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J.
Agric.
Food
Chem. 1992,
40,
431-434
43
1
Relative Solubility, Stability, and Absorptivity
of
Lutein and @-Carotene
in Organic Solvents
Neal
E.
Craft'
Organic Analytical Research Division, Chemical Science and Technology Laboratory, National Institute of
Standards and Technology, Gaithersburg, Maryland 20899
Joseph
H.
Soares,
Jr.
Nutritional Sciences Program, University of Maryland, College Park, Maryland 20742
The relative solubility, stability, and absorptivity of lutein and @-carotene were determined in 18 organic
solvents. The solubility of both carotenoids was greatest in tetrahydrofuran, while hexane exhibited
the least solubility for lutein; methanol and acetonitrile exhibited the least solubility for @-carotene.
Stability was monitored for 10 days at room temperature by measuring absorbance changes at the
wavelength maximum. In the majority of the solvents, initial absorbance decreased by less than 10%
during the 10-day period. Degradation was greatest for both carotenoids in cyclohexanone. The relative
absorptivities were determined by calculating the carotenoid concentration in a reference solvent using
a reference absorptivity, and then Beer's Law was applied to the measured absorbance of the same
carotenoid concentration in other organic solvents. Absorbance maxima and relative absorptivities
were in good agreement with available literature values.
Interest in carotenoids has increased during the past
decade. Carotenoids are not only natural pigments and
vitamin
A
precursors but have been proposed
as
cancer
prevention agents, ulcer inhibitors, life extenders, and
heart attack inhibitors (Peto et al., 1981; Colditz et
al.,
1985; Mozsik et al., 1984; Cutler, 1984; Gazianoet al., 1990).
Unfortunately, physical information about these com-
pounds in organic solvents is limited. The wavelength
maxima and absorptivity of carotenoids change with the
nature of the solvent in which they are dissolved.
For
example, the visible spectrum of @-carotene in ethanol
has little fine structure with absorbance maxima at 453
and 480 nm, while the visible spectrum in carbon disul-
fide has more fine structure and exhibits maxima at 484
and 512 nm (Davies, 1976). The molar absorptivities of
@-carotene at
A,,
in these two solvents are 140 700 and
107 800 L mol-' cm-l, respectively (Davies, 1976). In the
past, absorbance maxima have been compiled for caro-
tenoids in several solvents (Davies, 1976; De Ritter and
Purcell, 1981), but such tables supply limited absorptiv-
ities, and frequently the maxima in a given solvent vary
by several nanometers depending on the source of infor-
mation. These tables also provide no information re-
garding the solubility and stability of the carotenoids in
the solvents. The lack of information about carotenoid
solubilities and molar absorptivities in a variety of organic
solvents increases the difficulty associated with developing
analytical methods for carotenoid research. Such practical
information is important for the selection of solvents for
use in sample preparation and liquid chromatography (LC)
mobile phases and also for the identification and quan-
tification of carotenoids in diverse LC mobile phases.
The two most prominent cyclized carotenoids in human
serum and foods are lutein (&r-carotene-3,3'-diol) and
0-
carotene (@,@-carotene) (Bieri et al., 1985; Khachik et
al.,
1986). Not only are they the most prominent, but they
also span a wide polarity range and are representative of
the
@,E-
and the @,@-carotenoids, respectively. Herein we
describe the determination of the relative solubility,
*
Author to whom correspondence should be addressed.
stability, and absorptivity of these two biologically im-
portant carotenoids in various organic solvents.
MATERIALS
AND
METHODS
Reagents. Crystalline @-carotene
(type
I, Sigma Chemical
Co.,
St. Louis,
MO)
and lutein (provided as
a
gift
by
Kemin
Industries, Des Moines, IA)
used
throughout the
study
were
assessed
by
spectrophotometric and liquid chromatographic
techniques to
be
greater than
90%
trans-@-carotene and
90%
trans-lutein, respectively. The sources, descriptions, and
lot
numbers
of the
solvents used
are
listed
in
Table I.
