Identification of astaxanthin diglucoside diesters from snow alga Chlamydomonas nivalis by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

Article abstract of DOI:10.1016/j.phytochem.2007.06.025

A method is described for the identification of astaxanthin glucoside esters from snow alga Chlamydomonas nivalis by means of liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (LC-MS/APCI). The method is based on the us

Full text of DOI:10.1016/j.phytochem.2007.06.025

Available online at www.sciencedirect.com
PHYTOCHEMISTRY
Phytochemistry 69 (2008) 479–490
www.elsevier.com/locate/phytochem
Identification of astaxanthin diglucoside diesters from snow alga
Chlamydomonas nivalis by liquid chromatography–atmospheric
pressure chemical ionization mass spectrometry
a,
b,c
a
c
ˇ
*
´ˇ
´
´
Tomas Rezanka , Linda Nedbalova , Karel Sigler , Vladislav Cepak
a
Institute of Microbiology, Academy of Sciences of the Czech Republic, Vı´denˇska´ 1083, 142 20 Prague, Czech Republic
Charles University in Prague, Faculty of Science, Department of Ecology, Vinicˇna´ 7, 128 44 Prague 2, Czech Republic
b
c
Institute of Botany, Academy of Sciences of the Czech Republic, Centre for Bioindication and Revitalization,
Dukelska´ 135, 379 82 Trˇebonˇ, Czech Republic
Received 19 March 2007; received in revised form 20 June 2007
Available online 2 August 2007
Abstract
A method is described for the identification of astaxanthin glucoside esters from snow alga Chlamydomonas nivalis by means of liquid
chromatography–mass spectrometry with atmospheric pressure chemical ionization (LC–MS/APCI). The method is based on the use of
preparative HPLC and subsequent identification of astaxanthin diglucoside diesters by microbore LC–MS/APCI. The combination of
these two techniques was used to identify more than 100 molecular species. The astaxanthin diglucoside diester, i.e. (all-E)-[di-(6-O-
oleoyl-b-D-glucopyranosyloxy)]-astaxanthin, was also synthesized to unambiguously confirm its structure.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Chlamydomonas nivalis; Snow alga; Astaxanthin diglucoside diesters; Liquid chromatography–mass spectrometry–atmospheric pressure
chemical ionization
1. Introduction
Carotenoids can be divided into two main groups – hydro-
carbon carotenes and oxygenated xanthophylls.
Carotenoids, typically bright yellow, orange, red, or
purple polyisoprenoid compounds with absorption max-
ima in the range of 400–500 nm are found predominantly
in photosynthesizing organisms such as green plants, algae
and some bacteria (Britton et al., 2004). The number of
currently known carotenoids exceeds 700 (Dembitsky,
2005) and it keeps on rising – more than 1000 papers on
these compounds have been published in 2006 alone. One
of their main functions is to interact with chlorophyll dur-
ing photosynthesis to absorb light and transfer energy; they
are also able to quench singlet oxygen and free radicals and
thereby protect plants from photo-oxidative damage.
Because of the presence of conjugated polyene chains in
their molecule, they are usually unstable in the presence of
light, heat, or oxygen. Isolation, identification, and quanti-
tation of carotenoids are difficult and many compounds
interfere in their isolation. The use of gas chromatography
(GC) and GC–mass spectrometry (GC–MS) is not suitable
because carotenoids are heat labile. The only useful
method appears to be high-performance liquid chromatog-
raphy (HPLC) with UV/visible (UV/Vis) detection or,
much more conveniently, with mass spectrometry detection
(LC–MS). Reversed-phase HPLC (RP-HPLC) is a pre-
ferred method, which is frequently used with C18 station-
ary phase, usually with gradient elution. This procedure
ensures sufficiently fast elution and the chromatographic
peaks are sharper. UV/vis absorbance detectors lack the
selectivity and the possibility of identifying individual
*
Corresponding author. Tel.: +420 241 062 300; fax: +420 241 062 347.
ˇ
E-mail address: rezanka@biomed.cas.cz (T. Rezanka).
0031-9422/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2007.06.025

ˇ
T. Rezanka et al. / Phytochemistry 69 (2008) 479–490
480
molecular species is limited because individual carotenoids
are structurally highly similar. In contrast to other ioniza-
tion techniques, xanthophylls and carotenes form both
molecular ions and protonated molecules during positive-
ion APCI (atmospheric pressure chemical ionization).
Several LC–MS methods have been reported for carot-
enoid analysis, including particle beam, continuous-flow
fast atom bombardment, electrospray and APCI tech-
niques (van Breemen, 1996, 1997; Careri et al., 1999; Lac-
ker et al., 1999). The latter provide high sensitivity and
highly linear detector response, suggesting that this LC–
MS technique should become the standard method for
carotenoid analysis (van Breemen, 1997). The first LC–
MS/APCI analysis of carotenoids was published by van
Breemen et al. (1996). While LC–MS/APCI analysis of free
carotenoids has been comprehensively reported (van Bre-
emen, 1996, 1997; van Breemen et al., 1996; Breithaupt
and Schwack, 2000), the characterization of carotenoid
esters in plant tissues has only emerged in the last few
years.
by the accumulation of the astaxanthin, which plays a cen-
tral role in protection against high levels of UV and photo-
synthetically-active radiation (Bidigare et al., 1993; Gorton
et al., 2001; Gorton and Vogelman, 2003). The content of
astaxanthin in dark red cysts was about 20 times that of
chlorophyll a in samples collected in Austrian Alps
(Remias et al., 2005).
In this paper, based on our previous communications on
constituent compounds from the algae and lichens (Dembit-
skyet al., 1993; Rezankaand Podojil, 1984), we examined the
chemical composition of the snow alga C. nivalis collected
from high-altitude environments with the aim of separating
astaxanthin diglycoside diesters and identifying their struc-
ture by means of the LC–MS/APCI. Because reference
substances were not available commercially, astaxanthin
diglucoside diester {di-[(6-O-oleoyl-b-D-glucopyranosyl)-
oxy]-astaxanthin – 18:1-G-A-G-18:1} was synthesized inde-
pendently to facilitate peak assignment in complex mixtures.
