Identification of very long chain fatty acids from sugar cane wax by atmospheric pressure chemical ionization liquid chromatography-mass spectroscopy

Article abstract of DOI:10.1016/j.phytochem.2006.02.014

A method is described for the enrichment of very long chain fatty acids (VLCFAs) from total fatty acids of sugar cane wax 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 of 100 mg amounts and their subsequent identification by microbore APCI LC-MS. The combination of these two techniques was used to identify unusual saturated VLCFAs up to C₅₀.

Full text of DOI:10.1016/j.phytochem.2006.02.014

PHYTOCHEMISTRY
Phytochemistry 67 (2006) 916–923
www.elsevier.com/locate/phytochem
Identification of very long chain fatty acids from sugar cane wax
by atmospheric pressure chemical ionization liquid
chromatography–mass spectroscopy
ˇ
*
´ˇ
Tomas Rezanka , Karel Sigler
Institute of Microbiology, Vı´denˇska´ 1083, Prague 142 20, Czech Republic
Received 1 December 2005; received in revised form 13 February 2006
Available online 29 March 2006
Abstract
A method is described for the enrichment of very long chain fatty acids (VLCFAs) from total fatty acids of sugar cane wax 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 of 100 mg amounts and their subsequent identifi-
cation by microbore APCI LC–MS. The combination of these two techniques was used to identify unusual saturated VLCFAs up to C50.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: Sugar cane wax; Very long chain fatty acids; Liquid chromatography–mass spectrometry – atmospheric pressure chemical ionization
1. Introduction
anka, 1990), which could be used not only for enrichment
of total fatty acids with VLCFAs but also for separation
of individual compounds.
The detection of eluted components is complicated as
fatty acids hardly absorb in the UV region. It is therefore
necessary to either use the absorption of the terminal car-
boxylic group, of the double bond(s), or derivatize this car-
boxylic group.
Picolinyl esters are the only derivatives that can be
detected via UV absorption and, at the same time, by gas
chromatography–mass spectrometry (GC–MS) (Christie
and Stefanov, 1987; Christie, 1998).
Fatty acids are important constituents of waxes, in which
they occur in free and esterified forms. These compounds
are found in animal, plant, and microbial tissues and have
a variety of functions, such as energy stores and waterproof-
ing (Kolattukudy, 1976; Hamilton, 1995). These waxes
contain mostly very long chain fatty acids (VLCFAs), i.e.,
fatty acids with more than 20 carbon atoms. The main
problem in the identification of VLCFAs in natural materi-
als, i.e., in plants, animals and also in microorganisms is
their low concentration (Rezanka, 1989).
In previous papers (Rezanka and Sokolov, 1993; Rez-
anka, 2002), we published methods for enriching mixtures
of natural fatty acids. These methods are based predomi-
nantly on chromatographic techniques, i.e., on thin-layer
chromatography or HPLC, both in reversed phase mode.
The method of choice turned out to be RP-HPLC (Rez-
The individual components were resolved by mobile
phases based on methanol with a small amount of pyridine
in water (Christie and Stefanov, 1987). In our previous
paper (Rezanka, 1990), undesirable interactions between
the picolinyl moiety and the stationary phase were elimi-
nated by triethylamine. A column with phases based on
C8/C18 multi-alkyl bonding and exhaustive endcapping
minimizes the use of organic modifiers. It was also recently
successfully used by Christie (1998), pure acetonitrile being
used as mobile phase. Gas chromatography has recently
*
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 ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2006.02.014

