Identification of very long chain unsaturated fatty acids from Ximenia oil by atmospheric pressure chemical ionization liquid chromatography-mass spectroscopy

Article abstract of DOI:10.1016/j.phytochem.2006.11.034

A method is described for the enrichment of very long chain unsaturated fatty acids from total fatty acids of Ximenia oil and their identification as picolinyl esters by means of liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (LC-MS/APCI). The method is based on the use of preparative reversed phase HPLC and their subsequent identification by microbore LC-MS/APCI. The combination of these two techniques was used to identify unusual unsaturated VLCFAs up to tetracontenoic acid. All four positional isomers of tetratriacontenoic acid were also synthesized to unambiguously confirm their structure.

Full text of DOI:10.1016/j.phytochem.2006.11.034

PHYTOCHEMISTRY
Phytochemistry 68 (2007) 925–934
www.elsevier.com/locate/phytochem
Identification of very long chain unsaturated fatty acids from
Ximenia oil by atmospheric pressure chemical ionization
liquid chromatography–mass spectroscopy
ˇ
*
Tom a´ sˇ Rezanka , Karel Sigler
Institute of Microbiology, V ı´ de nˇ sk a´ 1083, Prague 142 20, Czech Republic
Received 18 September 2006; received in revised form 28 November 2006
Available online 16 January 2007
Abstract
A method is described for the enrichment of very long chain unsaturated fatty acids from total fatty acids of Ximenia oil and their
identification as picolinyl esters by means of liquid chromatography–mass spectrometry with atmospheric pressure chemical ionization
(
LC–MS/APCI). The method is based on the use of preparative reversed phase HPLC and their subsequent identification by microbore
LC–MS/APCI. The combination of these two techniques was used to identify unusual unsaturated VLCFAs up to tetracontenoic acid.
All four positional isomers of tetratriacontenoic acid were also synthesized to unambiguously confirm their structure.
ꢀ
2006 Elsevier Ltd. All rights reserved.
Keywords: Ximenia oil; Very long chain unsaturated fatty acids; Liquid chromatography–mass spectrometry–atmospheric pressure chemical ionization
1
. Introduction
to bright orange-scarlet. The edible pulp clings to a stone,
which contains an oil-rich, soft cream-colored seed.
Fatty acids with more than 22 carbon atoms (very long
chain fatty acids, VLCFA) are found only rarely in nature
Smith, 1970; Rezanka, 1989). Their plant sources include,
e.g., oils from seeds of plants from the Oleacaceae family,
which are also known as a rich source of unsaturated fatty
acids, whether acetylenic or monoenoic (Badami and Patil,
Ximenia is a key part of native African medicine. The
fruits, rich in vitamin C, form an important component
of the diet: eaten raw, made into jams, jellies, or syrup.
The seed oil is edible and used in cooking. However, its
principal use is as an emollient, hair oil, conditioner and
skin softener, in soap manufacture, and as component of
lipsticks and lubricants. The roots, bark and leaves are
used for medicinal purposes; the bark is used to treat tooth-
ache, mouth infections and stomach aches.
The seed fats of Ximenia caffra, X. caffra var.natalensis,
and X. americana var. microphylla were found to contain
large proportions of monoenoic acids of the hexacosenoic
(ximenic), octacosenoic, and triacontenoic (lumequic) ser-
ies (Ligthelm et al., 1954). X. americana was later recorded
to contain octadec-11-en-9-ynoic acid, named ximenynic
(santalbic) acid as well as icosenoic-triacontenoic acids,
all of which belong to the x-9 series (Mikolajczak et al.,
(
1
980). The degree of unsaturation of these compounds var-
ies depending on their source, but alkenoic VLCFA gener-
ally have the Z stereochemistry. Important representatives
of this family are plants of the genus Ximenia.
Ximenia americana (also known as seaside plum, wild
plum, sour plum, wild olive, monkey plum or hog plum)
is a spiny, deciduous shrub or small tree growing in south-
ern Africa and South America in low altitude areas, wood-
land and across grassy savannahs, in coastal areas or on
riverbanks. The species is drought resistant. The fruits
are similar in shape and size to a plum, dark reddish-brown
1
963).
Surprisingly, no identification of monoenoic VLCFA
was done by those authors who studied Ximenia seed oil
*
Corresponding author. Tel.: +420 241 062 300; fax: +420 241 062 347.
ˇ
E-mail address: rezanka@biomed.cas.cz (T. Rezanka).
0
031-9422/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2006.11.034

ˇ
T. Rezanka, K. Sigler / Phytochemistry 68 (2007) 925–934
926
(
Rovesti, 1979; Fatope et al., 2000; Eromosele and Eromo-
Since no monoenoic VLCFA is commercially available,
we synthesized all positional isomers of tetratriacontenoic
acid, presumed to be present in Ximenia oil, for complete
confirmation of the structure. A number of different proce-
dures have been described for the synthesis of monoenoic
VLCFA. We chose previously described syntheses that
make use of the Wittig reaction that produces Z-alkeno-
ates, and also an alternative method utilizing reactions
between x-iodo esters and complexes formed from methyl-
copper (1) and Grignard reagents.
Based on our previous experience with semi-preparative
RP–HPLC (Rezanka, 2002; Rezanka and Sigler, 2006) we
have now used its preparative mode for the rapid enrich-
ment of the VLCFA fraction with minimal losses. The
presence of VLCFAs in Ximenia oil was demonstrated by
a newly developed method, i.e. identification of picolinyl
esters of the VLCFAs by means of LC–MS/APCI. The
present study describes also the synthesis of four positional
isomers of dotriacontenoic acid.
sele, 2002; Khumalo et al., 2002).
Other seeds have been found to contain monoenoic
VLCFA (Rezanka, 1989). Also, 17-hexacosenoic, 21-hexa-
cosenoic, and 23-octacosenoic acids were identified in Chil-
ean hazelnut (Gevuina avellana) seed oil (Aitzetm u¨ ller,
2
004), major 15-tetracosenoic and 17-hexacosenoic acids
with minor octacosenoic acid in Tropaeolum speciosum
Litchfield, 1970), 15-tetracosenoic, 17-hexacosenoic, and
octacosenoic acids in T. majus (Carlson and Kleiman,
993), triacontenoic acid in Curupira tefeensis (Spitzer
(
1
et al., 1990); 8-dotriacontenoic acid with unusual position
of a double bond was identified in the plant Centella asiat-
ova (Srivastava and Shukla, 1996). Very long chain mono-
unsaturated fatty acids, including 19-octacosenoic and 23-
dotriacontenoic acids, were identified in the alga Botryo-
coccus collected in Lake Shira (Siberia, Russia) (Kalacheva
et al., 2002).