Major and also minor astaxanthin diglycoside diesters were
characterized in positive ion mode by their specific fragmen-
tation patterns, revealing a more complex profile than
described so far. This is the first report of analysis of native
carotenoid glycoside esters by LC–MS/APCI.
Carotenoids and carotenoid esters were extracted from
red pepper pods (Capsicum annuum L.) without saponifica-
tion. Among the 42 compounds detected, 4 non-esterified,
11 mono- and 17 diesters were characterized based on their
retention times, UV/vis spectra and their fragmentation
patterns in collision-induced dissociation experiments in
atmospheric pressure chemical ionization mass spectrome-
try (MS/APCI) (Schweiggert et al., 2005).
2. Results and discussion
The MeOH extract from the snow alga C. nivalis was
subjected to preparative normal phase HPLC to obtain
eight fractions. Table 1 gives the data obtained after RP-
HPLC from fraction 4, i.e. di-(6-O-acyl-glucopyranosyl-
oxy)-astaxanthins (FA-G-A-G-FA, where FA is fatty acid,
G is glucose, and A is astaxanthin), which had the highest
weight proportion. The yield referred to the wet weight of
cells was 185 mg (0.93%). A total of 69 peaks were sepa-
rated by LC–MS/APCI on narrow-bore columns con-
nected in series. The chromatogram is given in Fig. 1.
MS/APCI identified 105 molecular species. All astaxanthin
diglycoside diesters exhibited very similar, if not identical,
UV spectra (200–550 nm). Substitution by any acyl chain
or glucosyl moieties did not modify the chromophore
(Yamano et al., 2002; Yokoyama et al., 1995a, 1998; Giuff-
rida et al., 2006). All the above findings lead to the conclu-
sion that the UV spectra of all compounds are very similar
and do not provide a clue for identification of the appropri-
ate structures.
The components of astaxanthin diglycoside diesters
were also analyzed by GC after hydrolysis. Fraction 4 con-
tained astaxanthin as the aglycone moiety. Glucose was the
only sugar which was detected in fraction 4. The fatty acid
composition, which was identical with the results obtained
from APCI analysis, was also determined by GC–MS, see
Table 2.
Optimum APCI conditions were established by using a
synthetic sample, i.e. (18:1-G-A-G-18:1). The different frag-
menter voltage settings used throughout this study and
different cone voltage were shown to have a significant
LC–MS/APCI was used to identify carotenoid esters in
mandarin essential oil (Giuffrida et al., 2006). After first
being analyzed by HPLC, 4 free carotenoids, 15 astaxan-
thin monoesters, 12 astaxanthin diesters, and 3 astacin
monoesters in Haematococcus pluvialis were identified by
LC–MS/APCI (Breithaupt, 2004).
Takaichi et al. (2003) isolated fractions with astaxanthin
monoesters and diesters from Antarctic krill Euphausia
superba. Astaxanthin esters were separated by RP-HPLC
depending on the number of carbons and double bonds
of esterified fatty acid(s) and identified by field desorption
mass spectrometry.
A new carotenoid glycoside, astaxanthin-glucoside, was
isolated from the astaxanthin-producing marine bacterium
Agrobacterium aurantiacum, and its structure was deter-
mined by Yokoyama et al. (1995a). Another new caroten-
oid glucoside, astaxanthin-diglucoside plus the known
astaxanthin glucoside were isolated from two transformed
E. coli strains (Yokoyama et al., 1998).
The snow alga Chlamydomonas nivalis (Bauer) Wille
(Chlorophyta, Chlamydomonadales), forms striking red
patches in high mountain and polar regions all over the
world (Kol, 1968). Since the snow environment is extre-
mely harsh, with low temperature, high irradiance and fre-
quent freeze–thaw cycles, for most of its life cycle,
Chlamydomonas nivalis occurs as immotile spherical
thick-walled resting stage, which can be considered as
major adaptation to the extreme habitat. Flagellates are
rarely observed. The red coloration of the cells is caused

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T. Rezanka et al. / Phytochemistry 69 (2008) 479–490
481
Table 1
Quantitative distribution of astaxanthin glucoside esters from snow alga Chlamydomonas nivalis by means of LC–MS/APCI
a
Peak
no.
RT
(s)
Mol. spec.
%
Ratiob M+H MꢀAcyl1 MꢀAcyl2 MꢀAcyl1- MꢀAcyl2- MꢀAcyl1- MꢀAcyl2- MꢀAcyl1- MꢀAcyl2-
18
18
G
G
G-18
G-18
1
2
3
4
5
6
7
605 18:5 18:5 1.36
620 18:5 16:4 1.87
637 16:4 16:4 2.56
690 22:6 18:5 0.88
699 22:6 16:4 1.21
707 18:5 18:4 1.17
766 18:5 16:3 4.04 60
1433
1407
1381
1487
1461
1435
1409
1409
1383
1541
1489
1463
1463
1437
1437
1437
1411
1411
1411
1385
1385
1517
1491
1465
1465
1439
1439
1439
1413
1413
1413
1413
1387
1387
1387
1361
1493
1467
1441
1493
1467
1441
1441
1441
1441
1415
1415
1415
1415
1415
1389
1389
1389
1389
1363
1495
1469
1469
1443
1417
1443
