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A quantitative comparison of the commonly used methods
for extracting carotenoids from avian plasma
Kevin J. McGraw & Elizabeth A. Tourville &
Michael W. Butler
Received: 29 September 2007 / Revised: 23 April 2008 / Accepted: 2 July 2008 / Published online: 25 July 2008
# Springer-Verlag 2008
Abstract Interest in animal carotenoids, especially in birds,
has exploded in recent years, and so too have the methods
employed to investigate the nature and function of these
pigments. Perhaps the most easily and commonly performed
procedure in this work has been the determination
of carotenoid concentration from avian plasma. Over the
past 20 years of research on avian carotenoids, numerous
methods have been used to extract carotenoids from bird
plasma, all of which have differed in several important
parameters (e.g., number and types of solvents used, degree
of mixing/centrifugation). However, to date, no study has
systematically compared these methods to determine if any
of them are more effective than others for recovering any or
all types of carotenoids present. We undertook such an
investigation on plasma samples from two bird species
(house finch, Carpodacus mexicanus, and mallard, Anas
platyrhynchos) using five of the most commonly employed
methods for extracting carotenoids from avian plasma: (1)
acetone-only, (2) methanol-only, (3) ethanol-only, (4)
ethanol + hexane, and (5) ethanol + tert butyl methyl ether.
We also manipulated the amount of time that extracts were
centrifuged, which has varied tremendously in previous
studies, to evaluate its importance on carotenoid recovery.
We found that all methods equally recovered the polar
xanthophylls (lutein and zeaxanthin), but that the methanolonly
procedure poorly recovered non-polar carotenoids
(less β-carotene in both species and less β-cryptoxanthin
in house finches) compared to the other methods. These
results suggest that the data accumulated to date on
xanthophyll plasma carotenoids in birds should be comparable
across studies and species despite the different
chemical extraction methods used. However, care should be
taken to use relatively strong organic solvents for fully
recovering non-polar carotenoids. We also found no effect
of centrifugation duration (1 vs. 10 min at 10,000 rpm) on
carotenoid recoveries, demonstrating that researchers can
save considerable time by centrifuging for a much shorter
time period than is typically used.
Keywords Carotenoid pigments . Ethanol . House finch .
HPLC . Lutein . Mallard . Methanol . Zeaxanthin
Introduction
Carotenoid pigments have recently captured the attention of
biologists because of their unique and wide-ranging
controls and functions in animals (Vershinin 1999). From
sea urchins to birds, carotenoids play key roles in health,
mating, breeding, and development (McGraw 2006a). This
is typically because carotenoids are a dietarily (Grether et
al. 1999; Hill et al. 2002), physiologically (McGraw and
Parker 2006; Blas et al. 2006), or immunologically (Blount
et al. 2003a; McGraw and Ardia 2003) limiting but
valuable resource in animals. A major challenge to ecologists
and evolutionary biologists interested in carotenoids
has been to biochemically track these lipid-soluble pigments
in foods, flesh, feathers, and other tissues (Hudon
and Brush 1992; Stradi et al. 1995) so that we may determine
their limitations and allocation priorities.
One of the easiest and most common ways to determine
the carotenoid status of vertebrates has been to assay the
carotenoid content of blood plasma or serum, which
represents the current pool of pigments (ingested from food
Behav Ecol Sociobiol (2008) 62:1991–2002
DOI 10.1007/s00265-008-0622-4
K. J. McGraw (*) : E. A. Tourville : M. W. Butler
School of Life Sciences, Arizona State University,
Tempe, AZ 85287-4501, USA
e-mail: kevin.mcgraw@asu.edu
and/or retrieved from tissue stores) available for allocation
to various body functions (e.g., oxidative stress, immune
challenges, color acquisition/maintenance, yolk formation).
