step followed by an extraction with organic
solvents [12-15]. Losses of up to 40% of the polar
carotenoids lutein and zeaxanthin have been reported due
to alkaline hydrolysis. [12,13]. Internal standards such as
echininone have been used to correct for losses due to
hydrolysis [15]. We modified the carotenoids extraction
method developed by Schweigert et al. [12] wherein the
lutein was extracted prior to the saponification procedure
used to recover more lipophilic carotenoids and their
esters. The use of echininone as an internal standard was
also investigated during the method implementation. The
assay performance was demonstrated at two levels of
fortification: 25 mcg/L in term formula and 200 mcg/L in
follow on formula, and milks for toddlers and young
children. The method was found to be precise and accurate
for the measurement of lutein in infant formula and
milk product matrices.
2. Materials and Methods
Chemicals, Solvents and Solutions. Lutein was purchased
from Indofine Chemical Company (Hillsborough,
NJ). Hexane, acetone, methanol and methyl-tert-butylether
(MTBE) were HPLC grade obtained from EMD
(Darmstadt, Germany). Inhibitor free, anhydrous tetrahydrofuran
(THF), 99.9% purity, was from Sigma-Aldrich
(St. Louis, MO). Absolute Ethanol was purchased
from Rossville Gold Shield (Haywood, CA). Deionized
water was from an internal Millipore purification system,
(Billerica, MA). Extraction solvent solutions were: hexane:
THF, 80:20, v:v, ethanol:deionized H2O, 90:10, v:v.
Test tubes with Teflon® lined screw caps were used for
all extractions.
Standard Preparation. A stock standard solution was
prepared by dissolving 2.0 mg lutein in 25 mL absolute
ethanol, stored at –20°C for up to one month. A working
standard was prepared by diluting the stock standard solution
1 25 in a volumetric flask with ethanol. This
solution was stored in a refrigerator at 10°C and used
within 5 days. The working standard purity was determined
by HPLC with UV detection at 445 nm. The purity,
absorbance at 445 nm and the molar absorption coefficient
(E 1%
1 cm : 2,550 at 445 nm in ethanol) [13] were
used to calculate the concentration of the working standard.
Dilutions of the working standard solution in ethanol
were prepared daily to yield concentrations bracketing
the levels of fortification. Aliquots of the standard
dilutions were carried through the extraction protocol.
Sample Preparation. Formula powders were accurately
weighed (200 mg) into test tubes, mixed with 1.0
mL deionized H2O followed by mixing with 1.0 mL absolute
ethanol. For liquid formulas, 1.0 mL ethanol was
added to 1.0 mL aliquots of formula. Hexane:THF (2.0
mL) was added to the samples and the tubes were shaken
on a reciprocating shaker (Eberbach Corporation, Ann
Arbor, MI) for 15 minutes. The tubes were centrifuged
15 minutes at 3500 rpm and the upper organic layer was
transferred to another test tube. The extraction was repeated
and the organic phases combined. These combined
phases were extracted with ethanol: H2O (2.0 mL),
briefly mixed on a vortex (Digital Pulse Mixer, Glas-Col,
Terre Haute, IN), shaken again for 10 minutes and centrifuged
for 5 minutes at 3,500 rpm. The lower aqueous
layer was transferred to another test tube and the extraction
was repeated and the aqueous phases combined. The
combined aqueous phases were evaporated to dryness
with nitrogen and heat (30°C to 35°C).
Calibration Standards. Deionized H2O (1.0 mL) was
added to 1.0 mL aliquots of the working standard dilutions.
The standards were then carried through the extraction
protocol with the samples beginning with the
addition of 2.0 mL hexane:THF. Following evaporation
to dryness, the sample and standard extracts were dissolved
in 1.0 mL mobile phase, MeOH:MTBE, 85:15,
v:v. Samples were vortexed and allowed to stand until
clear. Samples were transferred to amber vials for HPLC
analyses, leaving behind any insoluble lipid material. The
areas of the standard lutein peaks were plotted against
lutein concentration; the regression equation was calculated
and used to calculate the amount of lutein in the
samples.
Chromatography conditions. The HPLC system was
an Agilent 1100 Series coupled with a Variable Wavelength
Detector (Little Falls, DE). Chromatographic
separation was performed using a C30 carotenoid column,
5μm, 4.6  250 mm, (YMC, Wilmington, NC). The mobile
phase was (A) MeOH and (B) MTBE. A step-wise
linear elution gradient was used: Initially 85% A to 70%
A at 18 min, then decreased to 10% A within 1 min, held
for 4 min, then increased to 85% A within 1 min. The
column was re-equilibrated at 85% A for 6 min. Analyses
were conducted with a flow rate of 1.0 mL/min and
lutein was quantified by measuring the absorbance at 445
nm. Total run time was 30 min. Representative chromatograms
of lutein reference material and extracts of infant
formula are shown in Figures 1 and 2, respectively.
3. Results and Discussion
Due to the high levels of lipids in milks and formulas,
carotenoids analyses typically utilize a saponification
step or an enzymatic digestion to separate the carotenoids
from the lipid fraction [14-16]. This alkaline hydrolysis
can lead to significant losses (up to 40%) of the polar
xanthophylls, including lutein [16]. Our method is a
modification of the Schweigert method for carotenoids
extraction which extracted the xanthophyll fraction with
A Method for the Measurement of Lutein in Infant Formula
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147
Figure 1. Chromatogram of lutein reference material, C30 carotenoid column, 5 μm, 4.6  250 mm, detection at 445 nm.