1159
1133
1133
1159
1133
1161
1135
1133
1135
1213
1161
1135
1159
1133
1163
1161
1137
1135
1133
1137
1135
1189
1163
1137
1161
1135
1165
1163
1139
1137
1135
1133
1113
1139
1137
1113
1165
1139
1113
1189
1163
1137
1167
1165
1163
1141
1139
1137
1135
1133
1113
1141
1139
1137
1113
1167
1141
1165
1139
1113
1169
1159
1159
1133
1213
1213
1159
1159
1161
1133
1213
1213
1213
1189
1189
1159
1161
1159
1161
1163
1133
1135
1213
1213
1213
1189
1189
1159
1161
1159
1161
1163
1165
1159
1133
1135
1133
1213
1213
1213
1189
1189
1189
1159
1161
1163
1159
1161
1163
1165
1167
1161
1133
1135
1137
1135
1213
1213
1189
1189
1189
1159
1141
1115
1115
1141
1115
1143
1117
1115
1117
1195
1143
1117
1141
1115
1145
1143
1119
1117
1115
1119
1117
1171
1145
1119
1143
1117
1147
1145
1121
1119
1117
1115
1095
1121
1119
1095
1147
1121
1095
1171
1145
1119
1149
1147
1145
1123
1121
1119
1117
1115
1095
1123
1121
1119
1095
1149
1123
1147
1121
1095
1151
1141
1141
1115
1195
1195
1141
1141
1143
1115
1195
1195
1195
1171
1171
1141
1143
1141
1143
1145
1115
1117
1195
1195
1195
1171
1171
1141
1143
1141
1143
1145
1147
1141
1115
1117
1115
1195
1195
1195
1171
1171
1171
1141
1143
1145
1141
1143
1145
1147
1149
1143
1115
1117
1119
1117
1195
1195
1171
1171
1171
1141
997
997
997
971
1051
1051
997
997
999
971
1051
1051
1051
1027
1027
997
999
997
999
1001
971
979
953
953
979
953
981
955
953
955
1033
981
955
979
953
983
981
957
955
953
957
955
1009
983
957
981
955
985
983
959
957
955
953
933
959
957
933
985
959
933
1009
983
957
987
985
983
961
959
957
955
953
933
961
959
957
933
987
961
985
959
933
989
979
979
953
1033
1033
979
979
981
953
1033
1033
1033
1009
1009
979
981
979
981
983
971
971
997
971
999
973
971
973
1051
999
973
997
971
1001
999
975
973
971
975
973
1027
1001
975
999
973
1003
1001
977
975
973
971
951
977
975
951
1003
977
951
1027
1001
975
1005
1003
1001
979
977
975
973
971
951
979
977
975
951
1005
979
1003
977
951
1007
18:4 16:4
40
8
9
786 16:4 16:3 3.35
804 22:6 22:6 0.57
822 22:6 18:4 0.75
881 22:6 16:3 1.59
909 20:4 18:5 0.99
919 20:4 16:4 1.36
946 18:5 18:3 2.06 50
18:4 18:4 50
972 18:5 16:2 4.40 20
10
11
12
13
14
15
16
18:4 16:3
18:3 16:4
45
35
994 16:4 16:2 5.56 80
953
955
16:3 16:3
20
973
17
18
19
20
21
22
1099 22:6 20:4 0.64
1124 22:6 18:3 0.68
1202 22:6 16:2 0.55
1220 20:4 18:4 0.85
1252 20:4 16:3 1.78
1283 18:5 18:2 1.68 45
18:4 18:3 55
1300 18:5 16:1 4.72 20
1051
1051
1051
1027
1027
997
999
997
999
1001
1003
997
971
973
1033
1033
1033
1009
1009
979
981
979
981
983
23
18:4 16:2
18:3 16:3
18:2 16:4
15
40
15
985
979
953
955
24
25
1328 18:5 14:0 0.27
1350 16:4 16:1 2.96 50
16:3 16:2
50
26
27
28
29
30
31
32
33
1475 16:4 14:0 0.38
1495 22:6 18:2 0.50
1503 22:6 16:1 0.66
1515 22:6 14:0 0.17
1527 20:4 20:4 0.72
1562 20:4 18:3 0.77
1581 20:4 16:2 0.62
971
953
1051
1051
1051
1027
1027
1027
997
999
1001
997
1033
1033
1033
1009
1009
1009
979
981
983
979
981
983
985
987
981
953
955
957
955
1610 18:5 18:1 3.44 55
18:4 18:2
18:3 18:3
20
25
34
1634 18:5 16:0 6.47 15
18:4 16:1
18:3 16:2
18:2 16:3
15
10
20
40
999
1001
1003
1005
999
971
973
18:1 16:4
35
36
1666 18:4 14:0 0.23
1689 16:4 16:0 3.53 35
16:3 16:1
16:2 16:2
50
15
975
973
37
38
39
40
41
42
43
1725 16:3 14:0 0.50
1816 22:6 18:1 1.27
1831 22:6 16:0 0.54
1855 20:4 18:2 0.56
1882 20:4 16:1 0.75
1905 20:4 14:0 0.20
1051
1051
1027
1027
1027
997
1033
1033
1009
1009
1009
979
1924 18:5 18:0 2.59 10
(continued on next page)

ˇ
T. Rezanka et al. / Phytochemistry 69 (2008) 479–490
482
Table 1 (continued)
a
Peak
no.
RT
(s)
Mol. spec.
%
Ratiob M+H MꢀAcyl1 MꢀAcyl2 MꢀAcyl1- MꢀAcyl2- MꢀAcyl1- MꢀAcyl2- MꢀAcyl1- MꢀAcyl2-
18
18
G
G
G-18
G-18
18:4 18:1
18:3 18:2
1953 18:4 16:0 5.97 10
65
25
1443
1443
1417
1417
1417
1417
1417
1391
1391
1391
1365
1497
1471
1445
1445
1445
1445
1419
1419
1419
1419
1393
1393
1393
1367
1341
1473
1447
1447
1421
1421
1421
1395
1395
1369
1449
1449
1423
1423
1397
1397
1451
1425
1453
1167
1165
1141
1139
1137
1135
1133
1141
1139
1113
1113
1169
1167
1141
1169
1167
1165
1141
1139
1137
1135
1113
1141
1139
1113
1113
1169
1169
1167
1141
1139
1137
1113
1141
1113
1169
1167
1141
1139
1113
1141
1169
1141
1169
1161
1163
1161
1163
1165
1167
1169
1135
1137
1163
1137
1213
1189
1189
1161
1163
1165
1163
1165
1167
1169
1165
1137
1139
1139
1113
1189
1163
1165
1165
1167
1169
1167
1139
1141
1165
1167
1167
1169
1169
1141
1167
1169
1169
1149
1147
1123
1121
1119
1117
1115
1123
1121
1095
1095
1151
1149
1123
1151
1149
1147
1123
1121
1119
1117
1095
1123
1121
1095
1095
1151
1151
1149
1123
1121
1119
1095
1123
1095
1151
1149
1123
1121
1095
1123
1151
1123
1151
1143
1145
1143
1145
1147
1149
1151
1117
1119
1145
1119
1195
1171
1171
1143
1145
1147
1145
1147
1149
1151
1147
1119
1121
1121
1095
1171
1145
1147
1147
1149
1151
1149
1121
1123
1147
1149
1149
1151
1151
1123
1149
1151