Particularly in birds, numerous researchers have used
solvent extraction techniques to isolate carotenoids from
serum/plasma of wild animals with the aim of comparing
such levels to factors such as diet, health, age, phylogeny,
coloration, and tissue carotenoid concentrations (Blount et
al. 2002; Tella et al. 2004; McGraw et al. 2006a; Costantini
et al. 2007a; Martinez-Padilla et al. 2007). However, this
has been done using a variety of chemical extraction
methods, none of which have been quantitatively compared
to date to determine their relative effectiveness. A survey of
recently published papers reveals that five different procedures
have mainly been used to extract carotenoids from
bird plasma (Appendix), which differ primarily in the type
(s) of solvent(s) used. Most common among these is an
acetone-only extraction first used by Ruff et al. (1974) and
Allen (1987a, b) with chickens. The next two most frequently
used methods involve a two-step procedure where
ethanol is used first to remove protein and then a second
organic solvent—either hexane (adapted from extraction
methods on human plasma carotenoids, i.e., Staciewicz-
Sapuntakis et al. 1987) or tert butyl methyl ether (TBME;
originally employed by McGraw et al. 2002 with zebra
finches)—is added to fully recover lipid-soluble carotenoids.
The fourth and fifth methods have been recently
added as single-solvent extractions using either ethanol
or methanol. All of these solvents differ considerably in
their polarity and thus their ability to solubilize different
types of carotenoids (e.g. polar xanthophylls versus nonpolar
cryptoxanthins and carotenes). If these methods
were to differ significantly in their carotenoid recoveries,
attempts to draw comparisons among types and amounts
of plasma carotenoids across studies and species would
be difficult. Thus, a systematic, quantitative comparative
study of plasma carotenoid extraction methods in birds is
warranted.
We undertook such an investigation by comparing
carotenoid recoveries among five aforementioned carotenoid
extraction methods using plasma from two free-ranging bird
species from North America—the house finch (Carpodacus
mexicanus) and the mallard (Anas platyrhynchos). We
performed this test on these two species as an attempt to
understand the generality of the results and because plasma
in both species were known to contain both polar and nonpolar
carotenoids (McGraw et al. 2006a; M. W. Butler and
K. J. McGraw, unpublished data). We could have tested
numerous procedural variables in this study (e.g., degree of
mixing between solvent and plasma, duration of plasma/
extract storage prior to analysis), but instead focused on the
two factors that seemed to show the highest and (to us)
most important variability in the literature: (1) solvent type
and (2) centrifugation time. We hypothesized that methods
using particularly weak solvent types, like ethanol and
methanol, would poorly recover carotenoids, especially
non-polar forms, from plasma compared to the other three
methods. Consistent with this, a previous study that
compared solvent extraction methods for plant carotenoids
showed that methanol failed to fully recover β-carotene
(Dunn et al. 2004). We also hypothesized that longer
centrifugation times could result in the loss of carotenoids
(if carotenoids are not wholly soluble in some solvents and
thus are spun down with the pellet) or add unnecessary
processing time for researchers if, for example, 1 min as
opposed to ten is sufficient for centrifuging the solutions (to
remove flocculant proteins). It is noteworthy that a few
other methods for extracting avian plasma have been
employed, but only once in the literature (acetonitrile-only:
Saino et al. 1999 in barn swallows; hexane-only: Zhao et al.
2006 in chickens; ethanol + ethyl ether: Khachik et al. 2002
in quail; three-step organic solvent extraction process:
Koutsos et al. 2003a, b in chickens; ethanol/chloroform/
methanol: Wang et al. 2007 in chickens), and thus for the
sake of feasibility, we did not include them here.
Materials and methods
We used heparanized capillary tubes to collect 80–200 μl
blood from 50 wild-caught house finches in Tempe, AZ on
20 May 2007 and to collect 120–320 μl blood from 30
wild-caught mallard ducklings in Tempe, AZ from May to
June 2007. We centrifuged the blood at 10,000 rpm for
2 min and saved plasma fractions in 1.5-ml screw-capped
Eppendorf tubes at −80°C for 4–6 weeks until analysis. To
obtain large enough samples on which we could conduct
controlled experimental tests, we pooled plasma samples
from four to six individuals to generate nine stock samples
of mallard plasma and 11 stock samples of house finch
plasma, each containing approximately 200 μl plasma.
These stocks were vortexed thoroughly at the original time
of mixing and before each test aliquot was drawn from it to
ensure homogenization.