ˇ
T. Rezanka, K. Sigler / Phytochemistry 67 (2006) 916–923
917
been used for the separation of VLCFA (Rezanka, 1989;
Van Pelt et al., 1999). This method is suitable for the anal-
ysis of compounds which are volatile as such or after deriv-
atization. It is known from the literature (Rezanka and
Mares, 1991), that the discrimination of higher homo-
logues increases with increasing number of carbons and
double bonds. The technique used in this paper, i.e., APCI,
avoids this shortcoming (Rezanka, 2000a,b; Rezanka and
Votruba, 2005).
Based on our previous experience with semi-preparative
RP-HPLC (Rezanka, 2002) we have now used its prepara-
tive mode for the rapid enrichment of the VLCFA fraction
with minimal losses. FA mixture containing VLCFAs pre-
pared from the sugar cane wax was utilized to demonstrate
the suitability of the method. The presence of VLCFAs in
this sample was demonstrated by a newly developed
method, i.e., identification of picolinyl esters of the VLC-
FAs by means of LC–MS/APCI.
A natural mixture of higher primary aliphatic acids
(24:0 to 36:0) was obtained from sugar cane (Saccharum
officinarum L.) wax (Gonzales et al., 2002). This product
was found to be responsible for some biological properties
related to health, such as antioxidant (Menendez et al.,
2002) and cholesterol-lowering effects, determined in both
in vivo and in vitro models (Gamez et al., 2000; Rodriguez
et al., 2004), beneficial effects on platelet aggregation in
healthy volunteers (Arruzazabala et al., 2005), as well as
antiplatelet and antithrombotic activities (Molina et al.,
2000; Mas, 2004). The nutritional significance and metabo-
lism of very long chain fatty alcohols and acids from sugar
cane has recently been reviewed (Hargrove et al., 2004). It
was also found that their mixture does not show any evi-
dence of cytotoxic or genotoxic activity on either somatic
or germ cells in rodents (Gamez et al., 2004).
2. Results and discussion
Fatty acids isolated from the sugar cane wax have
already been identified and quantified using a GC method
(Delange et al., 2002; Gonzales et al., 2002; Antolin et al.,
2004). As mentioned above, long chain fatty acids are
extremely sensitive to thermal degradation, i.e., to condi-
tions under which they are eluted from the GC column.
To minimize the possibility of thermal degradation, we
used two HPLC steps; the FA derivatives were first concen-
trated by preparative RP-HPLC (Fig. 1) and individual
molecular types of VLCFA derivatives were separated,
identified, and quantified by LC–MS.
The difficulty in identifying the VLCFA in the whole
complex of fatty acids was eliminated by separating shorter
chain fatty acids (up to C36) by RP-HPLC, i.e., enriching
longer chain FA. Although the formation of higher homo-
logues of VLCFA drops dramatically with proceeding
chain elongation, the C50 is still detectable even though it
constitutes only 0.028% of total fatty acids.
The enrichment of the individual fractions with VLCFA
enabled us to identify as yet unknown homologues (Fig. 2).
The total ion current was used for quantification, as shown
also in Fig. 2.
Table 1 shows the percent representation of VLCFA in
the enriched complex of fatty acids of sugar cane wax. The
fatty acids were saturated from C35 up to C50, odd and even
numbered with a straight chain.
Fig. 1. Preparative RP-HPLC chromatogram of FA derivatives from the sugar cane wax. Peak identification: 36:0 – derivative of the FA with chain length
36 carbon atoms. Broken line, chromatogram after the addition of a standard-picolinyl ester of 36:0 synthetic acid. The fraction within the interval 37.5–
90 min was used for further analysis.

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T. Rezanka, K. Sigler / Phytochemistry 67 (2006) 916–923
918
140
120
100
80
38
x10
46
48
41
60
40
35
50
20
0
2300
2500
2700
2900
3100
3300
3500
3700
3900
time (sec)
Fig. 2. LC–MS/APCI chromatogram of enriched fraction (after preparative RP-HPLC, see Fig. 1). Peak identification: 35 – picolinyl ester of
pentatriacontanoic acid, etc.
In contrast to electron impact ionization (Christie, 1997)
where the mass spectra of the picolinyl esters of straight
chain saturated fatty acids contain ions from the lower part
of mass spectra, i.e., ions m/z 92, 108, 151 and 164, these
ions are in minority in APCI as shown in Fig. 3A (e.g.,
46:0). Even-numbered ions differing by 14 amu and repre-
senting cleavage between methylene groups also have low
intensity. Quite a distinct situation can be found in the
M+ region. Here, we can observe the formation of two
abundant ions [M ꢀ 1]+ and [M + 1]+ including the gener-
ation of adducts with the mobile phase such as [M + 40]+
(M + C2H2N) and [M + 54]+ (M + C3H4N). In all investi-
gated VLCFA spectra, the [M + H]+ ion is the most abun-
dant one. Fig. 3B shows the APCI spectrum of
synthetically prepared 46:0 picolinyl ester; there is obvi-
ously hardly any difference between this compound and
the natural one.
The industrial process of sugar refining results in the
accumulation of by-products. One of them, mill mud, com-
prises crude wax and fats, fiber, sugar, crude protein, and
ash. Typical components of wax are described in US Patent
(Valix, 2004). The members of individual groups exhibit
mostly straight saturated chains. No chains with more than
36 carbon atoms were found in any of the cases mentioned
below. For instance, fatty acids with up to 34 carbon atoms
and hydrocarbons with up to 35 carbon atoms were identi-
fied in different fractions obtained by extraction of sugar
cane wax with different solvents (Nuissier et al., 2002).
Interestingly, in the hydrocarbon fraction pentatriacontane
accounted for 12% of the total alkane content. Alcohols up
to tetratriacontanol have been described in a US patent
(Laguna et al., 1999) and fatty acids up to hexatriaconta-
noic in another US patent (Gonzales et al., 2002). Alde-
hydes with chains of the same length were identified in
two studies (Purcell et al., 2005; Lamberton and Redcliffe,
1960).
Table 1
Total fatty acids from sugar cane wax longer than C34
Fatty acid
Molar% SD (n = 5)
24:0–34:0
35:0
36:0
37:0
38:0
39:0
40:0
41:0
42:0
43:0
44:0
45:0
46:0
47:0
48:0
49:0
50:0
98.723
0.130 0.099
0.398 0.054
0.087 0.023
0.096 0.012
0.073 0.007
0.070 0.031
0.043 0.015
0.025 0.018
0.044 0.017
0.041 0.010
0.038 0.013
0.063 0.021
0.032 0.018
0.050 0.016
0.047 0.009
0.040 0.015
In contrast to these data, we demonstrated two basic
features of sugar cane wax:
(1) Chain length of 36 carbon atoms is not final because,
as seen in Figs. 2 and 3, homologues containing up to
50 carbon atoms have been identified. The reasons for
their not being discovered earlier can include, e.g.,
insufficient sensitivity of the analytical techniques