1151
1005
1003
979
977
975
973
971
979
977
999
1001
999
1001
1003
1005
1007
973
987
985
961
959
957
955
953
961
959
933
933
989
987
961
989
987
985
961
959
957
955
933
961
959
933
933
989
989
987
961
959
957
933
961
933
989
987
961
959
933
961
989
961
989
981
983
981
983
985
987
989
955
957
983
957
1033
1009
1009
981
983
985
983
985
987
989
985
957
959
959
933
1009
983
985
985
987
989
987
959
961
985
987
987
989
989
961
987
989
989
44
18:3 16:1
18:2 16:2
18:1 16:3
18:0 16:4
15
10
60
5
45
1984 16:3 16:0 2.15 70
16:2 16:1
30
975
1001
975
46
47
48
49
50
51
2000 18:3 14:0 0.21
2032 16:2 14:0 0.17
2144 22:6 18:0 0.19
2166 20:4 18:1 1.43
2185 20:4 16:0 0.61
951
951
1007
1005
979
1007
1005
1003
979
977
975
973
951
979
977
951
951
1007
1007
1005
979
977
975
1051
1027
1027
999
1001
1003
1001
1003
1005
1007
1003
975
2214 18:4 18:0 2.22 10
18:3 18:1
18:2 18:2
2249 18:3 16:0 3.01 20
70
20
52
18:2 16:1
18:1 16:2
18:0 16:3
20
40
20
53
54
2275 18:2 14:0 0.15
2290 16:2 16:0 1.30 40
16:1 16:1
60
977
977
951
55
56
57
58
2355 16:1 14:0 0.21
2377 14:0 14:0 0.05
2548 20:4 18:0 0.22
2592 18:3 18:0 1.34 20
18:2 18:1 80
2640 18:2 16:0 2.15 20
1027
1001
1003
1003
1005
1007
1005
977
59
18:1 16:1
18:0 16:2
70
10
60
61
62
63
2669 18:1 14:0 0.40
2699 16:1 16:0 0.63
2724 16:0 14:0 0.16
2883 18:2 18:0 3.00
18:1 18:1
951
979
951
1007
1005
979
977
951
979
5
95
1003
1005
1005
1007
1007
979
1005
1007
1007
64
2933 18:1 16:0 1.44 95
18:0 16:1
5
65
66
67
68
69
a
3001 18:0 14:0 0.05
3021 16:0 16:0 0.51
3162 18:1 18:0 0.44
3233 18:0 16:0 0.18
3497 18:0 18:0 0.06
979
1007
979
1007
Abundance.
b
The ratio of isomers, determined solely from the detector response to each one of the compounds and from the mass spectra of the respective
astaxanthin glucoside esters.
impact on the fragmentation patterns. The fragmentation
of the astaxanthin diglycoside diester required two different
voltage values. If the cone voltage was set at 30 V in the
positive mode, all compounds exhibited strong [M+H]+
peaks (data not shown) but no fragmentation was
observed, whereas a higher cone voltage (70 V) gave rise
to fragmentation and individual daughter ions can be used
for identification of molecular species. LC–MS analysis at
m/z 400–1700 of the extract using an APCI showed
[M+H]+ (pseudomolecular ions) for the peaks characteris-
tic for astaxanthin diglycoside diesters and allowed the
attribution of their molecular weights. A similar situation
has already been observed with astaxanthin esters (Bre-
ithaupt, 2004). The following discussion of the APCI mass
spectra of the astaxanthin diglycoside diesters studied here
includes speculative assignments of fragmentation path-
ways for each compound analyzed.
Some fragmentation of the peak at 2883 s (No. 63, 18:1-
G-A-G-18:1) was evident with a fragmenter voltage of
70 V (Fig. 2 and Table 1). The parent ion at m/z1449
[M+H]+ dissociates to six ions m/z 1167 [M+HꢀFA1]+,
identical in this case with [M+HꢀFA2]+ m/z 1149
[M+HꢀFA1ꢀH2O]+ = [M+HꢀFA2ꢀH2O]+, m/z 1005
[M+HꢀGlcꢀFA1]+ = [M+HꢀGlcꢀFA2]+,
m/z
987
[M+HꢀGlcꢀFA1ꢀH2O]+ = [M+HꢀGlcꢀFA2ꢀH2O]+,
and ions common to all astaxanthin diglycoside diesters

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T. Rezanka et al. / Phytochemistry 69 (2008) 479–490
483
34
16000
12000
8000
4000
0
44
16
23
7
52
63
3
59
11
68
500
1000
1500
2000
2500
3000
3500
time (s)
Fig. 1. Separation of astaxanthin glucoside esters from snow alga Chlamydomonas nivalis by means of LC–MS/APCI. For peak assignment, see Table 1.
[M+HꢀFA1]+ and/or [M+HꢀFA2]+ ions for determining
Table 2
Fatty acid composition observed by GC–MS
peaks inseparable by HPLC. The results are documented in
Fatty acid
%
Table 1. Fig. 3 illustrates an excision of mass spectra of
peak no. 44.
14:0
16:0
16:1
16:2
16:3
16:4
18:0
18:1
18:2
18:3
18:4
18:5
20:4
22:6
1.1
5.2
6.6
The number of papers, which described the positive soft
ionization of astaxanthin derivatives is very low. It should
be noted that we did not find any papers describing the MS
of astaxanthin diglycoside diesters. Different xanthophyll
glycoside esters have been identified in several studies,
but the published data describe only the identification of
pseudomolecular ions (Takaichi et al., 2001, 2003), usually
in a high-resolution mode (Burgess et al., 1999; Yokoyama
et al., 1995b). Several papers dealt with the analysis of
acyls. The first of these studies (Breithaupt and Bamedi,
2002) describes the pseudomolecular ion [M+H]+,
[M+HꢀH2O]+ and other ions which we also identified
and described (see above), i.e. [M+HꢀFA1]+, [M+Hꢀ
FA1ꢀH2O]+, [M+HꢀFA1ꢀFA2]+, [M+HꢀFA1ꢀFA2ꢀ
H2O]+. The splitting was complicated by the two epoxidic
oxygen atoms present in the major xanthophyll–violaxan-
thin – described by the authors.