A 5×2 factorial design was used to extract carotenoids
from each of the stock plasma samples: five solvent combinations
(see above and Appendix) and two centrifugation
durations (1 and 10 min, both at 10,000 rpm). We chose
10,000 rpm as a standard speed because of recent trends in
the literature by other authors (publications from 2004–
present in Appendix), because of previous work by the first
author (McGraw 2005), and because it should work more
effectively than slower speeds (e.g., Bortolotti et al. 1996).
We chose these two durations for centrifugation because
10 min is the typical time allotted for this step in the
literature (Appendix) and because we recently began trying
1992 Behav Ecol Sociobiol (2008) 62:1991–2002
shorter spin durations such as 1 min with no apparent ill
effects on carotenoid recovery (personal observations).
Samples from each stock were all extracted and run (using
high-performance liquid chromatography; see more below)
simultaneously to avoid inter-assay variation; within each
assay, we also randomized the order in which samples from
different treatments were processed to ensure no sequence
biases. Into each of ten different tubes for each stock, we
transferred 15 μl plasma, followed by 150 μl of the
appropriate solvent(s). A 1:10 dilution (plasma-to-each
solvent) was used throughout because this is the most
frequently reported ratio in the literature (Appendix). We
vortexed tubes for 5 s after each solvent was added,
centrifuged them for their allotted times (in a Beckman-
Coulter Microfuge® 18 centrifuge, Fullerton, CA, USA),
and then transferred each solution to a fresh tube. Solvents
were immediately evaporated to dryness under a stream of
nitrogen and samples prepared for analysis via highperformance
liquid chromatography (HPLC).
Following prior work by the first author (McGraw et al.
2006a), we used a Waters Alliance HPLC instrument
equipped with a Waters Carotenoid C-30 column to
determine types and amounts of carotenoids present. The
gradient method previously employed (McGraw et al.
2006a) was modified slightly to cut down on run times:
initial isocratic conditions were maintained until minute 11,
at which point we began running the linear gradient until
minute 21. Final gradient conditions were then held until
minute 25 and then returned to initial isocratic conditions
until minute 29.5. Pigment concentrations were calculated
based on external curves constructed from known amounts
of purified reference carotenoids.
We extracted and ran 35% of all samples in duplicate to
calculate coefficients of variation for the two carotenoids
that consistently appeared in all samples, lutein and
zeaxanthin. The overall coefficient of variation among all
duplicate runs was 6% for lutein and 11% for zeaxanthin,
the difference in them being mostly due to lower mean
values (denominators) for zeaxanthin. Degrees of assay
variation did not differ as a function of solvent extraction
method or centrifuge time, nor did absolute carotenoid
recoverability [which we tested using an internal standard,
anhydrolutein, and which averaged 88.5±2.2%; two-way
analyses of variance (ANOVAs), all P>0.13].
We compared carotenoid recoveries using mixed-model
ANOVA, with solvent method, centrifugation time, and
their interaction as fixed factors and sample as a random
factor. Analyses were run using SAS 9.1 software for
Windows (SAS Institute, Cary, NC, USA). Separate models
were run for each carotenoid type in each species. We made
post hoc pairwise comparisons among methods using
Tukey’s honestly significant difference (HSD) tests.
Table 1 Mixed-model
ANOVA table depicting the
effects of solvent extraction
method, centrifugation time,
and their interaction on the
recovery of different
carotenoids from the plasma
of wild house finches
Significant terms are in bold.