ˇ
T. Rezanka, K. Sigler / Phytochemistry 67 (2006) 916–923
919
100
75
50
25
0
178
768
108
151
+H
O
N
(CH2)40
92
O
164
766
109
808
93
164
151
822
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
m/z
Fig. 3A. Mass spectrum of picolinyl ester of 46:0 natural acid; the structures of major ions are shown. For explanation of values see the text.
100
768
75
50
109
93
164
25
808
151
822
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
m/z
Fig. 3B. Mass spectrum of picolinyl ester of 46:0 synthetic acid.
used for the purpose (all relevant studies used gas
chromatography). The LC–MS method with APCI
used in our study is much more efficient in identifying
compounds with very long chains. The identification
was also greatly facilitated by the enrichment of the
samples by VLCFAs that we used.

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T. Rezanka, K. Sigler / Phytochemistry 67 (2006) 916–923
920
(2) Table 1 clearly implies that differences in proportion
and, on top of it, permits the use of any soft ionization
method in the mass spectrometer. This method has as yet
been developed for the analysis of relatively saturated com-
pounds but it can be readily extended to compounds with
more than 10 double bonds and a chain of corresponding
length, for which GC is not applicable.
(in molar%) between chains with odd and even num-
bers of carbon atoms are obliterated. Although this
phenomenon has been observed repeatedly (Rezanka,
2002), we have no satisfactory explanation.
Although conventional theory suggests successive chain
elongation C16 and C18 acids as a possible mechanism for
the biosynthesis of this VLCFA, further research into the
pathways of long chain fatty acid biosynthesis in marine
organisms is needed.
Even though the presence of VLCFA in cane wax has
not yet been described, the probability of their detection
is likely to increase due to the advances in physico-chemical
separation and identification methods as well as improve-
ment of methods of sample collection, and also due to
the currently rising interest in higher plants. Like other lip-
ids, waxes and hydrocarbons, very long chain fatty acids
act predominantly as components of the hydrophobic layer
on the surface of leaves, which decreases their wettability. It
can be speculated that they could participate in the forma-
tion of surface structures responsible for the so-called lotus
effect (Furstner et al., 2005; Neinhuis and Barthlott, 1997).
The presence of a homologous series of very long chain
fatty acids in the material used in this study, i.e., cane wax
from Egypt, contrasts with the absence of similar com-
pounds documented by previous studies (Delange et al.,
2002; Gamez et al., 2000). It is difficult to say whether this
striking difference is caused by differences in the geograph-
ical origin of the analyzed cane (Egyptian cane in our case,
Cuban cane in the case of Gonzales et al., 2002), or if it can
be ascribed to different instrumentation and methodology.
The geographical factors might include different types of
climate (dry weather with very low amounts of precipita-
tions in Egypt versus a much more rainy weather in Cuba).
The more abundant precipitations in Cuba could, e.g.,
wash the VLCFA off the leaf surface. Also, due to these
different environmental conditions, the cane cultivars
grown in the two regions might differ in metabolic patters
resulting in different proportions of VLCFA being pro-
duced – plants growing in the arid regions might need these
acids more than those from more rain-exposed localities.
In conclusion, we succeeded in identifying some of the
longest fatty acids found in nature, and extending the
knowledge of the sugar cane wax composition. The combi-
nation of preparative 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.
GC–MS can be recommended as a method of a first
choice for fatty acid analysis. It is well suited for the anal-
ysis of a sample that contains VLCFA with chain lengths
exceeding to common length to a certain degree – to about
C34. On the other hand, the more expensive LC–MS tech-
nique is more convenient when it is necessary to analyze
longer-chain VLCFA. This method may spur further devel-
opment of VCLFA analysis and extend the options in the
exploration of metabolism of recently examined organisms.
When analyzing VLCFA with chain length identified
here in sugar cane wax one should keep in mind that these
compounds are much more like triacylglycerols than fatty
acids. An example is, e.g., the C50 acid that we identified,
when compared with the common plant triacylglycerol 1,3-
dioleoyl-2-palmitoylglycerol (POO). The acid derivative
has 56 carbon atoms, POO has 55 and the degree of unsat-
uration of both compounds is five. The analysis of triacyl-
glycerols and VLCFA therefore poses similar problems (see
below).
High-temperature GC has until recently been used lar-
gely to separate molecular species of simple lipids accord-
ing to the combined chain-lengths of the fatty acid
moieties. It is an analytical technique that is capable of a
high degree of precision, and can be married well with mass
spectrometry. However, the technique may be nearing the
technical limits before pyrolytic breakdown both of sam-
ples and of stationary phases occurs and a greater attention
must be paid to instrumental parameters than with other
types of gas chromatography.
The first problem concerns the injection system – the
injected compound can be very easily degraded. The same
holds for the passage of the compound through the col-
umn, during which both the eluted compound and the sta-
tionary phase may be degraded. Still another vulnerable
point is the entry into the MS and the mass spectrometer
itself. Probably for these reasons the GC–MS has seldom
been used for the analysis of triacylglycerols – see, e.g.,
Buchgraber et al., 2004; Laakso, 2002. As mentioned by
Christie (2003), ‘‘although useful separations of intact tria-
cylglycerols by high-temperature GC can be achieved rou-
tinely, the technique can still be fraught with difficulties.
The conditions necessary to elute lipids of such high molec-
ular weight from the columns approach the limits of ther-
mal stability both of the stationary phases and of the
compounds themselves’’. On the other hand, HPLC does
not suffer from these problems with sample thermal lability
3. Experimental
3.1. Standards and isolation
Standards of fatty acids were prepared as described
below. All solvents were double-distilled and degassed
before use. 1-Bromotetracosane (1) and 12-bromododeca-
noic acid (2) were obtained from Sigma–Aldrich, Prague.
The sugar cane wax from the 2003 sugar cane harvest
(imported from Egypt) was obtained from a local sugar
refinery.