Equipment. Spectral measurements were
made
using
a
pho-
todiode array scanning spectrometer (H-P 8450, Hewlett-Pack-
ard, Palo Alto,
CA).
The spectrophotometer
provided
a
1-nm
spectral band
pass
from
200
to
400
nm and
a
2-nm
spectral
band
pass
from
400
to
800
nm.
Wavelength accuracy
was
checked
using
a
holmium
oxide glass filter and found
to
be
correct
at
the
279-,
361-,
460-,
and
536-nm
absorption
maxima. Solutions
were
dispensed with calibrated
pipets
or
gas-tight syringes.
Caro-
tenoid
purity
was
determined as
previously
described (Craft
et
al., 1991).
Relative Solubility
of
Lutein and @-Carotene in Organic
Solvents. Approximately
10
mg
of
lutein
or
8-carotene was added
to
3
mL
of
each
of
the solvents listed in Table I.
Vials
were
ultrasonically agitated
for
5
min.
If
a
clear solution with
no
residual
crystals
resulted, additional carotenoid
was
added
until
crystalline material remained undissolved. Each solution
was
then
filtered
through
a
0.2-pm
membrane, and appropriate
dilutions
were
made
unt,il the
absorbance at
the wavelength
maximum
was
between
0.5
and
1.0
absorbance unit
at
ambient
temperature. The background absorbance
of
each
solution
was
subtracted using
the
appropriate
solvent
containing
no
caro-
tenoid.
Carotenoid
concentration
was
calculated
using
Beer's
law
and the
relative
absorptivities
determined
below
(Determi-
nation
of
Relative Absorptivity). Measurements
were
performed
in
triplicate
and
the calculation used
is
(absorbance,
at
X,,)(dilution
factor)/molar
absorptivity,
where
the
subscript
s
is
a
given
solvent. The measured
values
were
rounded
to
one
significant
figure
since
this
experiment
was
not
designed
to
determine
absolute
solubility
but
rather
to
indicate
solubility
relative
to
other
solvents.
Determination
of
Relative
Absorptivity.
Concentrated
solutions
(approximately
3
g/L)
of lutein
and
@-carotene
were
prepared
in
tetrahydrofuran
(THF)
containing butylated
hy-
0027-8561 /92/7440-0437t03.00/O
0
1992
American Chemical Society
492
J.
Agric.
Food
Chem.,
Vol.
40,
No.
3,
1992
Table
I.
List
of
Solvents, Sources, and Lot Numbers
Craft and Soares
solvent source, grade lot safety hazardsa
acetone Mallinckrodt, SpectAR 2438 1,295
acetonitrile
J.
T.
Baker, HPLC C28108 2,335
benzene
J.
T.
Baker, Photrex (214603 395
chloroform EM Science, Omnisolve 5102 3
cyclohexane EM Science, Omnisolve 6041
1,
2,5
cyclohexanone Kasai, GR FAVOl
1,
2
dichloromethane
J.
T.
Baker, HPLC D25082, D18131 2, 3
dimethylformamide (DMF) Burdick and Jackson, HPLC AK285
1,
2
dimethyl sulfoxide (DMSO) Mallinckrodt, SpectAR KMCD
1,
2,4
ethanol, absolute Warner-Graham
1,
5
ethyl acetate
EM
Science, Omnisolve 7278
1,
2,5
ethyl ether
EM
Science, GR 9130
1,
2,4,5
hexane
J.
T.
Baker, HPLC D33095
1,
295
2-propanol Mallinckrodt,
AR
3037KDEV, 3035KCAY
1,
5
methanol
EM
Science, Omnisolve 8352 3, 5
methyl tert-butyl ether (MTBE)
EM
Science, reagent llP23 2,5
tetrahydrofuran
(THF)
+
BHT
J.
T.
Baker, HPLC C24654 295
to
1
u e n e Burdick and Jackson, HPLC AK80
1,
295
0
1,
harmful when entering the body; 2, irritant to skin, eyes, and respiratory organs; 3, toxic (harmful if inhaled, ingested, or absorbed through
the skin); 4, explosive; 5, flammable.