Source Lutein Zeaxanthin β-cryptoxanthin β-carotene
Solvent extraction method F4,90=1.98 F4,90=1.89 F4,63=4.54 F4,81=14.05
P=0.10 P=0.12 P=0.003 P<0.0001
Centrifugation time F1,90=1.29 F1,90=0.89 F1,63=1.50 F1,81=0.56
P=0.26 P=0.35 P=0.23 P=0.46
Solvent method × centrifugation time F4,90=0.35 F4,90=0.65 F4,63=0.82 F4,81=0.14
P=0.84 P=0.63 P=0.51 P=0.97
0
0.1
0.2
0.3
0.4
Lutein
Zeaxanthin
β -cryptoxanthin
β -carotene
Plasma carotenoid concentration (μg/ml)
A A A A B
AB AB B A B
0
2
4
6
8
10
0
0.4
0.8
1.2
1.6
0
0.1
0.2
0.3
A E EH ET M
Fig. 1 Bar charts depicting carotenoid recoveries (means+SE) for
different solvent extraction methods and centrifuge times in house finch
plasma. A acetone, E ethanol, EH ethanol + hexane, ET ethanol +
TBME, M methanol. Dark bars denote extracts that were centrifuged
(to remove flocculant protein) for 1 min; light bars signify samples spun
for 10 min. Unshared letters atop the panels (e.g., β-carotene) denote
significant differences (p<0.05) among solvent types using Tukey’s
HSD tests
Behav Ecol Sociobiol (2008) 62:1991–2002 1993
Results
House finch and mallard plasma during spring contained four
main carotenoid types: lutein, zeaxanthin, β-cryptoxanthin,
and β-carotene (McGraw et al. 2006a). Lutein was the
dominant plasma pigment in house finches (approximately
80% of total), followed by zeaxanthin (approximately 10%
of total), and both were present in every sample. These
were accompanied by small amounts of β-cryptoxanthin
(present in all but three samples) and β-carotene (present in
all but one sample). Lutein (approximately 66% of total)
and zeaxanthin (approximately 25% of total) were also
dominant in mallard duckling plasma, and while β-
cryptoxanthin and -carotene combined to make up a similar
remaining amount of total plasma carotenoids compared to
house finches, they were more often absent from mallard
plasma (from three and six of the samples, respectively).
The mean and range of total carotenoid concentrations
were slightly higher in mallard samples (11.3 μg/ml and
4.4–34.1 μg/ml, respectively) than in house finch samples
(10.3 μg/ml and 2.2–28.8 μg/ml, respectively).
The five solvent extraction methods employed did not
differ significantly in the recovery of the two polar
xanthophylls, lutein and zeaxanthin, in either finches
(Table 1, Fig. 1) or mallards (Table 2, Fig. 2). However,
we did find significant differences among methods in the
recovery of β-cryptoxanthin in house finches and of β-
carotene in both species (Tables 1 and 2). Methanol
recovered significantly less of these carotenoids than all
other methods (Figs. 1 and 2). In fact, methanol failed to
recover any β-carotene in any mallard sample and only
recovered trace amounts (0.01 μg/ml) in two of the 11
house finch samples. In addition, in house finch plasma,
methanol and ethanol–hexane recovered significantly less
β-cryptoxanthin, and ethanol–TBME significantly more,
than the other methods. We failed to find any effect of
centrifugation duration on carotenoid recoveries for any
pigment type in either species (Tables 1 and 2).
Discussion
We tested the relative extent to which published methods
for chemically extracting carotenoid pigments from avian
plasma recovered both polar and non-polar carotenoids. A
diversity of extraction methods have been used with avian
plasma, so it was important at this phase of investigation in
the field to compare methods from different labs and
determine if there is an optimal procedure or if all are
equally effective. We found no statistically significant
differences in polar carotenoid (xanthophyll) recovery
among the methods used. This result is not wholly
surprising, as all methods contained a solvent or mixture
in which xanthophylls should be highly miscible (Britton
1985) and as prior comparisons of chemical methods for
extracting carotenoids from plant leaves yielded no sig-
Table 2 Mixed-model
ANOVA table depicting the
effects of solvent extraction
method, centrifugation time,
and their interaction on the
recovery of different
carotenoids from the plasma
of wild mallard ducklings
Significant terms are in bold
Source Lutein Zeaxanthin β-cryptoxanthin β-carotene
Solvent extraction method F4,72=1.7 F4,72=0.96 F4,45=0.54 F4,18=13.74
P=0.16 P=0.44 P=0.71 P<0.0001
Centrifugation time F1,72=0.58 F1,72=1.12 F1,45=0.11 F1,18=0.45
P=0.45 P=0.29 P=0.74 P=0.51
Solvent method × centrifugation time F4,72=0.72 F4,72=0.38 F4,45=0.85 F4,18=0.22
P=0.58 P=0.82 P=0.50 P=0.92
A A A A B
0
2
4
6
8
0
1
2
3
0
0.4
0.8
1.2
0
0.1
0.2
0.3
Plasma carotenoid concentration (μg/ml)
Lutein
Zeaxanthin
β-cryptoxanthin
β-carotene
A E EH ET M
Fig. 2 Bar charts depicting carotenoid recoveries for different solvent
extraction methods and centrifuge times in mallard duckling plasma.