Figure 2. Chromatogram of lutein extracted from infant formula, C30 carotenoid column, 5 μm, 4.6  250 mm, detection at 445
nm.
hexane prior to the saponification step [12].
Several protocols were investigated before the optimal
method was achieved. Results for the preliminary methods
are presented in Table 1. Initially, quantitation was
by external standardization. Recoveries of lutein were
low. The method was modified to incorporate THF into
the hexane extraction step to improve xanthophyll extraction
efficiency and recoveries improved. Echininone
was added as an internal standard and initially recoveries
were acceptable. Subsequent studies demonstrated the
echininone internal standard was not recovered reproducibly
at a level similar to the analyte of interest, lutein
(data not shown). The difference in extraction efficiency
resulted in recovery values for lutein that were in excess
of 120%. This was not acceptable in this application.
Ultimately, the calibration curve was constructed using
lutein reference material diluted to concentrations bracketing
the two levels of fortification and carried through
the extraction protocol. The correlation coefficient, R2, of
the standard calibration curve was an average 0.997 over
8 runs.
Precision. Selected samples of lutein fortified products
were analyzed in replicate and the relative standard
deviation, RSD r, of the results was determined. The acceptability
criteria for within-day repeatability were
RSDr < 0.5 of RSDH, where RSDH is defined by the Horwitz
equation: RSDH, % = 2C–0.1505, where C is the concentration
as a decimal fraction in g/g [17]. The predicted
Relative Standard Deviations (< 0.5 of RSDH) were <
10.2% and < 7.5% for the 25 mcg/L and 200 mcg/L fortification
levels, respectively. Repeatability results for
both the low and high fortification levels are presented in
Table 2. The within day repeatability results met the
acceptability criteria, RSDr < 0.5 of RSDH.
A Method for the Measurement of Lutein in Infant Formula
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148
Acceptance criteria for intermediate precision (iRSDR)
over three separate days of analyses was defined as
Table 1. Comparison of recovery of lutein between three
trial methods.
Calibration Extraction solvent % Recoverya n
External standardization Hexane 65.8 ± 8.7 6
External standardization Hexane: THF 89.1 ± 6.5 15
Internal standardization Hexane: THF 98.3 ± 7.7 12
a mean ± relative standard deviation.
Table 2. Results of lutein analyses for intraassay precision
(RSDr) %.
Product Category Meanª
mcg/L RSDr % RSDH
% HORRAT
ratiob
S26 gold Infant
formula 49.3 2.9 20.4 0.14
Promil Follow-on
formula 259.8 2.1 14.9 0.14
Progress Toddler
milk 280.6 3.3 14.9 0.22
Promise Milk for young
children 281.2 2.2 14.9 0.15
ª N = 6 each matrix; b Horrat ratio defines the acceptability criteria,
RSDr/RSDH < 0.5.
Table 3. Results of lutein analyses for within-lab intermediate
precision (iRSDR).
Brand Category Meanª
mcg/L iRSDR % RSDH % HORRAT
ratiob
S26 gold Infant formula 49.2 18.6 20.4 0.91
Promil Follow-on
formula 247.8 9.9 14.9 0.66
Progress Toddler milk 273.2 11.5 14.9 0.77
Promise Milk for young
children 299.1 10.2 14.9 0.68
ª 3 days, duplicate analyses; b Horrat ratio defines the acceptability criteria,
iRSDR/RSDH < 1.0.
Table 4. Results for lutein recovery.
Fortification level 25 mcg/L 200 mcg/L
Level of spike 50% 100% 150% 50% 100% 150%
Innate (mcg/L) 21.4 21.4 21.4 24.4 24.4 24.4
Spike (mcg/L) 13.2 24.7 37.8 107.2 198.5 301.0
Theoretical (mcg/L) 34.6 46.1 59.1 107.4 198.9 301.6
Measured (mcg/L) 33.0 44.5 60.6 132.4 233.7 294.5
Recovery (%) 87.9 93.9 103.9 100.8 105.5 89.8
Acceptance criteria, +/- 2σ 100% +/- 28.5% 100% +/- 21.0%
iRSDR < RSDH. Predicted iRSDR were 20.4% and 14.9%
for 25mcg/L and 200 mcg/L fortification levels, respectively.
Intermediate precision results met the acceptability
criteria of iRSDR < RSDH and are presented in Table 3.
Recovery. Lutein was spiked into the sample matrix
and carried through the extraction protocol. Recoveries
of reference lutein spiked into samples at three levels
(50%, 100% and 150%) of the low fortification level, 25
mcg/L, ranged from 87.9% to 103.9%. Recoveries of
reference lutein spiked into samples at three levels (50%,
100% and 150%) of the high fortification level, 200
mcg/L, ranged from 89.8% to 105.5%. Recovery results
for both the low and high lutein fortification levels are
presented in Table 4.
Ruggedness. This method for the measurement of lutein
in fortified infant formulas and toddler milks was
successfully transferred and validated at several quality
assurance and contract laboratories.
In conclusion, this method was developed to measure
the lutein content of fortified term and follow on formula
as well as toddler milk and milk for young children. It
was shown to be rugged, precise and accurate at lutein
fortifications of 25 mcg/L and 200 mcg/L.
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A Method for the Measurement of Lutein in Infant Formula
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