In previous papers (Rezanka, 2002; Rezanka and Sigler,
2
006) we published a method suitable for enriching mix-
tures of natural fatty acids. This method is based on RP–
HPLC, which could be used not only for enrichment of total
fatty acids with VLCFAs but also for separation of individ-
ual compounds. The individual components were resolved
by mobile phases based on methanol with a small amount
of triethylamine in water. A column with phases based on
C8/C18 multi-alkyl bonding and exhaustive endcapping
minimizes the use of organic modifiers. The technique used
for identification in this paper, i.e. APCI, avoids the dis-
crimination against higher homologues, which increases
with increasing number of carbons and double bonds (Rez-
anka, 2000a,b; Rezanka and Votruba, 2005).
2. Results and discussion
The RP–HPLC method is very useful for both analytical
and preparative purposes. In an analytical mode and in
combination with soft ionization mass spectrometry, this
method gives excellent results, as has been repeatedly docu-
mented in our studies (Rezanka, 2002; Rezanka and Sigler,
2006). As mentioned above, the fatty acids from Ximenia oil
have been identified and quantified by gas chromatography
many times. One wonders, however, why the analyses done
in the last century have not been repeated in the last 40
1
8:1
7950
5950
3950
1950
1
8:2
28:1
26:1
30:1
24:1
26:0
18:0
20:1
2
2:1
1
7:1
1
6:1
2
4:0
1
8:3
19:1
9:0
20:0
21:1
28:0
29:1
32:1
2
2:0
34:1
1
6:0
25:1
27:1
36:1
1
23:1
-
50
100
1
1300
1500
1700
1900
2100
2300
2500
time (s)
Fig. 1. LC–MS/APCI chromatogram of Ximenia oil. Peak identification, see Table 1.

ˇ
T. Rezanka, K. Sigler / Phytochemistry 68 (2007) 925–934
927
years. This can be explained by the fact that, in recent years,
Table 1
Total fatty acids from Ximenia oil
all researchers have concentrated on identifying fatty acids
with a triple bond (and/or a hydroxyl) in the molecule, com-
pletely neglecting the identification of VLCFA. In view of
our long-term research concerning the presence of VLCFA
in natural materials, we decided to fill this gap. Long-chain
FA having double bond(s) tend to be thermally degraded
during GC analysis (Rezanka, 1990; Rezanka and Mares,
a
Acid
Ratio of positional isomers in %
Total %
9
5
n ꢀ 7
n ꢀ 9
D
D
1
6:1
7
93
0
0
1.88
0.43
16:0
18:3n ꢀ 3
–
5
–
6
–
95
–
–
–
–
–
0.42
0.09
21.34
32.51
2.54
0.11
0.12
0.57
1
1
1
1
7:1
0
0
0
0
0
0
0
0
8:2n ꢀ 6
1
991). The problems associated with the separation and
b
8:1
94
identification of VLCFA by gas chromatography, i.e. the
thermal degradation under the conditions used for the elu-
tion from a GC column, and with their low proportion in
the analyzed materials can be solved by using RP–HPLC,
which eliminates the risk of thermal degradation, semipre-
parative RP–HPLC making it possible to enrich the FA
fraction above 28:0. Identification and quantification can
then be performed with fatty acid picolinyl esters by using
LC–MS/APCI.
Although higher homologues are present in minor
amounts, we succeeded in identifying in Ximenia oil FA
up to tetracontenoic acid, including the position of the
double bond. Fig. 1 shows the chromatogram of total
FA from Ximenia oil up to 36:1 and the data are also given
in Table 1. In contrast to previous studies (Rovesti, 1979;
Fatope et al., 2000; Eromosele and Eromosele, 2002;
Khumalo et al., 2002) we found the oil to contain both
odd numbered monoenoic FA and VLCFA with chain
length C34 and C36 that have not yet been identified in
the oil. The formation of higher homologues of VLCFA
drops dramatically with increasing chain elongation but
the FA 40:1 is still detectable even though it constitutes
only 0.0033% of total fatty acids. The enrichment of the
individual fractions with VLCFA enabled us to identify
previously unknown homologues. The total ion current
was used for quantification, as shown also in Fig. 1. Table
8:0
19:1
19:0
6
5
94
95
2
2
2
2
0:1
0:0
1:1
2:1
0.23
7
6
93
94
0
0
0
0
2.43
2.27
22:0
23:1
0.67
5
8
95
92
0
0
0
0
0.18
4.68
2
2
2
2
4:1
4:0
5:1
6:1
1.16
8
10
92
90
0
0
0
0
0.32
6.77
26:0
27:1
3.84
9
10
91
90
0
0
0
0
0.27
9.68
2
2
2
3
30:0
31:1
8:1
8:0
9:1
0:1
0.97
11
12
89
85
0
3
0
0
0.096
5.355
0.023
0.054
0.891
0.033
0.027
0.003
0.114
0.017
0.014
0.032
0.012
0.013
0.003
c
14
15
84
80
2
5
0
0
32:1
32:0
33:1
33:0
c
c
c
14
80
6
0
34:1
34:0
15
17
15
12
16
19
7
65
62
58
61
57
52
93
7
8
9
10
8
13
13
18
17
19
18
0
c
c
3
3
3
3
5:1
6:1
7:1
8:1
c
c
c
11
0
2
shows the percent representation of VLCFA in the
40:1
enriched complex of fatty acids of Ximenia oil. The fatty
acids were monounsaturated from 29:1 up to 40:1 and sat-
urated, odd and even numbered with a straight chain.