See Fig. 1 legend for additional details
1994 Behav Ecol Sociobiol (2008) 62:1991–2002
nificant differences for lutein (Dunn et al. 2004). The
similar performance of the procedures, however, is comforting
and suggests that prior data collected on polar
xanthophylls using any of these five methods should be
comparable across studies and species, at least for HPLCbased
measurements; we await comparable examinations
for those studies that use absorbance spectrophotometry to
quantify carotenoid content. This is especially important
because lutein and zeaxanthin are the most common and
concentrated carotenoids in circulation in birds (reviewed in
McGraw 2006a). Further tests are now needed to understand
how these methods comparatively recover metabolically
derived plasma xanthophylls like anhydrolutein or
ketocarotenoids like astaxanthin or canthaxanthin, though
we predict a similar outcome as to the one uncovered in this
study. We also recognize that the methods tested here still
varied in xanthophyll carotenoid recoveries (as is evident
from the fact that not all means are identical within a panel
in Figs. 1 and 2), but this variability fell within ranges of
measurement error (see “Materials and methods”).
In contrast to the xanthophylls, recovery of non-polar
carotenoids was dependent upon the type of solvent(s)
used. The primary methods employed in the literature
performed equally well at extracting β-cryptoxanthin and -
carotene from finch and mallard plasma, but a relatively
recently added method, using methanol alone, proved
weak at recovering β-carotene in both bird species.
Methanol alone failed to fully recover β-carotene from
plant leaves as well (Dunn et al. 2004). Methanol, along
with another method (ethanol–hexane), also poorly recovered
β-cryptoxanthin in house finches, where it was more
common and concentrated than in mallards. Thus, the use
of a broad generalized solvent (or a mix of a hydrophilic
and hydrophobic solvent) that captures both polar and nonpolar
carotenoids is recommended over the use of a more
aqueous solvent like methanol alone. The same recommendations
are made for human plasma and food (Khachik
et al. 1992a, b). This recommendation is especially true
when nothing is known of the carotenoid content in the
focal species and in bird species where these non-polar
pigments are key (e.g., β-cryptoxanthin for attaining
maximal plumage redness in house finches; McGraw et
al. 2006a) or in high concentration (e.g., common moorhen,
Gallinula chloropus; American coot, Fulica americana;
lesser black-backed gull, Larus fuscus; Surai et al. 2001).
The other main variable tested here was centrifugation
time, and we failed to find any significant effect of spinning
extracts for 1 vs. 10 min on the recovery of any type of
carotenoid in house finch or mallard plasma. Thus, at least
in these two species, substantial time can be saved by
centrifuging the plasma extract for a shorter amount of time
prior to solvent recovery and analysis. The only apparent
benefit we can see to retaining a long centrifuge time might
be to allow the formation of a smaller, more solid protein
pellet at the bottom of tubes, which is harder to disturb
during solvent removal and thus less likely to contaminate
the extract with flocculant protein.
In conclusion, we have performed the first comparative
test of carotenoid extraction methods in birds and found
some important differences among them. Future studies
might consider adding additional variables (i.e., vortexing
times, plasma/solvent volumes) to further understand the
optimal methods for recovering polar and non-polar
carotenoids from plasma or serum. We also suggest similar
studies of procedures that remove carotenoids from tissues
(i.e., thermochemical vs. mechanochemical extractions
from feathers and bare parts like bill or leg).
Acknowledgments We thank S. Quinn for assistance in capturing
ducklings as well as two anonymous referees for providing helpful
comments on the manuscript. Financial support for this study was
provided by the School of Life Sciences and College of Liberal Arts
and Sciences at Arizona State University. Birds from both species
were captured and sampled under university (protocol nos. 05-764R
and 07-910R), state (SP797514), and federal (MB088806-0) permits.