Table
11.
Relative Solubility and Absorptivity
of
Lutein and @-Carotene in Organic Solvents
lutein @-carotene
solvent
acetone
acetonitrile
benzene
chloroform
cyclohexane
cyclohexanone
dichloromethane
DMF
DMSO
ethanol
ethyl acetate
ethyl ether
hexane
2-propanol
methanol
MTBE
THF
toluene
solubility, absorptivity molar absorptivity,a
mg/L
A,,,"
nm
E'%,
cm-' L mol-' cm-1
800 446 2540 144 500
100
446 2559 145 600
600 456(458) 2350
133
700 (127 200)
6000 454(458) 2369 134
800
50 448 2520 143 400
4000 454 2359 134 200
800 452 2320 132
000
1000 454 2390 136
000
1000 460 2369 134
800
300 444(445) 2550 145
100b
800
446 2529 143 900
2000 444 2629 149 600
20 444(445) 2589 147 300
400 444 2599 147 900
200 442 (444) 2629 149 600
2000 444 2589 147 300
8000 450 2469 140 500
500 456 2290 130 300
solubility, absorptivity molar absorptivity?
mg/L
x,,,a
nm
El%,
cm-l L mol-' cm-1
200 452(452) 2559 137 400
10 452 2540 136 400
4000 462 (462) 2304 124 000 (125 500)
2000 462 (461) 2330 125 100 (128 600)
2000 454 (457) 2508 134 700 (134 500)
2000 462 2359 126 700
6000
460 2369 127 200
200 460 2389 128 300
30 466 2259 121 300
30 450 (449) 2529 135
800
(140 700)
500 452 2520 135 300
1000 448 2659 142 800
600 448 (453,450) 2592 139 2WC
40 450 2508 134 700
10 450 2540 136 400
1000
450 2588 139
000
10000 456 2399 128 800
4000 462 (463) 2270 121 900
Calculated molar absorptivities and
A,,
in parentheses are taken from Davies (1976). Reference absorptivity for lutein. Reference
absorptivity for @-carotene.
droxytoluene (BHT)
as
an antioxidant and filtered through 0.2-
pm membranes. To 30 mL of each of the solvents listed in Table
I
was added
30
pL of concentrated lutein
or
@-carotene in THF.
We were cautious to work well within the carotenoid solubility
limits (determined above) of the solvenh being examined to avoid
precipitation of the carotenoid compounds. Sealed vials were
ultrasonically agitated for
3
min to assure dissolution. Spec-
trophotometric scans were performed from 250 to 550 nm, and
absorbance
at
the wavelength maximum was determined. The
concentrations
of
the lutein and @carotene solutions were
determined using the most widely accepted molar absorptivity
forlutein
inethanolandp-caroteneinhexane
(145 100and 139
200
L mol-' cm-1, respectively) (Davies, 1976). Relative absorptiv-
ities in the different solvents were determined on the basis of the
calculated concentrations of lutein and @-carotene determined
in ethanol and hexane, respectively, and the absorbance of the
carotenoid solutions at the wavelength maximum in a given
solvent using Beer's law. Absorbance measurements were
performed in triplicate, and the combined error (mean standard
deviation of absorbance measurements and estimated limits of
bias) associated with the measurements was approximately 1%.
Lutein and &Carotene Stability. The solutions prepared
under Determination of Relative Absorptivity were stored in
amber glass vials with Teflon-lined screw caps at room temper-
ature. The UV-vis absorbance spectrum from 250 to 550 nm
was monitored over a 10-day period. Decreases in absorbance
and shifts in wavelength maxima were indicators of carotenoid
degradation. Degradation is reported as percent of initial ab-
sorbance at
Amax.
RESULTS AND DISCUSSION
Both carotenoids tested were most soluble in THF
(Table
11).