When analyzing monoenoic acids in the form of picoli-
nyl derivatives, analysis of mass spectra revealed that most
of the chromatographic peaks were not homogeneous but
were formed by several positional isomers. An example
of analysis of positional isomers is given for peak 34:1 of
the original mixture, which constituted 0.114% of total
fatty acids. Analysis of spectra led to the preliminary con-
clusion that the peak contains four (cf. Fig. 2) positional
isomers of tetratriacontenoic acid in an approximate ratio
a
The ratio of positional isomers, determined solely from the detector
response to each one of the compounds and from the mass spectra of the
respective picolinyl esters.
b
9
In this case n ꢀ 9 and D positional isomer are identical, and represent
oleic acid.
c
Determined only after sample enrichment, see Section 3.
less conspicuous. In addition, six positional isomers of 20:1
acid and five positional isomers of 22:1 acid were found.
Chemical ionization tandem mass spectrometry was used
to isolate FA from different sources and localize the posi-
tion of the double bonds in their molecules; among these
FA were, e.g., 15–24:1 and 17–24:1 acids and also their
lower homologues (Michaud et al., 2002).
We synthesized esters 1–4 to confirm the structure of all
four presumed positional isomers. Methyl (Z)-tetratria-
cont-27-enoate (1) and methyl (Z)-tetratriacont-25-enoate
(2) were synthesized as depicted in Schemes 1 and 2, respec-
tively. The key reaction in both cases was the Grignard
reaction performed in the presence of cuprous iodide. When
synthesizing acid 1, we did not succeed in finding an
5
9
25
27
of 13:7:65:15, their sequence being D , D , D , and D .
The presence of multiple positional isomers of monoenoic
acids in natural samples has been described several times
(
Rezanka, 1989, 2002). An illustrative example is the GC
analysis of the 24:1 concentrate from flathead flounder lip-
ids that showed four peaks having different ECL values
(
2
Ota et al., 1995). These peaks agreed with 11–24:1, 13–
4:1, 15–24:1 and 17–24:1 isomers. The 24:1 isomers were
dominated by 15–24:1 (83%), the other isomers being much

ˇ
T. Rezanka, K. Sigler / Phytochemistry 68 (2007) 925–934
928
available supplier of x-bromostearic acid (11) and had to
synthesize it according to the literature (Kling et al., 1993)
from two commercially available compounds (7 and 9),
again by using Grignard reaction in the presence of cuprous
iodide. The subsequent reaction step, i.e. the joining of two
long chains (compounds 6 and 12) was analogous to that
described above. In the case of acid 2, both long chain com-
pounds (i.e. 13 and 14) were commercially available.
A different route (Scheme 3), based essentially on the
study of Hahn et al. (1988), was followed for the synthesis
of methyl (Z)-tetratriacont-9-enoate (3) and (Z)-tetratria-
cont-5-enoate (4). The key step for the synthesis of both
esters (Scheme 3) was Wittig reaction of nonacosanal or
pentacosanal with triphenylphosphonium bromides (22a
or 23a, respectively), affording the Z-configured esters 3
and 4. Since the compounds having suitable position of
As seen from Tables 2–5, the spectra of all four posi-
tional isomers are virtually identical and, based on analysis
of the spectra, we can only state that positional isomers
cannot be distinguished solely by NMR. The isomeric pur-
ity of the esters 1–4 was determined using C NMR spec-
troscopy (Table 2). Allylic carbons of straight chain Z-
alkenes give rise to NMR signals in the range d 27.2–
27.4, while those of the corresponding E-alkenes appear
in the range d 32.6–32.9.
1
3
As we published earlier, the APCI mass spectra of the
picolinyl esters of straight chain saturated fatty acids con-
tain ions from the lower part of mass spectra in minor con-
centrations – see also Fig. 3. An entirely different situation
+
arises in the M region, which features two abundant ions
+
+
[Mꢀ1] and [M+1] including the generation of adducts
+
with the mobile phase such as [M+40] , (M + C H N)
2
2
+
(
Z) double bond and a chain length of 25 and 29 carbon
and [M+54] (M + C H N). In all investigated VLCFA
3 4
spectra, the ion [M+H] is the most abundant one. Scheme
4
+
atoms are not commercially available, we used a different
reaction sequence illustrated in Scheme 3. Both aldehydes
needed for the Wittig reaction were prepared by a reduc-
tion of commercially available acids. x-Bromo acids were
also purchased (9-bromononanoic acid (21) from Matrix
Scientific, Columbia, USA).
With monoenoic derivatives (34:1, see Fig. 3) the detec-
tion revealed also a minority group of ions with m/z 93,
109, 151 and 164. The same spectrum features ions that dif-
fer from others by a value of 14 Daltons, as well as other
Table 2
1
H NMR spectral data of the methyl esters 1–4
CH
3
CH
2
3-H
2
–CH
2
–C@
CH
2
COO
OCH
3
–CH@
1
2
3
4
0.89, t, 6.2
0.89, t, 6.3
0.88, t, 6.5
0.88, t, 6.4
1.23 m
1.24 m
1.25 m
1.24 m
1.62, m
1.61, m
1.61, m
1.60, m
2.02, m
2.01, m
2.00, m
2.03, m
2.29, t, 7.3
2.30, t, 7.2
2.31, t, 7.4
2.32, t, 7.3
3.64, s
3.65, s
3.64, s
3.65, s
5.35, m
5.34, m
5.36, m
5.34, m
32:1
4
3
2
1
0000
0000
0000
0000
0
3
0:1
x 10
34:1
3
0:0
1:1
3
32:0
33:1
33:0
36:1
2
9:1
31:0
34:0 35:1
37:1
38:1
40:1
1000
1200
1400
1600
1800
2000
time (s)
Fig. 2. LC–MS/APCI chromatogram of enriched fraction after preparative RP–HPLC. Peak identification, see Table 1.

ˇ
T. Rezanka, K. Sigler / Phytochemistry 68 (2007) 925–934
929
Br
I
COOCH3
COOCH3
(
CH2)10
7
Br
(
CH2)5
(CH2)10
9
8
COOCH3
(CH2)15
1
0
Br
I
COOCH
3
(
CH2)
(CH )
OH
Br
5
5
2 7
(CH2)17
1
1
(
CH2)
(CH )
2 7
COOCH
3
5
6
(
CH2)17
1
2
COOCH
3
(
CH2)5
(CH )
2 25
1
Scheme 1.
abundant ions with a gap of 26 Daltons (i.e. ions at m/z
Chromatography of the residual oil, with light petroleum
as eluent gave the title bromide as a colorless oil (285 mg,
+
+
+
1
78, 204 and 428). The ions [MꢀH] , [M+H] , [M+40]
+ +
+
+
and [M+54] were also very abundant in the M region.