Appendix List of the 98 published studies that we were able to locate that used one of five main chemical methods (acetone, ethanol + hexane,
ethanol + TBME, ethanol, and methanol) to extract carotenoids from avian plasma
Citation Method Species Plasma/solvent
ratio
Vortexing description Centrifugation
rate
Ruff et al. (1974) Acetone-only Chicken (Gallus
gallus domesticus)
1:9 Not mentioned 1,000×g for
10 min
Ruff and Fuller
(1975)
Acetone-only Chicken (Gallus
gallus domesticus)
Cited Ruff et al.
(1974) for method
Augustine and
Thomas (1979)
Acetone-only Turkey (Meleagris
gallopavo)
1:9 Not mentioned Not mentioned
Augustine and Ruff
(1983)
Acetone-only Turkey (Meleagris
gallopavo)
1:9 Not mentioned Not mentioned
Allen (1987a) Acetone-only Chicken 1:9 Vortexed 1,500×g for
10 min
Behav Ecol Sociobiol (2008) 62:1991–2002 1995
Appendix (continued)
Citation Method Species Plasma/solvent
ratio
Vortexing description Centrifugation
rate
Allen (1987b) Acetone-only Chicken Cited Wilson (1956)
for method
Lillehoj and
Ruff (1987)
Acetone-only Chicken Cited Ruff et al.
(1974) for method
Augustine (1988) Acetone-only Turkey 1:9 Twice for 10 s 1,000×g for
10 min
Allen (1992a) Acetone-only Chicken 1:10 Mixed well Not mentioned
Allen (1992b) Acetone-only Chicken Cited Allen (1987a)
for method
Allen (1992c) Acetone-only Chicken Cited Allen (1987b)
for method
Conway et al. (1993) Acetone-only Chicken Cited Allen (1987a)
for method
Allen et al. (1996) Acetone-only Chicken 1:10 Not mentioned Not mentioned
Bortolotti et al. (1996) Acetone-only American kestrel
(Falco sparverius)
1:10 Not mentioned 1,500×g for
10 min
Loggerhead shrike
(Lanius ludovicianus)
Allen (1997a) Acetone-only Chicken 1:10 Not mentioned Not mentioned
Allen (1997b) Acetone-only Chicken 1:10 Not mentioned Not mentioned
Allen et al. (1997) Acetone-only Chicken Cited Allen (1992a)
for method
Matthews et al. (1997) Acetone-only Chicken 1:4 Vortexed 2,800×g for
10 min
Allen and Danforth
(1998)
Acetone-only “ Cited Allen et al.
(1996) for method
Negro et al. (1998) Acetone-only American kestrel 1:10 Mixed well 1,500×g for
10 min
Tella et al. (1998) Acetone-only 26 bird sp. from
Mexico
1:10–1:40 Well mixed 1,500×g for
10 min
Gray et al. (1998) Acetone-only Chicken 1:4 Vortexed 1,500×g for
10 min
Allen (2000) Acetone-only Chicken Cited Allen et al.
(1996) for method
Allen and Fetterer (2000) Acetone-only Chicken Cited Allen et al.
(1996) for method
Bortolotti et al.
(2000)
Acetone-only American kestrel 1:10 Cited Tella et al. (1998) for
method
Fetterer and
Allen (2000)
Acetone-only Chicken Cited Allen et al.
(1996) for method
Matthews and
Southern (2000)
Acetone-only Chicken Cited Allen (1987a)
and Matthews et
al. (1997) for
method
Negro and Garrido-
Fernandez (2000)
Acetone-only White stork
(Ciconia ciconia)
1:3 Not mentioned 13,000×g for
10 min
Negro et al. (2000) Acetone-only White stork
(Ciconia ciconia)
1:3 Not mentioned 13,000×g for
10 min
Zhu et al. (2000) Acetone-only Chicken Cited Allen (1997a)
for method
Fernie and Bird (2001) Acetone-only American kestrel Cited Bortolotti et
al. (1996) for
method
Negro et al. (2001) Acetone-only Red-legged partridge
(Alectoris rufa)
1:10 Cited Tella et al. (1998)
for method
Allen and Fetterer
(2002a)
Acetone-only Chicken Cited Allen et al.
(1996) for method
Allen and Fetterer
(2002b)
Acetone-only Chicken Cited Allen et al.