Another study identified astaxanthin esters, including
both monoesters and diesters, in aquatic organisms (Bre-
ithaupt, 2004). In contrast to our approach, the authors
used APCI in the negative mode. Among the metabolites
in phytoplankton described by Frassanito et al. (2005) only
adonirubin among the many xanthophylls present was
esterified by two different polyene fatty acids (18:4 and
20:5). However, the method of identification has not been
described. Two other studies (Schweiggert et al., 2005;
Miao et al., 2006) devoted to carotenoid esters in red
5.3
16.9
12.7
1.3
13.4
4.7
6.8
7.6
9.0
6.3
5.5
m/z 885 [M+HꢀFA1ꢀFA2]+; m/z 867 [M+HꢀFA1ꢀFA2ꢀ
H2O]+; m/z 849 [M+HꢀFA1ꢀFA2ꢀ2·H2O]+; m/z 723
[M+HꢀFA1ꢀFA2ꢀGlc]+; m/z 705 [M+HꢀFA1ꢀFA2ꢀ
GlcꢀH2O]+; m/z 561 [M+HꢀFA1ꢀFA2ꢀ2·Glc]+; m/z
543 [M+HꢀFA1ꢀFA2ꢀ2·GlcꢀH2O]+; m/z 525 [M+Hꢀ
FA1ꢀFA2ꢀ2·Glc-2·H2O]+. Fig. 2 shows a tentative
pathway for the mass spectral fragmentation of 18:1-G-
A-G-18:1. The other 104 compounds had very similar mass
spectra, viz. Table 1. We illustrate this on the peak with
retention time 1953 s (peak no. 44), which is a mixture of
five molecular species: 18:4–16:0, 18:3–16:1, 18:2–16:2,
18:1–16:3, and 18:0–16:4. Its semiquantitation was done
by using a previously described method (Rezanka and
Sigler, 2006, 2007), which makes use of the abundance of

ˇ
T. Rezanka et al. / Phytochemistry 69 (2008) 479–490
484
100
M+H
561
1449
O
1005
O
HO
O
1167
885
OH
OH
O
885
6
O
O
O
O
O
OH
HO
O
HO
561
M-FA
1
1167
50
M-FA -G-18
1
M-FA -FA -G-G-18-18
1
2
M-FA -FA -18-18
M-FA -G
1
1
2
M-FA -FA -G-G-18
M-FA -FA -18
1
2
1
2
1005
987
525 M-FA -FA -G-G
M-FA -FA
1
2
1 2
M-FA -18
1
M-FA -FA -G-18
885
M+H-18-18
M+H-18
561
543
1
2
1149
1431
1413
M-FA -FA -G
705
1
2
723
849
867
0
400
600
800
1000
m/z
1200
1400
1600
Fig. 2. APCI spectrum of astaxanthin glucoside ester in peak 63 and major fragmentation pathway of protonated astaxanthin glucoside esters di-[(6-O-
oleoyl-b-D-glucopyranosyl)oxy]-astaxanthin, i.e. 18:1-G-A-G-18:1 in Fig. 1.
pepper pods and in mandarin essential oil also made use of
MSn APCI and identified the appropriate ions, i.e. [M+H]+
and [M+HꢀFA1]+.
preparation of astaxanthin diglucoside esters appeared
to be the sequence of reactions described in the paper
by Yamano et al. (2002). Although it does not provide
high yields, it involves simple reactions, low number
of reaction steps and easy availability of commercial
reagents.
The structure of astaxanthin-b-D-diglucoside was deter-
mined based on the identity of the data obtained by mea-
suring the spectra of our compound (4) with the data
published previously (Yokoyama et al., 1995a, 1998). The
1H and 13C NMR data, see Table 3.
In order to fully confirm the structure, peak no. 63 (i.e.
peak with retention time 2883 s) was separated by prepara-
tive HPLC. The reasons for separating this peak were man-
ifold. Firstly, according to mass spectral analysis this peak
contains only two molecular species, the symmetrical
molecular species being the major component (about 95%
of the total content). Secondly, this peak can be relatively
easily separated from other neighboring peaks; thirdly, its
abundance is among the highest, and fourthly oleic acid
and/or its acylchloride is commercially available for the
partial synthesis of di-(6-O-oleoyl-b-D-glucopyranosyl-
oxy)-astaxanthin (Scheme 1).
The UV–vis absorption spectrum of 6 in MeOH showed
a broad absorption maximum at around 489 nm and small
maxima at 300 and 263 nm, indicating the presence of a
conjugated system, which was comparable to that of asta-
xanthin-b-D-glucoside (Yokoyama et al., 1995a). The
molecular formula of 6 was determined to be C88H136O16
by HRFABMS. The 1H and 13C NMR data of 6 in
CD3OD (Table 4) indicated again the presence of astaxan-
thin as the carotenoid moiety, two hexose moieties, and
The first paper concerning the synthesis of glycosides
of xanthophyll was published by Pfander and Hodler
(1974). These authors described the synthesis of zeaxan-
thin b,b-D-diglucoside using a reaction of 2,3,4,6-tetra-
O-acetyl-a-D-glucopyranosyl bromide and zeaxanthin
under catalysis with silver carbonate. Another study
(Yamano et al., 2000) described the preparation of zea-
xanthin- and cryptoxanthin-b-D-glucopyranosides from
appropriate aglycones, with tetra-O-benzoyl-a-D-glucopyr-
anosyl bromide as a glycosyl donor and silver triflate as
an activator. However, the most suitable method for the
1
two monounsaturated fatty acids. The H signals of the
hexose moieties showed two anomeric protons at dH 4.50
(d, J = 7.8 Hz). Further, we compared the 13C NMR data
and found that the two hexoses were b-D-glucopyranose
based on the coupling constants of the anomeric protons,
3
1
0
0
0
0
i.e. JH1 ꢀH2 ¼ 7:8 Hz and JH1 ꢀC1 ¼ 169:8 Hz (Podlasek

ˇ
T. Rezanka et al. / Phytochemistry 69 (2008) 479–490
485
astaxanthin molecules with fatty acids allows this chro-
mophore to be localized within cytoplasmic globules to
maximize its photoprotective efficiency. Composition of
red cysts similar to that found by us was described by
Bidigare et al. (1993). According to their data, the fatty
acid pattern of the astaxanthin fraction differs from the
total fatty acid pattern of the fat extract of the alga;
the astaxanthin diester fraction contained as dominant
acids with one or more double bonds, i.e. more than
60% of all fatty acids were unsaturated. Although, in
contrast to our findings, these acids were found also in
astaxanthin diesters, astaxanthin with unsaturated fatty
acids can be safely assumed to be main components of
the cysts (see below).