Semiquantification of all positional isomers (i.e. D , D ,
94%); EIMS (m/z) 304 [M] , 302 [M] , 150, 148, 111, 97,
5
9
1
83, 69, 55; H NMR d 0.89 (3H, t, J = 6.6, CH ), 1.65
3
2
5
27
D , and D ) was based on counting the total abundance
of all above-mentioned ions.
(24H, m, CH ), 3.32 (2H, t, J = 6.5, CH Br), 5.25 (2H,
3
2
m, CH).
However, although the separation efficiency of the two
columns (2 · 250 mm) was estimated at about 100,000
theoretical plates per meter, it was insufficient for the
full separation of all positional isomers of monoenoic
VLCFA.
The combination of semipreparative RP–HPLC and
microbore LC–MS thus extends the analytical possibilities
of both methods and contributes to the acquisition of new
information on biological materials.
Methyl 10-iododecanoate (8). A solution of the com-
mercially obtained (7) (180 mg, 0.65 mmol) and sodium
iodide (300 mg, 2 mmol) in acetone (10 ml) was heated
under reflux for 16 h and then cooled to room temperature
and poured onto water (200 ml). The mixture was extracted
with dichloromethane and the combined extracts were
washed with 10% aqueous sodium thiosulfate, dried, and
concentrated under reduced pressure. Chromatography of
the residual oil, with light petroleum as eluent, gave the
title iodide. It was obtained as a colorless oil (189 mg,
+
1
90%), EIMS (m/z) 326 [M] , 294, 265, 247, 155;
H
3
. Experimental
NMR d 1.60 (14H, m, CH ), 2.31 (2H, m, CH CO), 3.18
2
2
(
2H, t, J = 6.5, CH I), 3.67 (3H, s, OCH ).
2
3
3
.1. Standards and isolation
Methyl octadec-17-enoate (10). This compound, pre-
pared from the iodo ester (8) (144 mg, 0.44 mmol) and bro-
mide (9) (163 mg, 0.92 mmol) as described for the synthesis
of the ester 1, was obtained as a colorless oil (102 mg, 78%),
Standards of fatty acids were prepared as described
below. All solvents were double-distilled and degassed
before use. All chemicals except for x-bromononanoic acid
were obtained from Sigma–Aldrich, Prague. Ximenia oil was
purchased from Augustus Oils Ltd. Hampshire, England.
+
m.p. 24–26 ꢁC; EIMS (m/z) 296 [M] , 265, 264, 222, 87, 74
1
(base peak); H NMR d 1.3–1.4 (26H, m, CH ), 2.12 (2H,
2
m, CH COO), 2.17 (2H, m, CH CH), 3.63 (3H, s, OCH ),
2
2
3
(
Z)-Hexadec-9-enyl bromide (6). Carbon tetrabromide
4.96 (1H, dd, J = 1.8, 9.2, cis-H C = C), 5.09 (1H, dd,
2
(
332 mg, 1 mmol) was added in small portions to a solution
J = 1.8, 15.3, trans-H C = C), 5.81 (1H, ddt, J = 15.3,
2
of the alcohol (5) (240 mg, 1 mmol) and triphenylphos-
phine (265 mg, 1.01 mmol) in dichloromethane (3 ml),
cooled in ice. The mixture was stirred at room temperature
for 16 h and then concentrated under reduced pressure.
The residual solid was extracted with light petroleum and
the extracts were concentrated under reduced pressure.
9.2, 6.4, H C = CH).
2
Methyl 18-bromooctadecanoate (11). Hydrogen bro-
mide gas was passed through a solution of acid (10)
(102 mg, 0.34 mmol) and bis-azoisobutyronitrile (ca.
5 mg) in light petroleum (10 ml), for 15 min whilst the mix-
ture was illuminated with a 300 W UV lamp. The mixture

ˇ
T. Rezanka, K. Sigler / Phytochemistry 68 (2007) 925–934
930
Br
COOH
COOH
(
2 15
CH )
1
4
I
(
CH
2
)
15
1
5
I
COOCH3
(
CH2)15
16
(
CH2)
(CH
2
)
7
Br
7
13
COOCH3
(
CH
2
)
7
(CH
2
)
23
2
Scheme 2.
COOH
was irradiated for a further 15 min and then cooled to
(
CH2)y
ꢀ10 ꢁC. The resulting precipitate was filtered off and
1
1
7 y = 23
8 y = 27
recrystallized from light petroleum to give the title bromide
BrH C
COOH
2
(CH2)x
1
(
11) as colorless crystals (77 mg, 59%), m.p. 36–37 ꢁC; H
2
1 x = 7
NMR d 1.45 (30H, m, CH ), 2.28 (2H, m, CH COO),
2
2
CHO
BrH
2
C
COOCH
3
3
.38 (2H, t, J = 6.5, CH Br), 3.65 (3H, s, OCH ); EIMS
2 3
+
(
CH2)
x
(
CH2)
+
y
19 y = 23
0 y = 27
(m/z) 378 [M] , 376 [M] , 346, 344, 296, 264, 199, 143,
2
2
2 x = 7
3 x = 3
2
87, 74 (base peak).
Methyl 18-iodooctadecanoate (12). This compound,
prepared from the bromo ester (11) (75 mg, 0.20 mmol)
as described for the synthesis of the iodide (8), was
obtained as colorless crystals after recrystallization from
COOCH3
(
CH
2
)
y
(CH
2
)
x
1
methanol (59 mg, 70%), m.p. 44–45 ꢁC; H NMR d 1.3–
3
4
x = 7, y = 23
x = 3, y = 27
1
.5 (26H, m, CH ), 2.30 (2H, m, CH COO), 3.23 (2H, t,
2 2
J = 6.4, CH I), 3.68 (3H, s, OCH ); EIMS (m/z) 424
2
3
+
Scheme 3.
[M] , 392, 293, 265, 247, 155.