6-Carotene was least soluble in methanol and
acetonitrile, while lutein was least soluble in hexane. Many
existing extraction techniques partition carotenoids into
hexane
or
petroleum ether from aqueous alcoholor acetone
(Bieri et al., 1985; De Ritter and Purcell, 1981; Simpson
et
al.,
1985). Given the poor solubility of dihydroxy and
more polar carotenoids in hexane, this may lead
to
losses.
Diethyl ether has also been used to partition carotenoids
from aqueous/polar organic mixtures (Britton, 1985; De
Ritter and Purcell, 1981; Rodriquez-Amaya, 1989); on the
basis of the solubilities listed in Table
11,
this may present
a more effective approach. One possible disadvantage is
the solubility of fatty acid soaps in ether which must be
thoroughly removed with water (Britton, 1985). Although
THF is subject to peroxide formation, it has found
increased use (Bureau and Bushway, 1986; Khachik et al.,
1986; Peng et al., 1987) for carotenoid extractions due to
the high solubility of
a
wide polarity range of Carotenoids.
We are unaware of published absorptivities for carotenoids
in THF. This lack of information may hamper its use as
a solvent and result in the introduction of errors associated
with evaporation and solvent-transfer steps due to the
use of less appropriate solvents with published absorp-
tivities. The information listed in Table
I1
should prove
Lutein and &Carotene in Organic Solvents
Table
111,
Relative Lutein and &Carotene Degradation in Organic Solvents
J.
Agric.
Food
Chem.,
Vol.
40,
No.
3,
1992
433
%
of
initial absorbance
of
lutein at
A,,,
%
of initial absorbance of p-carotene at
A,,
time, days
Amax
cis time, days
Amax
cis
solvent
1
3 6 10
shift,anm peakb
1
3 6 10
shift,anm peakb
acetone
99 96 95 95
0
+
98 96 96 93
0
+
acetonitrile
98 97 94 94 -2
+
99 96 93 92
0
+
benzene
100 99 100 97
0
87 77 71 67
0
-
chloroform
97 96 93 90 -2
+
97 91 92 91 -2
+
cyclohexane
100 100 98 99
0
98 98 93 91
0
+
cyclohexanone
88 70 49 37 -2
+++
86 67 45 32 -4
+++
dichloromethane
95 91 88 83
0
-
77 59 47 34 -30
+
100 100 97 97
0
96 94 93 90
0
-
DMSO
100 99 99 97
0
94 90 86 80
0
-
DMF
ethanol
96 96 93 91
0
+
98 94 92 91 -2
+
ethyl acetate
98 97 95 96
0
99 97 96 95
0
-
ethyl ether
96 88 79 65 -2
++
94 78 70 69 -2
+
hexane
99 100 100 98
0
-
96 95 94 92
0
+
2-propanol
99 99 99 95
0
97 94 94 89
0
-
methanol
97 95 95 90 -2
++
97 92 89 88
-4
++
97 90 82 76
0
++
96 89 82 74 -2
++
100 100 100 99
0
99 98 99 97
0
-
MTBE
to
1
u
e n e
99 98 100 97
0
90 80 76 71
0
-
THF
+
BHT
-
-
-
-
-
-
-
-
a
Indicates direction and amount
of
spectral shift at day
10.
*
Indicates presenceiabsence
of
cis peak. Number
of
+'s
indicates intensity
of cis peak.
valuable in the selection of solvents employed for caro-
tenoid extractions and dissolutions.
The wavelength maximum for @-carotene in the various
solvents ranged from
448
to
466
nm. The
A,,,
in hexane
was
448
nm, which is
4-5
nm below the referenced value
(Davies,
19761,
although the same reference lists other
citations reporting
A,,,
in hexane at
449
nm but without
an absorptivity. The difference in the reference
A,,
and
the
A,,
obtained in this work
can
be attributed to aromatic
contaminants in some solvents, the presence of cis isomers
in the p-carotene employed, and differences in the
wavelength calibration or spectral band pass of the spec-
trophotometers used for the measurements. For the
purposes of this work, the maximum absorbance was used
to determine relative solubilities and absorptivities. The
wavelength maximum for lutein ranged from
442
to
460
nm. The
A,,,
of lutein in ethanol was within
1
nm of the
reported value of
445
nm (Davies,
1976).