As described in the green alga Haematococcus pluvialis,
which forms similar amounts of secondary carotenoids as
C. nivalis, but grows at mesophilic temperatures, astaxan-
thin derivatives also have antioxidant activity (Kobayashi
and Okada, 2000; Boussiba, 2000).
In C. nivalis they may act as multipurpose protective
compounds, screening the chloroplast and nucleus from
potentially damaging UV and VIS radiation, quenching
singlet oxygen and protecting against superoxide. Astaxan-
thin derivatives may also provide cryoprotection because
the large volume filled by astaxanthin ester rich lipid drop-
lets reduces the amount of cellular water and may thus
reduce damage from ice crystals (Hoham, 1992). The poly-
enoic fatty acid moieties aid in preserving fluidity of mem-
branes even at very low temperatures. Thus, carotenoids
with their bipolar, linear, and rigid structures may be
responsible for general membrane stabilization (Ourisson
et al., 1987; Yokoyama et al., 1996).
In addition to all these functions, compounds with
characteristic ‘‘hydrophobic–hydrophilic–hydrophobic’’ struc-
tures such as thermozeaxanthins and thermocryptoxanthins
are assumed to stabilize membranes even at high tempera-
ture, a function considered essential, e.g., for the growth of
thermophilic bacteria. Studies on thermophilic bacteria pro-
ducing carotenoid glucoside esters, e.g. zeaxanthin mono-
and diglucoside esters (Thermus thermophilus) (Yokoyama
et al., 1995a, 1996) showed that membrane-associated
carotenoids in most cases are monocyclic or dicyclic, with
one or two C-6 acylated glucosyl units involved with charac-
teristic acyl groups (Yamano et al., 2002).
We have developed a new LC–MS/APCI methodology
that uses a single RP-HPLC gradient separation and differ-
ent MS experimental set-up (positive ion mode, APCI ioni-
zation). It is suitable both for the screening of pigments and
for the detection of other interesting natural products from
raw extracts of microalgae such as snow algae, and will aid
in identifying xanthophyll derivatives in natural sources.
Analysis of carotenoids in C. nivalis showed the presence
of astaxanthin diglucoside diesters. The C-3 hydroxyl group
of astaxanthin is glucosylated to yield carotenoid glucoside;
then the C-6 hydroxyl group of the glucosyl moiety is
esterified with the specific fatty acids. After analyzing the
LC–MS/APCI data, we identified astaxanthin derivatives
Fig. 3. Part of APCI spectrum of astaxanthin glucoside ester in peak 44
containing five molecular species: 18:4-G-A-G-16:0, 18:3-G-A-G-16:1,
18:2-G-A-G-16:2, 18:1-G-A-G-16:3, and 18:0-G-A-G-16:4.
et al., 1995). The HMBC correlations prove that the glu-
cose moieties were attached to the hydroxyl groups at C-
3 of the astaxanthin moiety and also that oleoyl moieties
are bound by an ester bond to the 6-hydroxy group of
glucose. The assignment of signals in the olefinic region
of the spectra of 6 revealed an (E)-configuration for all of
the double bonds, which meant that the aglycone of this
compound could be identified as (all-E)-astaxanthin. For
the determination of the absolute configuration of the car-
bohydrate, it was cleaved from the carotenoid by mild
methanolysis and subsequently perbenzylated. The CD
spectrum in the region from 200 to 250 nm of modified
b-glucose in 6 belongs to the D-series because it was in
accordance with published data for b-D-glucose (Kalu-
arachchia and Bush, 1989). Comparison of UV, MS and
1H and 13C NMR data of both, i.e. synthetic and natural
compounds showed that the compounds are completely
identical, having the structure of di-(6-O-oleoyl-b-D-gluco-
pyranosyloxy)-astaxanthin.
The survival of snow algae in high altitude regions is
dependent on the synthesis of specific lipid components
with multiple physiological functions. Secondary carote-
noids function as passive photoprotectants minimizing
the amount of light available for absorption by cells
(Gorton et al., 2001; Gorton and Vogelman, 2003).
Esterification of the relatively polar hydroxyl containing

O
OH
1
HO
1) α-D-glucopyranosyl bromide tetrabenzoate + AgOTf stirred at 0 ˚C/2 h
O
2) MeOH + NaOMe
O
O
19
20
HO
17
16
7
15
OH
OH
O
1
3
HO
HO
O
4
O
HO
18
6´
1´
O
OH
HO
oleoyl chloride + pyridine-CH2Cl2
3´
O
O
HO
OH
OH
2´´
O
O
O
6
O
O
O
O
OH
O
HO
HO
18´´
Scheme 1. Scheme of the synthesis of di-[(6-O-oleoyl-b-D-glucopyranosyl)oxy]-astaxanthin.
9´´ 10´´

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T. Rezanka et al. / Phytochemistry 69 (2008) 479–490
487
Table 3
Table 4
1H and 13C NMR data of astaxanthin diglucoside (4) (in CD3OD)
1H and 13C NMR data of diacyl diglucoside astaxanthin (6) (in CD3OD)
No.
1H
13C
No.