Table 3
1
3
C NMR spectral data of the methyl esters 1–4
x-1 C
x-2 C
C-3
–CH
2
–C@
CH
2
x-3 C
C-2
OCH
3
–CH@
C-1
1
2
3
4
14.3
14.4
14.2
14.3
22.8
22.9
22.7
22.8
25.0
25.1
24.9
24.9
27.2
27.2
27.1
27.0
29.0–30.0
29.0–30.0
29.0–30.0
29.0–30.0
32.0
32.1
31.9
32.0
34.0
33.9
34.1
34.0
51.6
51.5
51.5
51.6
129.9
129.8
130.0
130.1
174.5
174.5
174.4
174.3
Table 4
1
H NMR spectral data of the picolinyl esters 1a–4a
0
0
0
0
CH CH
3
2
CH
2
CON
NCH
2
–CH@
H-5
H-4
H-6
H-2
1a
2a
3a
4a
0.91, t, 6.8
0.90, t, 6.8
0.89, t, 6.8
0.90, t, 6.8
1.20–1.44, m
1.21–1.43, m
1.20–1.47, m
1.22–1.44, m
2.23, m
2.21, m
2.22, m
2.21, m
5.10, m
5.09, m
5.08, m
5.09, m
5.30, m
5.31, m
5.30, m
5.29, m
7.14, m
7.15, m
7.14, m
7.16, m
7.50, m
7.51, m
7.49, m
7.51, m
8.35, m
8.37, m
8.35, m
8.36, m
8.40, m
8.41, m
8.39, m
8.41, m

ˇ
T. Rezanka, K. Sigler / Phytochemistry 68 (2007) 925–934
931
Table 5
1
3
C NMR spectral data of the picolinyl esters 1a–4a
0
0
0
0
0
x-1
x-2
C-3
CH
2
–C=
CH
2
x-3
C-2
–CH@
C-1
NCH
2
H-5
H-4
H-6
H-2
H-3
1
2
3
4
a
a
a
a
13.2
13.0
13.0
13.1
19.9
19.8
20.0
20.0
24.6
24.6
24.7
24.8
27.2
27.2
27.1
27.0
28.9–29.7
28.9–29.7
28.9–29.7
28.9–29.7
32.0
32.1
31.9
31.9
34.4
34.3
34.3
34.2
129.9
129.8
130.0
130.1
174.1
174.2
174.2
174.1
65.4
65.5
65.6
65.5
124.5
124.6
124.4
124.5
135.8
135.9
135.8
135.7
147.8
148.1
148.0
147.9
147.3
147.3
147.4
147.5
136.4
136.3
136.5
136.4
Methyl (Z)-tetratriacont-27-enoate (1). A solution of
methyllithium in ether (1.6 mol/l; 0.25 ml, 0.4 mmol) was
added slowly to a suspension of cuprous iodide (76 mg,
reduced pressure. The yield was obtained as colorless crys-
tals after recrystallization from methanol (276 mg, 82%),
+
m.p. 37.5–38 ꢁC; EIMS m/z 396 [M] , 364, 259, 226, 208;
1
0
.4 mmol) in tetrahydrofuran (5 ml), while the temperature
H NMR d 1.62 (26 H, m, CH ), 2.31 (2H, m, CH COO),
2
2
was maintained between ꢀ60 and ꢀ78 ꢁC. The resultant
mixture was stirred at ꢀ78 ꢁC for 1 h after which it was
slowly allowed to warm to 0 ꢁC, whereupon a bright yellow
suspension formed. The mixture was immediately cooled to
3.22 (2H, t, J = 6.5, CH I), 3.70 (3H, s, OCH ).
2
3
Methyl (Z)-tetratriacont-25-enoate (2). This compound,
prepared from the oleyl bromide (13) (450 mg, 1.36 mmol)
and the x-iodo ester 16 (270 mg, 0.68 mmol) as described
for the synthesis of the ester (1), was obtained as a colorless
ꢀ
78 ꢁC and a solution of magnesium bromide [formed by
ꢀ
1
the addition of bromide (6) (85 mg, 0.28 mmol) to magne-
sium (24.3 mg, 1 mmol) in tetrahydrofuran (5 ml), under an
atmosphere of nitrogen] was added, while the temperature
was maintained below ꢀ60 ꢁC. The mixture thus obtained
was stirred at ꢀ78 ꢁC for 1 h and then allowed to warm to
wax (52 mg, 14.6%), m.p. 45.5–46 ꢁC; IR (cm ) 3004,
2928, 2848, 1742, 1650, 1468, 1172; EIMS m/z 521
+
+
[M+l] , 520 [M] , 489, 488, 446, 415, 97, 83, 74 (base
+
peak), 69, 57, 55; HREIMS m/z 520.5217 [M] , calc. for
m/z 520.5219 [C H O ] ; H and C NMR data are
+
1
13
3
5
68
2
0
–10 ꢁC, whereupon a distinct purple coloration appeared.
shown in Tables 2 and 3.
The mixture was then cooled to ꢀ78 ꢁC and a solution of
the x-iodo ester (12) (58 mg, 0.14 mmol) in tetrahydrofu-
ran (15 ml) was added, while the temperature was main-
tained below ꢀ60 ꢁC. That mixture was stirred at ꢀ78 ꢁC
for 1 h and then allowed to warm to room temperature
whereupon it was stirred for 2 h, before being poured into
saturated aqueous ammonium chloride (20 ml). The layers
that formed were separated and the aqueous layer was
extracted with ether. The organic layer and the ether
extracts were combined and the mixture was washed with
brine, dried, and concentrated under pressure. Chromatog-
raphy of the residual oil on silica gel, with ether-light petro-
leum as eluent gave the title ester (1), was obtained as a
Pentacosanal (19). An oven-dried, 100-ml flask, fitted
with a side arm and a vent adapter connected to a mer-
cury bubbler, was flushed with nitrogen and charged with
122 mg (1 mmol) of 9-borabicyclo[3.3.1]nonane (9-BBN)
and pentacosanoic acid (17) (383 mg, 1 mmol) (Chaz
et al., 1987). To this mixture was added 1 ml of THF
and the slurry was stirred at room temperature until
hydrogen evolution ceased. After completing the hydro-
gen evolution, 2.1 ml of 0.5 M solution of Li 9-BBNH
(Brown et al., 1984) (1.05 mmol, 5% excess) in THF was
injected and the reaction mixture was stirred for 1 h at
room temperature. The reaction mixture was hydrolyzed
with 20 ml of water for 1 h at room temperature. The
mixture was then saturated with NaCl and the organic
layer was separated, dried, and subjected to crystalliza-
tion. The yield was colorless crystals (334 mg, 91%),
ꢀ
1
colorless wax (8.6 mg, 12%), m.p. 46–47 ꢁC; IR (cm
)
3
1
4
008, 2924, 2852, 1742, 1650, 1468, 1262, 1172, 1116 and
+
+
014; EIMS (m/z) 521 [M+1] , 520 [M] , 489, 488, 446,
ꢀ
1
1
04, 143, 125, 111, 97, 83, 74, 69, 57, 55 and 43; HREIMS
m.p. 55–57 ꢁC; IR (cm ) 2780 (CHO), 1720 (C@O); H
+
+ 1
m/z 520.5218 [M] , calc. for m/z 520.5219 [C H O ] ; H
and C NMR data are shown in Tables 3 and 4.