(1996) for method
Allen (2003) Acetone-only Chicken 1:10 Not mentioned Not mentioned
1996 Behav Ecol Sociobiol (2008) 62:1991–2002
Appendix (continued)
Citation Method Species Plasma/solvent
ratio
Vortexing description Centrifugation
rate
Bortolotti et al. (2003a) Acetone-only Red-legged partridge Cited Bortolotti et
al. (1996) and
Tella et al. (1998)
for method
Bortolotti et al. (2003b) Acetone-only American kestrel 1:10 Not mentioned 1,500×g for
10 min
Fetterer et al. (2003) Acetone-only Chicken 1:9 Vortexing 1,000×g for
10 min
Zhu et al. (2003) Acetone-only Chicken Cited Allen (1997b)
for method
Allen et al. (2004) Acetone-only Chicken 1:10 Not mentioned Not mentioned
Peters et al. (2004) Acetone-only Mallard (Anas
platyrhynchos)
1:3–1:7 Not mentioned 1,500×g for
10 min
Tella et al. (2004) Acetone-only 80 bird sp. from
Mexico
1:9 Mixed 10,000 rpm for
10 min
Allen et al. (2005) Acetone-only Chicken Cited Allen et al.
(2004) for method
Blanco et al. (2005) Acetone-only Linnet (Carduelis
cannibina)
1:5 Shaken/sonicated
for 1 min
12,000×g for
5 min
Figuerola et al. (2005) Acetone-only Greylag goose
(Anser anser)
1:1 Not mentioned 16,249×g for
10 min
Peters et al. (2005) Acetone-only Mallard 1:3–1:7 Not mentioned 1,500×g for
10 min
Tummeleht et al. (2006) Acetone-only Great tit (Parus major) 1:10 Not mentioned 1,500×g for
10 min
Blas et al. (2006) Acetone-only Red-legged partridge Cited Bortolotti et
al. (1996) for
method
Yang et al. (2006) Acetone-only Chicken 1:1:4 Not mentioned 1,000×g for
10 min
Casagrande et al. (2006) Acetone-only Eurasian kestrel
(Falco tinnunculus)
1:40 Not mentioned 14,000×g for
5 min
Horak et al. (2006) Acetone-only Greenfinch (Carduelis
chloris)
1:10 Not mentioned 16,800×g for
10 min
Aguilera and Amat
(2007)
Acetone-only Greenfinch (Carduelis
chloris)
1:20 Mixed with a vortex 10,000 rpm for
10 min
Perez-Rodriguez
et al. (2007)
Acetone-only Red-legged partridge 1:10 Vortexed 10,000 rpm for
10 min
Martinez-Padilla
et al. (2007)
Acetone-only Red grouse (Lagopus
lagopus)
1:10 Vortexed 10,000 rpm for
10 min
Isaksson and
Andersson (2008)
Acetone-only Great tit 1:19 Not mentioned Not mentioned
Surai and Speake
(1998)
Ethanol/H2O +
hexane
Chicken Not mentioned Shaken vigorously
for 5 min
Not mentioned
Slifka et al. (1999) Ethanol + hexane 14 sp. of zoo birds Not mentioned Not mentioned Not mentioned
Surai (2000) Ethanol + hexane Chicken 1:1:2.5 Shaken vigorously
for 5 min
Not mentioned
Surai and Sparks
(2001)
Ethanol + hexane Chicken 1:1:2.5 Shaken vigorously
for 5 min
Not mentioned
Surai et al. (2001) Ethanol + hexane Chicken 1:1:2 Stirred vigorously
on a vortex
2,000 rpm for
5 min
Blount et al. (2002) Ethanol/H2O +
hexane
Lesser black-backed
gull (Larus fuscus)
Cited Surai and
Speake (1998) for
method
Breithaupt et al.
(2003)
Ethanol + hexane Chicken 1:2:2 Stirred vigorously
on a vortex
2,000 rpm ×
5 min
Surai et al. (2003) Ethanol/H2O +
hexane
Chicken Cited Surai et al.
(2001) for method
Blount et al. (2003a) Ethanol + hexane Zebra finch
(Taeniopygia guttata)
1:2:35 Vortexed Not mentioned
Behav Ecol Sociobiol (2008) 62:1991–2002 1997
Appendix (continued)
Citation Method Species Plasma/solvent
ratio
Vortexing description Centrifugation
rate
Blount et al. (2003b) Ethanol + hexane Zebra finch
(Taeniopygia guttata)
1:2:35 Vortexed 20 s
per solvent
Not mentioned
Horak et al. (2004) Ethanol + hexane Great tit Cited Surai et al.