1H
13C
1
2
–
37.9 s
45.6 t
1
2
–
37.9 s
45.6 t
1.95 (2H, t, J = 14.0, 14.0 Hz); 2.18
(2H, dd, J = 14.0, 5.5 Hz)
4.56 (2H, dd, J = 14.0, 5.5 Hz)
–
–
–
6.19 (2H, d, J = 15.8 Hz)
6.42 (2H, d, J = 15.8 Hz)
–
6.31 (2H, d, J = 10.9 Hz)
6.64 (2H, m)
6.47 (2H, d, J = 14.8 Hz)
–
6.28 (2H, d, J = 11.1 Hz)
6.63 (2H, m)
1.95 (2H, t, J = 14.0, 14.0 Hz); 2.18
(2H, dd, J = 14.0, 5.5 Hz)
4.56 (2H, dd, J = 14.0, 5.5 Hz)
–
–
–
6.19 (2H, d, J = 15.8 Hz)
6.42 (2H, d, J = 15.8 Hz)
–
6.31 (2H, d, J = 10.9 Hz)
6.64 (2H, m)
6.47 (2H, d, J = 14.8 Hz)
–
6.28 (2H, d, J = 11.1 Hz)
6.63 (2H, m)
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
10
20
30
40
50
60
78.1 d
200.5 s
128.8 s
163.4 s
123.3 d
143.1 d
135.1 s
135.8 d
124.6 d
137.8 d
136.5 d
134.0 d
131.7 d
26.7 q
30.8 q
14.3 q
12.6 q
12.7 q
105.3 d
73.5 d
76.8 d
70.0 d
75.9 d
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
10
20
30
40
50
60
78.1 d
200.5 s
128.8 s
163.4 s
123.3 d
143.1 d
135.1 s
135.8 d
124.6 d
137.8 d
136.5 d
134.0 d
131.7 d
26.7 q
30.8 q
14.3 q
12.6 q
12.7 q
104.3 d
74.2 d
75.7 d
69.6 d
75.1 d
1.35 (6H, s)
1.21 (6H, s)
1.90 (6H, s)
1.98 (6H, s)
1.35 (6H, s)
1.21 (6H, s)
1.90 (6H, s)
1.98 (6H, s)
2.00 (6H, s)
2.00 (6H, s)
4.50 (2H, d, J = 7.8 Hz)
3.61 (2H, dd, J = 7.8, 9.4 Hz)
3.54 (2H, t, J = 9.4)
3.59 (2H, t, J = 9.4)
3.38 (2H, m)
4.50 (2H, d, J = 7.8 Hz)
3.63 (2H, dd, J = 7.8, 9.4 Hz)
3.58 (2H, t, J = 9.4)
3.56 (2H, t, J = 9.4)
3.46 (2H, m)
4.25 (2H, dd, J = 12.0, 5.1 Hz); 4.40
(2H, dd, J = 12.0, 2.9 Hz)
–
2.28 (4H, t, J = 7.1 Hz)
1.60 (4H, m)
1.23-1.48 (16H, m)
1.95-2.03 (8H, m)
5.31 (4H, m)
3.75 (2H, dd, J = 12.0, 5.2 Hz); 3.85
(2H, dd, J = 12.0, 3.4 Hz)
62.6 t
61.2 t
100
173.5 s
34.5 t
25.2 t
29.0-29.8 t
27.3 t
130.1 d
29.0-29.8 t
32.1 t
200
300
in C. nivalis according to molecular mass and characteristic
fragmentation patterns. Positive ion APCI mass spectra
produced structurally significant fragment ions that reflect
the main structure information of astaxanthin derivatives
and the rule of fission. This approach is convenient for ana-
lyzing the structure and composition of astaxanthin dig-
lucoside diesters in the snow alga.
400-700
800, 1100
900, 1000
1200-1500
1600
1.23-1.48 (16H, m)
1.23-1.48 (4H, m)
1.23-1.48 (4H, m)
0.87 (6H, t, J = 7.0 Hz)
1700
1800
22.8 t
14.2 q
3. Experimental
double distilled and degassed before use. All chemicals
were obtained from Sigma–Aldrich, Prague.
3.1. Plant material
Carotenoids were extracted with MeOH several times
from the wet cell pellets (ca. 20 g) using an ultrasonicator
for several seconds. The extract was partitioned with
CH2Cl2 and H2O, and then the CH2Cl2 layer was evapo-
rated. Major carotenoid classes were purified by prepara-
tive HPLC. The yield of the astaxanthin diglycoside
diesters was 185 mg. All procedures were performed under
dim light and argon.
The astaxanthin diglycoside diesters (10 mg) were
saponified in 10% KOH–MeOH at room temperature over-
night. A fatty acid fraction obtained from saponification
was partitioned between alkali solution (pH 9) and Et2O
to remove basic and neutral components. The aqueous
phase, containing fatty acids, was acidified to pH 2 and
extracted with hexane. The fatty acid fraction was methyl-
ated using CH2N2. GC–MS of a FAME mixture was done
on a Finnigan 1020 B in EI mode. Splitless injection was at
Red snow was aseptically collected on July 28th, 2006
from surface layers of a snow field in the Pirin Mountains
(south western Bulgaria). The sampling site was situated in
the alpine zone near the lake Tevno ezero at an altitude of
2546 m a.s.l. (41ꢁ4109300N, 23ꢁ2901500E). The snow cover at
the site is seasonal. Snow sample was transported into lab-
oratory in a thermos bottle, identified under microscope
and the algal biomass was kept frozen until further analy-
sis. The red bloom was formed entirely by spherical cysts of
Chlamydomonas nivalis (Bauer) Wille, no flagellates were
observed in the sample.
3.2. Standards and isolation
Standard of astaxanthin diglucoside diester (18:1-G-A-
G-18:1) was prepared as described below. All solvents were

ˇ
T. Rezanka et al. / Phytochemistry 69 (2008) 479–490
488
100 ꢁC, and a fused silica capillary column (Supelcowax 10;
60 m · 0.25 mm ID, 0.25 mm film thickness; Supelco,
Prague) was used. The temperature program was as fol-
lows: 100 ꢁC for 1 min, subsequently increasing at 20 ꢁC/
min to 180 ꢁC and at 2 ꢁC/min to 280 ꢁC, which was main-
tained for 1 min. The carrier gas was helium at a linear
velocity of 60 cm/s. All spectra were scanned within the
range m/z 70–500. The structures of fatty acids were con-
firmed by comparison of retention times and fragmentation
patterns to those of the standard fatty acid methyl esters
(Supelco, Prague).
the organic layer was washed with 5% HCl, saturated
NaHCO3, brine and dried. Evaporation of the solution
provided
a residue, which was purified by HPLC
(CH2Cl2–MeOH, 95:5) to afford the di-oleate 6 (29 mg,
23%). HRFABMS (m/z) 1449.9911 [M+H]+, calcd. for
[C88H136O16+H]+ 1449.9906. UV kmax (MeOH) nm (loge)
489 (4.76), 300 (4.07), 254 (4.16). NMR data see Table 4.
