Chemistry Reference
In-Depth Information
25.3.2 M
ASS
S
PECTROMETRY
LC-MS was carried out using a ThermoSeparations HPLC composed of the Spectra System P4000
pump and the Spectra System AS3000 auto-injector coupled to a ThermoQuest Finnigan naviga-
tor mass spectrometer, and was run in the positive atmospheric pressure chemical ionization mode
(APCI+). The cone voltage was set to 5 V and the data were collected in both the full-scan mode and
single-ion monitoring mode observing the lutein parent
M
+
1 (569
e
/
z
) and principle
M
+
1 − H
2
O
(551
e
/
z
) ions and the parent
M
+
1 (391
e
/
z
) ion for 3-hydroxy-10
′
-apo-b-carotenal. The chromatog-
raphy was carried out using a 150
4.6 mm Luna 3 mm C-18 column (Phenomenex) under elution
conditions identical to those described above but without the triethyl amine. (Triethyl amine inter-
feres the carotenoid ionization in the mass sensitive detector.)
×
25.3.3 C
AROTENOID
I
DENTIFICATION
Tissue samples obtained from the different colored regions of the larvae were separately analyzed
by HPLC. The white, black, and yellow bands of Monarchs all contained a single, major carotenoid
component, lutein (all
E
-3,3
-carotene), Figure 25.3a. The amount of lutein present
in the black and white bands was markedly lower (
′
-dihydroxy-b,
ε
15x) than that in the yellow bands, see below. A
small quantity of 13-
cis
-lutein and zeaxanthin were observed to elute immediately following lutein
in the chromatogram and the lutein peak was preceded by a unique metabolite that is formed by the
cleavage of lutein, see Section 25.4.
The HPLC analysis of milkweed, the food-plant source for Monarch butterl ies, demonstrates
that it contains a complex mixture of carotenoids including lutein, several other xanthophylls,
xanthophyll epoxides, and b-carotene, Figure 25.3b. There is a component in the leaf extract that
is observed to elute near 8 min, which has a typical carotenoid spectrum but is not identical to
that of the lutein metabolite observed at near the same retention time in the extracts from larval
tissue.
The identity of lutein extracted from samples was coni rmed by its retention time, by co-injec-
tion with an authentic lutein standard, by the measurement of the characteristic UV-visible absorp-
tion spectrum, and mass spectra. Figure 25.4a shows the photodiode array spectrum of the lutein
peak, which possesses a principal maximum at 447 nm and the distinctive clear separation of the
central, principal maximum from the long wavelength maximum at 475 nm. The ratio of these two
peaks as measured from valley to peak is equal to 0.6, consistent with the presence of the
∼
ε
-ionone
ring. The mass spectrum of the lutein peak, Figure 25.4b, shows the 569
e
/
z
parent
M
1 ion and
the principle 551
e
/
z
ion resulting from the loss of water from the parent ion. In addition to lutein,
an unidentii ed component was detected that eluted prior to lutein in the extracts from larvae. Its
spectrum exhibits a single maximum at 435 nm in acetonitrile/methanol, see Figure 25.5a. The
UV/visible spectrum, retention time, and mass spectrum of this component are consistent with the
structure, 3-hydroxy-10
+
carbon-
carbon double bond. The mass spectrum of this component (see Figure 25.5b) has a strong parent
ion at 391
e
/
z
, consistent with the assignment. This component was not detected in the leaves of
the food plant.
′
-apo-b-carotenal, a product of the cleavage of lutein at the 9
′
-10
′
25.3.4 C
OMPARISON
OF
C
AROTENOID
C
ONTENT
IN
D
IFFERENT
C
OLORED
R
EGIONS
OF
L
ARVAE
25.3.4.1 Monarchs
The different colored bands within individual animals were found to contain signii cantly different
amounts of lutein. Punches of the black, white, and yellow bands were analyzed from each of nine
different animals. Because of the challenge in handling and analyzing the small punches of tissue,
three to eight punches were collected, pooled, and analyzed as a single sample. This procedure
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