6-Iodohexadecanoic acid (15). This compound, pre-
NMR d 0.90 (3H, t, J = 6.8, CH ), 1.00–1.90 (44H, m,
3
5
68
2
3
1
3
CH ), 2.35 (2H, t, J = 7.2, CH CHO), 9.75 (1H, t,
2
2
+
1
J = 1.95, CHO); EIMS m/z 366 [M] , 365, 348, 336,
322. These data are in agreement with the values given
by Marukawa et al. (2001).
pared from the bromo acid (14) (335 mg, 1 mmol) as
described for the synthesis of the iodo derivative (8), was
obtained as colorless crystals (332 mg, 87%), m.p. 71–
Nonacosanal (20). This compound was prepared from
the nonacosanoic acid (18) (439 mg, 1 mmol) as described
for the synthesis of the aldehyde (19). The yield of aldehyde
(20) as colorless crystals was 90% (381 mg), m.p. 84–86 ꢁC;
+
1
7
3 ꢁC; EIMS m/z 382 [M] , 364, 143, 125, 97, 83, 55; H
NMR d 1.60 (26H, m, CH ), 2.37 (2H, m, CH COO),
2
2
3
.21 (2H, t, J = 6.5, CH I).
2
ꢀ1
1
Methyl 16-iodohexadecanoate (16). Iodo acid (15)
325 mg, 0.85 mmol) was added to methanol (20 ml) that
IR (cm ) 2780 (CHO), 1720 (C@O); H NMR d 0.91 (3H,
t, J = 6.8, CH ), 1.00–1.90 (52H, m, CH ), 2.35 (2H, t,
(
3
2
had been pretreated with thionyl chloride (1 ml), and the
mixture was stirred at room temperature for 4 h. The sol-
vent was then removed under reduced pressure and the res-
idue was dissolved in dichloromethane (10 ml). The
resultant solution was washed with saturated aqueous
sodium hydrogen carbonate, dried, and concentrated under
J = 7.2, CH CHO), 9.73 (1H, t, J = 1.8, CHO); EIMS m/
z 422 [M] , 421, 404, 392, 378.
2
+
Methyl 9-bromononanoate (22). Bromoester (22), pre-
pared from the bromoacid (21) (286 mg, 1.21 mmol) as
described for the synthesis of the ester (16), was obtained
1
as a colorless oil (251 mg, 83%). H NMR d 1.3–1.8

ˇ
T. Rezanka, K. Sigler / Phytochemistry 68 (2007) 925–934
932
100
5
98
COO
N
(
CH2)27
(CH )
2 3
4
a
1
78
204
50
1
3
09
6
38
9
1
64
1
78 204
652
1
51
0
5
0
0
0
0
100 150 200 250 300 350 400 450 500 550 600 650 700
m/z
1
1
1
00
5
98
3
a
5
0
1
09
9
3
638
1
64
1
51
234 260
652
0
5
100 150 200 250 300 350 400 450 500 550 600 650 700
m/z
00
5
98
2a
5
0
1
3
09
9
638
6
1
64
1
51
458 484
52
0
5
100 150 200 250 300 350 400 450 500 550 600 650 700
m/z
00
5
98
1a
5
0
1
09
638
9
3
1
64
4
86 512
1
51
652
0
5
100 150 200 250 300 350 400 450 500 550 600 650 700
m/z
Fig. 3. Mass spectra of picolinyl esters of positional isomers of synthetic 34:1 acid. For explanation of values see the text.

ˇ
T. Rezanka, K. Sigler / Phytochemistry 68 (2007) 925–934
933
1
2
3
4
x = 25, y = 5
x = 23, y = 7
x = 7, y = 23
x = 3, y = 27
Methyl (Z)-tetratriacont-5-enoate (4). This compound,
prepared from the bromide (23a) (215 mg, 0.5 mmol) and
aldehyde (20) (106 mg, 0.25 mmol) as described for the syn-
thesis of the ester (4), was obtained as colorless crystals
COOCH
3
(
(
CH
2
)
y
(CH
N
2 x
)
ꢀ
1
t-BuOK,
(74 mg, 57%), m.p. 44–46 ꢁC; IR (cm ) 2995, 2930,
850, 1734, 1465, 1442, 1175; HREIMS m/z 520.5220
OH
2
+
+
1
13
[
M] , calc. for m/z 520.5219 [C H O ] ; H and
C
3
5
68
2
COOCH
2
NMR data are showed in Tables 2 and 3.
CH2)y
(CH )
N
2 x
1
2
3
4
a x = 25, y = 5
Picolinyl esters of 1a–4a. A solution of potassium tert-
butoxide in tetrahydrofuran (0.5 ml, 1.0 M) was added to
nicotinyl alcohol (1 ml). After mixing, the appropriate
fatty acid methyl ester (ꢁ30 mg) in dry dichloromethane
a x = 23, y = 7
a x = 7, y = 23
a x = 3, y = 27
Scheme 4.
(
1 ml) was added, and the mixture was held at 40 ꢁC for
30 min in a closed vial. After cooling to room tempera-
ture, water and hexane were added, and the organic phase
was collected, dried over anhydrous sodium sulfate, and
evaporated.
(
10H, m, CH ), 1.91 (2H, m, CH CH Br) 2.28 (2H, t,
2 2 2
J = 7.4 Hz, CH CO), 3.40 (2H, t, J = 7.3 Hz, CH Br),
3
2
2
+
+
.66 (3H, s, OCH ); EIMS (m/z) 252 [M] , 250 [M] .