It may be that,
in the lutein used, the presence of
-6
%
zeaxanthin, which
has a higher
A,,,
offset the wavelength lowering due to
cis isomers or differences in wavelength calibration. The
spectrophotometer used for this work was limited to a
2-nm spectral band pass in the visible region. These two
observations may explain why the measured
Amax
values
were consistently
1-2
nm lower than reported values.
Because the absorptivity and
A,,,
of carotenoids vary
in different solvents and these values are only published
for a few solvents, relative absorptivities and
A,,
were
determined for lutein and @carotene in the solvents listed
in Table
I.
These values, given in Table 11, were used to
calculate the solubility of the carotenoids, also reported
in Table
11.
Relative absorptivity values listed in Table
I1 are in good agreement with previously published values
(Davies,
1976;
De Ritter and Purcell,
1981).
The primary
advantage of the determination of the spectral maxima
and absorptivities is that carotenoid concentrations can
be determined directly in a wide range
of
solvents. In
addition, data obtained using diode array detectors in
conjunction with
LC
can be better interpreted when the
wavelength shifts that occur in different solvents are
known.
Finally, the stability
of
these two carotenoids in the
various solvents was monitored spectrophotometrically
over
a
period of
10
days. Carotenoid degradation was
accompanied by decreases in the absorbance and, in some
cases, a downward shift in the
A,,,
(Table
111).
We are
aware that some degradation products (e.g., geometric
isomers and carotenals) contribute to the absorbance in
the visible region; however, all degradation products
exhibit lower absorptivity at the wavelength maximum of
the parent compound. This also implies that changes in
absorbance are not necessarily proportional to the con-
centration of lutein or p-carotene in the solution. The
definitive measure of degradation would have been to
monitor the trans isomer
of
both carotenoids by HPLC;
however, while attempting to do this, we encountered
technical difficulties. First, it was not possible to make
all of the measurements by HPLC at the appointed times
without staggering the experiments; second, few of the
solvents could be injected directly into the HPLC system;
and third, complete redissolution of carotenoids was
questionable if a solvent evaporation was included. For
these reasons we opted to record the UV-vis spectra to
monitor major changes in the analytes. When the ab-
sorbance expressed as percent of initial absorbance at
A,,
was plotted against time, the degradation function was
similar in all solvents but proceeded at different rates
(Figure
1).
Stability was poorest for both lutein and
8-
carotene in cyclohexanone, retaining only
37
5%
and
32
%
,
respectively, of their initial absorbance by day
10.
The
degradation of P-carotene in cyclohexanone was followed
closely by degradation in dichloromethane with
-
34
5%
absorbance remaining at day
10.
In general, the rate of
lutein degradation was slower than p-carotene degradation
as illustrated by the curves shown in Figure
1
representing
the average rate of degradation in all solvents. Even on
day
10,
the carotenoid absorbance in most solvents
was
clustered above
90%
of the initial absorbance. The
conditions incorporated were selected to exacerbate the
degradation process so that stability/instability would be
evident. In a laboratory setting, greater efforts would be
made to stabilize carotenoid solutions, e.g., by the incor-
poration of antioxidants and use of lower storage tem-
peratures.
Little information of this type has been reported
previously. In comprehensive reviews (Davies,
1976;
De
Ritter and Purcell,
1981),
references to factors important
to
solvent selection are mentioned, but specific information
about the influence of solvents on carotenoid stability is
lacking. Information presented in Table
I11
should be
used for comparative purposes since stability is also de-
pendent on solvent supplier and lot number. The solvents
434
J.
Agric.
Food
Chem.,
Vol.
40,
No.
3,
1992
LUTEIN DEGRADATION IN ORGANIC SOLVENTS
Craft
and
Soares
from a dissertation submitted to the Graduate School,
University of Maryland, by N.E.C. in partial fulfillment
of the requirement for the Ph.D. degree in Nutritional
Sciences.
A
EE
.