(2001) for method
Møller et al. (2005) Ethanol + hexane Barn swallow
(Hirundo rustica)
1:2:25 Vortexed Not mentioned
Ewen et al. (2006a) Ethanol + hexane Hihi (Notiomystis
cincta)
1:2:13.3 Homogenized Not mentioned
Ewen et al. (2006b) Ethanol + hexane Hihi (Notiomystis
cincta)
1:25:20 Vortexed Not mentioned
Biard et al. (2006) Ethanol + hexane Blue tit (Cyanistes
caeruleus)
1:2:25 Mixed Not mentioned
McGraw et al. (2002) Ethanol + TBME Zebra finch 1:8:8 Vortexed 3 min
(RPMs set)
McGraw et al. (2003a) Ethanol + TBME Zebra finch 1:8:4 Vortexed 4 min
(RPMs set)
McGraw et al. (2003b) Ethanol + TBME Yellow warbler
(Dendroica petechia)
1:10:10 Vortexed 5 s
per solvent
4 min
(RPMs set)
Common yellowthroat
(Geothlypis trichas)
McGraw and
Ardia (2003)
Ethanol + TBME Zebra finch Cited McGraw et al.
(2002, 2003a) for
method
McGraw and
Ardia (2004)
Ethanol + TBME Zebra finch 1:7.5:7.5 With each
solvent added
3 min
(RPMs set)
McGraw and
Nogare (2004)
Ethanol + TBME 5 parrot species 1:7.5:7.5 With each
solvent added
16,000×g for
4 min
McGraw et al. (2004) Ethanol + TBME American goldfinch
(Carduelis tristis)
Cited McGraw et al.
(2002) for method
Zebra finch
McGraw and
Schuetz (2004)
Ethanol + TBME 3 estrildid finch
species
1:10:10 With each
solvent added
3 min
(RPMs set)
McGraw and
Gregory (2004)
Ethanol + TBME American goldfinch 1:7.5:7.5 5 s with each
solvent added
3 min
(RPMs set)
McGraw (2004) Ethanol + TBME 11 songbird species Cited McGraw et al.
(2002) for method
McGraw et al. (2005) Ethanol + TBME American goldfinch 1:7.5:7.5 5 s with each
solvent added
3 min
(RPMs set)
McGraw and
Ardia (2005)
Ethanol + TBME Zebra finch Cited McGraw et al.
(2002) for method
McGraw (2005) Ethanol + TBME Society finch
(Lonchura domestica)
1:10:10 Vortexed 10,000 rpm for
4 min
House finch
(Carpodacus
mexicanus)
McGraw (2006b) Ethanol + TBME Zebra finch Cited McGraw et al.
(2002) for method
McGraw and
Parker (2006)
Ethanol + TBME Zebra finch Cited McGraw et al.
(2003a) for
method
McGraw and
Klasing (2006)
Ethanol + TBME Red junglefowl
(Gallus gallus)
Cited McGraw et al.
(2002) for method
McGraw et al. (2006a) Ethanol + TBME House finch Cited McGraw et al.
(2002) for method
McGraw et al. (2006b) Ethanol + TBME Zebra finch Cited McGraw et al.
(2003a) for
method
McGraw et al. (2006c) Ethanol + TBME Society finch Cited McGraw
(2005) for method
1998 Behav Ecol Sociobiol (2008) 62:1991–2002
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5 min
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5 min
Costantini et al. (2007b) Methanol-only Eurasian kestrel 1:8 Not mentioned 12,000×g for
5 min
Costantini et al. (2007c) Methanol-only Eurasian kestrel 1:8 Not mentioned 12,000×g for
5 min.
Casagrande et al. (2007) Methanol-only Eurasian kestrel 1:8 Not mentioned Not mentioned
Studies using each extraction method are organized in chronological order. Other method parameters, such as plasma/solvent ratio, vortexing, and
centrifugation rate, are also reported for comparison and for justification of some of our procedures (see text).
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