1a-4a as colorless, waxy compounds, LC–MS/APCI: m/
3
+
Methyl 5-(triphenylphosphonium bromide) nonanoate
z
598 [M+H] ; HREIMS m/z 597.5486–597.5491
+
+
1
(
22a). A mixture of the bromoester (22) (251 mg, 1 mmol),
triphenylphosphine (524 mg, 2 mmol) and acetonitrile
5 ml) was heated under reflux for 16 h. The resultant mix-
C H NO [M] , calculated for [M] 597.5484; for H
40 71 2
1
3
and C NMR data see Tables 4 and 5.
(
ture was cooled and concentrated under reduced pressure
to afford a colorless solid, which was washed several times
with ethyl acetate and then recrystallized from dichloro-
methane-light petroleum to give the title salt as colorless
3.2. Preparative chromatography
A Chromatospac Prep 100 preparative chromatograph
(Jobin-Yvon, Longjumeau, France) with axial compression
of the chromatographic bed (61.2 · 8 cm ID) was used for
the isolation of the fraction enriched with picolinyl esters.
+
crystals (251 mg, 55%). HREIMS m/z 456.0849 [M] , calc.
+
for [C H BrO P] 456.0854.
2
4
26
2
Methyl 5-(triphenylphosphonium bromide) valerate
The column efficiency was 6510 theoretical plates, V was
0
(
1
(
23a) was prepared from the bromoester (23) (195 mg,
mmol) as described for the synthesis of the bromide
1185 ml and tR of the picolinyl ester of octacosanoic acid
(28:0) was 23.0 min. The mobile phase flow rate was
55 ml/min. The mean particle diameter of the Separon
C18 reversed-phase packing (formerly Laboratorn ´ı p rˇ ´ı s-
troje, Prague, Czechoslovakia) was 15 lm. A Holochrome
H/MD variable-wavelength detector (Gilson, France) with
a 40-ll flow-through cell was set at 235 nm. All experi-
ments were carried out at room temperature.
The amount of 290 mg picolinyl esters (see below) was
dissolved in 15 ml of the mobile phase (injection volume)
(methanol-isopropanol-triethylamine, 87:13:0.05). In the
time interval 0–35 min the column was eluted with mobile
phase that was later replaced with diethyl ether (60 min).
The column was again conditioned with mobile phase for
15 min. The fraction up to 23.0 min was discarded and that
obtained within the interval 23.0–83 min was used for fur-
ther analysis. After evaporation of the mobile phase, the
total yield of a mixture of fatty acids with the chain longer
than 28 carbon atoms was 19.3 mg. These were separated
and identified by LC–MS.
22a). It was obtained as colorless crystals (266 mg, 52%).
+
+
HREIMS m/z 512.1481 [M] , calc. for [C H BrO P]
5
2
8
34
2
12.1479.
Methyl (Z)-tetratriacont-9-enoate (3). A solution of lith-
ium hexamethyldisilazide, generated by the addition of
butyllithium in hexane (1.5 M; 0.34 ml, 0.51 mmol) to hex-
amethyldisilazane (0.11 ml, 0.51 mmol) in tetrahydrofuran
(
(
2 ml) at 0 ꢁC, was added to a suspension of bromide
22a) (236 mg, 0.5 mmol) in tetrahydrofuran-hexamethyl-
phosphoramide (4:1; 3 ml) cooled to 0 ꢁC. The resultant
orange solution was stirred for 10 min at 0 ꢁC and then
cooled to ꢀ78 ꢁC when a solution of the aldehyde (19)
(
92 mg, 0.25 mmol) in THF (3 ml) was added to it at that
temperature. The mixture was subsequently allowed to
warm to 0 ꢁC and was stirred at that temperature for 1 h;
it was then poured into saturated aqueous ammonium
chloride and extracted with ethyl acetate. The combined
organic extracts were washed with water, dried, and con-
centrated under reduced pressure to give oil, which was
chromatographed with hexane as eluent. After reaction
with diazomethane the title ester 3 was obtained as color-
less crystals after recrystallization from acetone (74 mg,
3.3. LC–MS/APCI
HPLC equipment consisted of a 1090 Win system, PV5
ternary pump and automatic injector (HP 1090 series,
Hewlett–Packard, USA) and two Hichrom columns
HIRPB-250AM 250 · 2.1 mm ID, 5 lm phase particle, in
series. This setup provided us with a high-efficiency column
ꢀ
1
5
1
7%), m.p. 44–46 ꢁC; IR (cm ) 2995, 2930, 2850, 1734,
+
465, 1442, 1175; HREIMS m/z 520.5220 [M] , calc. for
+
1
13
m/z 520.5219 [C H O ] ; H and C NMR data are
shown in Tables 2 and 3.
3
5
68
2

ˇ
T. Rezanka, K. Sigler / Phytochemistry 68 (2007) 925–934
934
–
approximately ꢁ26,000 plates/250 mm. A quadrupole
Khumalo, L.W., Majoko, L., Read, J.S., Ncube, I., 2002. Characterization
of some under utilized vegetable oils and their evaluation as starting
materials for lipase-catalyzed production of cocoa butter equivalents.
Ind. Crops Prod. 16, 237–244.
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 tem-
perature 220 ꢁC, corona current 5 lA, sheath gas – high-
purity nitrogen, pressure 0.38 MPa, and auxiliary gas (also
nitrogen) flow rate 15 ml/min). Positively charged ions with
m/z 70–900 were scanned with a scan time of 0.5 s. The
whole HPLC flow (0.37 ml/min) was introduced into the
APCI source without any splitting. Fatty acid picolinyl
esters were separated using a gradient solvent program
with acetonitrile (ACN), dichloromethane (DCM) and pro-
pionitrile (EtCN) as follows: initial ACN/EtCN/DCM
Kling, M.R., Easton, C.J., Poulos, A., 1993. Synthesis of very long-chain
fatty-acid methyl-esters. J. Chem. Soc., Perkin Trans. I, 1183–1189.
Ligthelm, S.P., Horn, H.S., Schwartz, H.M., von Holdt, M.M., 1954. A
chemical study of the fruits of three South African Ximenia species,
with special reference to the kernel oils. J. Sci. Food. Agric. 5, 281–
2
88.