The yield of sequence 1 ! 3 ! 4 ! 6 was 6.6%.
3.3. Preparative HPLC
For establishment of the configuration of the glycosides,
the astaxanthin diglycoside diesters (10 mg) were subjected
to a mild methanolysis in 0.5 ml of 0.1 M methanolic HCl
at room temperature overnight. The solvent was removed
under Ar gas. To dried sample was added 0.5 ml of freshly
prepared benzoic anhydride (5% benzoic anhydride and
10% (dimethylamino)pyridine in pyridine). The reaction
was carried out overnight at room temperature, and the
solvent removed again under Ar. The sample was dissolved
in EtOAc and subjected to preparative TLC (Si 60, hex-
ane–EtOAc, 1:2). The perbenzylated glycoside was eluted
from the TLC plates, desorbed with EtOAc, filtered, and
evaporated to dryness.
The liquid chromatograph was the semipreparative Gra-
dient LC System G-1 (Shimadzu, Kyoto, Japan) with two
LC-6A pumps (0.5 ml/min), an SCL-6A system controller,
an SPD ultraviolet detector an SIL-1A sample injector and
a C-R3A data processor, with preparative column Supelco-
sil LC-Si HPLC column 5 lm particle size, length · ID
25 cm · 21.2 mm and/or discovery C18 HPLC column
(5 lm particle size, length · ID 25 cm · 21.2 mm). UV
detection at 487 nm.
3.4. LC–MS/APCI
HPLC equipment consisted of a 1090 Win system, PV5
ternary pump and automatic injector (HP 1090 series, Agi-
lent, USA) and two Hichrom columns HIRPB-250AM
250 · 2.1 mm ID, 5 lm particle size, in series. This setup
provided us with a high-efficiency column – approximately
ꢁ26,000 plates/250 mm. A quadrupole mass spectrometer
system Navigator (Finnigan MAT, San Jose, CA, USA)
was used for analysis. The instrument was fitted with an
atmospheric pressure chemical ionization source (vaporizer
temperature 400 ꢁC, capillary heater temperature 250 ꢁC,
corona current 6 lA, sheath gas – high-purity nitrogen,
pressure 0.4 MPa, and auxiliary gas (also nitrogen) flow rate
17 ml/min. Positively charged ions with m/z 100–1700 were
scanned with a scan time of 0.5 s. The whole HPLC flow
(0.4 ml/min) was introduced into the APCI source without
any splitting. Astaxanthin diglucoside diesters were sepa-
rated using a gradient solvent program with acetonitrile
(ACN), dichloromethane (DCM) and methanol (MeOH)
as follows: initial ACN/MeOH/DCM (50:25:25, vol/vol/
vol); linear from 500 s to 3500 s ACN/MeOH/DCM
25:50:25, vol/vol/vol); held until 3600 s; the composition
was returned to the initial conditions over 300 s. A peak
threshold of 0.05% intensity was applied to the mass spectra.
Data acquisition and analyses were performed using PC
with MassLab 2.0 for Windows XP applications/operating
software.
3.2.1. Preparation of di-[(6-O-oleoyl-b-D-
glucopyranosyl)oxy]-astaxanthin
To a stirred suspension of astaxanthin (1) (238.4 mg,
400 lmol, mw 596), b-D-glucopyranosyl bromide tetra-
benzoate 2 (791 mg, 1.2 mmol, mw 659), and powdered
˚
molecular sieve 4 A (2 g) in dry CH2Cl2 (10 ml) was added
silver trifluormethanesulfonate (AgOTf) (411.2 mg, 1.6
mmol, mw 257) at 0 ꢁC. After being stirred at 0 ꢁC for
2 h, the reaction mixture gave a residue, which was purified
by HPLC (CH2Cl2–MeOH, 99:1) to yield the octabenzoate
3 (217.2 mg, 124 lmol, 31%, mw 1752) as a pale yellow
solid. HRFABMS (m/z) 1753.7101 [M+H]+, calcd. for
[C108H104O22+H]+ 1753.7097.
To a solution of the mixture of octabenzoate 3 (200 mg,
114.2 lmol) in MeOH (10 ml) was added NaOMe (1 M in
MeOH; 150 ll, 150 lmol) and the mixture was stirred at
room temperature for 40 min. To this mixture was added
Dowex 50W-X8 (50-100 mesh; H+) (800 mg) and stirring
continued at room temperature for a further 30 min. After
Dowex was filtered off, the filtrate was evaporated to give a
residue which was purified by HPLC (CH2Cl2–MeOH, 9:1)
to yield the diglucoside 4 (97.6 mg, 106 lmol, 93%) as a yel-
low foam. HRFABMS (m/z) 921.5004 [M+H]+, calcd. for
[C52H72O14+H]+ 921.5000, negative FABMS m/z 919
[MꢀH]ꢀ, m/z 757 [MꢀHꢀ162]ꢀ and m/z 595
[MꢀHꢀ162ꢀ162]ꢀ; UV kmax (MeOH) nm (loge) 487
(4.91), 299 (4.11), 252 (4.25). NMR data see Table 3.
A solution of the oleoyl chloride 5 (60.2 mg, 200 lmol,
mw 301) in CH2Cl2 (1 ml) was added to a solution of the
diglucoside 4 (80 mg, 87 lmol) and pyridine–CH2Cl2
(3 ml, 1:1). After being stirred at room temperature for
30 min, the reaction mixture was diluted with ethyl acetate,
1
The compounds were dissolved in CD3OD and the H
and 13C NMR spectra were measured using a Bruker
AMX 500 spectrometer (Bruker Analytik, Karlsruhe, Ger-
many) at 500.1 MHz (1H) and 125.7 MHz (13C). Circular
dichroism (CD) measurement was carried out under dry
N2 on a Jasco-500A spectropolarimeter at 24 ꢁC.
High- and also low-resolution MS were recorded using a
VG 7070E-HF spectrometer (70 eV). HRFABMS (positive

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matrix.
Kobayashi, M., Okada, T., 2000. Protective role of astaxanthin against
UV-B irradiation in the green alga Haematococcus pluvialis. Biotech-
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