40
1
,
, , ,
,
,
,
, ,
TCH
0
2
4
6
8
10
TIME (days)
@CAROTENE DEGRADATION IN ORGANIC SOLVENTS
E
50
:
::I,
,
0
,
, ,
, ,
-iM
30
0246810
TIME (days)
Figure
1.
Percent initial absorbance
at
A,,
of lutein and
@-
carotene monitored in
18
organic solvents over a period of
10
days at ambient temperature. Solid line represents the average
rate of degradation in all solvents. Actual values for a given
solvent are listed in Table
111.
A specific symbol is used to
illustrate the degradation in a given solvent; however, duplicates
of most symbols were necessary to account for all solvents. As
such, legends are not given for each symbol; however, solvents
with less than
85%
of initial absorbance at day
10
are indicated
by solvent abbreviations. Abbreviations: BZ, benzene; CH, cy-
clohexanone; DCM, dichloromethane; EE, ethyl ether; MTBE,
methyl tert-butyl ether; TL, toluene.
used may
or
may not be representative of current lots
from a given supplier. No attempt was made to sample
various sources of each solvent, and no additional antiox-
idants were added to the solvents used. However, our
experience with 2-propanol and dichloromethane indicates
that the source and lot of solvent used substantially
influence the stability of carotenoids in solution.
While developing methods for the extraction and de-
termination of carotenoids, we found that critical infor-
mation was frequently missing from tabulated data and
found it necessary to fill in some gaps. The information
provided in this paper supplements published absorp-
tivities and
A,,
values for solvents for which information
is currently unavailable. This information should also
aid in the selection of solvents to be used for carotenoid
research by giving an indication of stability and solubility
of two carotenoids, which vary greatly in polarity. Finally,
since the chromophore is not strongly influenced by the
presence of hydroxyl groups outside the conjugated double
bond system, the molar absorptivity values
for
lutein can
be used for estimating concentration values of other
@,e
carotenoids such as a-cryptoxanthin and a-carotene; the
molar absorptivity values for @,@-carotene can be used for
@-cryptoxanthin and zeaxanthin.
ACKNOWLEDGMENT
We are grateful to Chris Nelson and Kemin Industries
for the generous gift of lutein. The work is taken in part
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Analysis and stability of
retinol in plasma.
J.
Natl. Cancer Inst.
1987, 78, 95-99.
Peto, R.; Doll, R.; Buckley,
J.
D.;
Sporn, M. B. Can dietary (3-
carotene materially reduce human cancer rates? Nature
1981,
Rodriquez-Amaya, D. Critical Review of Provitamin A Deter-
mination in Plant Foods.
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Micronutr. Anal.
1989,
5,
191-
225.
Simpson, K. L.; Tsou,
S.
C.
S.;
Chichester, C.
0.
Carotenes. In
Methods
of
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Klein, B. P.,
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B., Eds.; Wiley: New York,
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1985,111, 113-149.
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3 15-318.
290,
201-208.
Received for review June
27,1991.
Accepted December
2,1991.
Certain commercial equipment, instruments, or materials are
identified in this paper to specify adequately the experimental
procedure. Such identification does not imply recommendation
of endorsement by NIST, nor does it imply that the equipment
or materials identified are necessarily the best available for the
purpose.
Registry
No.
Lutein,
127-40-2;
@-carotene,
7235-40-1;
acetone,
67-64-1;
acetonitrile,
75-05-8;
benzene,
71-43-2;
chloroform,
67-
66-3;
cyclohexane,
110-82-7;
cyclohexanone,
108-94-1;
dichlo-
romethane,
75-09-2;
dimethylformamide,
68-12-2;
dimethyl sul-
foxide,
67-68-5;
ethanol,
64-17-5;
ethyl acetate,
141-78-6;
ethyl
ether,
60-29-7;
hexane,
110-54-3;
2-propanol,
67-63-0;
methanol,
67-56-1;
methyl tert-butyl ether,
1634-04-4;
tetrahydrofuran,
109-
99-9;
toluene,
108-88-3.