Litchfield, C., 1970. Tropaeolum speciosum seed fat: A rich source of cis-
15-tetracosenoic and cis-17-hexacosenoic acids. Lipids 5, 144–146.
Marukawa, K., Takikawa, H., Mori, K., 2001. Synthesis of the enanti-
omers of some methyl-branched cuticular hydrocarbons of the ant,
Diacamma sp. Biosci. Biotech. Biochem. 65, 305–314.
Michaud, A.L., Diau, G.Y., Abril, R., Brenna, J.T., 2002. Double bond
localization in minor homoallylic fatty acid methyl esters using
acetonitrile chemical ionization tandem mass spectrometry. Anal.
Biochem. 307, 348–360.
(
60:30:10, vol/vol/vol); linear from 10 min to 40 min
ACN/EtCN/DCM 30:40:30, vol/vol/vol); held until
0.5 min; the composition was returned to the initial condi-
Mikolajczak, K.L., Earle, F.R., Wolff, I.A., 1963. The acetylenic acid in
Comandra pallida and Osyris alba seed oil. J. Am. Oil Chem. Soc. 40,
6
tions over 8 min. A peak threshold of 0.3% 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
42–343.
Ota, T., Ando, Y., Nakajima, H., Shibahara, A., 1995. C-20–C-24
monounsaturated fatty-acid isomers in the lipids of flathead
flounder, Hippoglossoides dubios. Comp. Biochem. Physiol. B 111,
1
95–200.
Rezanka, T., 1989. Very-long-chain fatty acids from the animal and plant
kingdoms. Progr. Lipid Res. 28, 147–187.
Acknowledgement
Rezanka, T., 1990. Identification of very long polyenoic acids as picolinyl
esters by Ag ion-exchange high-performance liquid chromatography,
reversed-phase high-performance liquid chromatography. J. Chroma-
togr. 513, 344–348.
This work was supported by the Institutional Research
Concept AV0Z50200510.
Rezanka, T., 2000a. Analysis of very long chain polyunsaturated fatty
acids using high-performance liquid chromatography–atmospheric
pressure chemical ionization mass spectrometry. Biochem. Syst. Ecol.
28, 847–856.
References
Rezanka, T., 2000b. Analysis of polyunsaturated fatty acids using high
performance liquid chromatography–atmospheric pressure chemical
ionization mass spectrometry. J. High Res. Chromatogr. 23, 338–342.
Rezanka, T., 2002. Identification of very long chain fatty acids by
atmospheric pressure chemical ionization liquid chromatography–
mass spectroscopy from green alga Chlorella kessleri. J. Sep. Sci. 25,
1332–1336.
Aitzetm u¨ ller, K., 2004. Chilean hazelnut (Gevuina avellana) seed oil. J.
Am. Oil Chem. Soc. 81, 721–723.
Badami, R.C., Patil, K.B., 1980. Structure and occurrence of unusual fatty
acids in minor seed oils. Progr. Lipid Res. 19, 119–153.
Brown, H.C., Mathew, C.P., Pyun, C., Son, J.C., Yoon, N.M., 1984.
Selective reductions. 36. Reaction of lithium 9-boratabicy-
clo[3.3.1]nonane with selected organic-compounds containing repre-
sentative functional-groups. J. Org. Chem. 49, 3091–3097.
Carlson, K.D., Kleiman, R., 1993. Chemical survey and erucic acid
content of commercial varieties of nasturtium, Tropaeolum majus L. J.
Am. Oil. Chem. Soc. 70, 1145–1148.
Rezanka, T., Mares, P., 1991. Determination of plant triacylglycerols
using capillary gas chromatography, high-performance liquid
chromatography and mass spectrometry. J. Chromatogr. 542,
145–159.
Rezanka, T., Sigler, K., 2006. Identification of very long chain fatty acids
from sugar cane wax by atmospheric pressure chemical ionization
liquid chromatography–mass spectroscopy. Phytochemistry 67, 916–
923.
Chaz, J.S., Kim, J.E., Oh, S.Y., Kim, J.D., 1987. One-pot conversion of
carboxylic acids to aldehydes through treatment of acyloxy-9-borabi-
cyclo[3.3.1]nonanes with lithium 9-boratabicyclo[3.3.1]nonane. Tetra-
hedron Lett. 28, 4575–4578.
Rezanka, T., Votruba, J., 2005. Analysis of fatty acids by APCI–MS. In:
Byrdwell, W.C. (Ed.), Modern Methods for Lipid Analysis by LC–MS
and Related Techniques. AOCS Press, Champaign, IL, USA, pp. 242–
275.
Eromosele, C.O., Eromosele, I.C., 2002. Fatty acid compositions of seed
oils of Haematostaphis barteri and Ximenia americana. Bioresour.
Technol. 82, 303–304.
Fatope, M.O., Adoum, O.A., Takeda, Y., 2000. C-18 acetylenic fatty acids
of Ximenia americana with potential pesticidal activity. J. Agric. Food
Chem. 48, 1872–1874.
Rovesti, P., 1979. Use of Ximenia americana Linnaeus oil and flowers in
cosmetics. Rivista Italiana 61, 190–193.
Smith, C.R., 1970. Occurrence of unusual fatty acids in plants. In:
Holman, R.T. (Ed.), Progress in the Chemistry of Fats and other
Lipids, vol. 11. Pergamon Press, Oxford, pp. 137–177.
Spitzer, V., Marx, F., Maia, J.G.S., Pfeilsticker, K., 1990. Curupira
tefeensis (Olacaceae) – a rich source of very long-chain fatty-acids. Fat
Sci. Technol. 92, 165–168.
Hahn, S., Stoilov, I.L., Ha, T.B.T., Raederstorff, D., Doss, G.A., Li, H.T.,
Djerassi, C., 1988. Biosynthetic-studies of marine lipids. 17. The course
of chain elongation and desaturation in long-chain fatty-acids of
marine sponges. J. Am. Chem. Soc. 110, 8117–8124.
Kalacheva, G.S., Zhila, N.O., Volova, T.G., 2002. Lipid and hydrocarbon
compositions of a collection strain and a wild sample of the green
microalga Botryococcus. Aquat. Ecol. 36, 317–331.
Srivastava, R., Shukla, Y.N., 1996. Some chemical constituents from
Centella asiatica. Ind. Drugs 33, 233–234.