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T. Rezanka, K. Sigler / Phytochemistry 67 (2006) 916–923
921
3.1.1. Standards (Fig. 4)
The acid (4) (40.1 mg; 0.07 mmol) was dissolved in 50 ml
of dry ether. Fifteen milliliters of a 10% solution of LiAlH4
(3.8 mg; 0.1 mmol) in 5 ml of dry diethyl ether was added
and the mixture refluxed for 1 h. Water was added drop-
wise until the evolution of gas ceased. 10% Sulfuric acid
was then added until the white precipitate had dissolved.
The mixture was transferred to a separating funnel. The
ether layer was washed twice with water (2 · 15 ml) and
dried by means of sodium sulfate and the ether was
removed by distillation. A solution of alcohol (31.9 mg,
0.061 mmol) and carbon tetrabromide (50 mg, 0.15 mmol)
in dry acetonitrile (2 ml) was heated under reflux with stir-
To the stirred mixture containing a large excess of mag-
nesium turnings (20 mg; 0.8 mmol) and 15 ml of dry diethyl
ether was added dropwise a solution of 1-bromotetraco-
sane (1) (91.6 mg; 0.22 mmol) in 25 ml of dry diethyl ether.
At the end of the addition (2 h), the mixture was stirred for
1 h, whereupon the Grignard reagent solution was trans-
ferred under nitrogen into a dropping funnel (see Fig. 4).
To the solution of 12-bromododecanoic acid (2)
(55.6 mg; 0.2 mmol) in 8 ml ethanol was added sodium eth-
anolate (from 5 mg Na; 0.22 mmol) in 8 ml ethanol and the
mixture was stirred for 1 h. Ethanol was evaporated away
and the sodium salt was dried at a pressure of 1 Torr
(Zakharkin et al., 1988).
ring, and
a solution of triphenylphosphine (58 mg,
0.22 mmol) in dry acetonitrile was added dropwise over
2 min. The mixture was heated under reflux for 2 h and
cooled, the solvent was removed in vacuo, and the residue
was extracted with benzene (3 · 10 ml). The combined
extracts were filtered, and the solvent was removed in
vacuo. The yield of product (6) was 85.5% (30.6 mg), MS
m/z (relative intensity) 726 ([M]+, 1), 646 ([M ꢀ HBr]+, 19).
Bromide 6 (37.3 mg; 0.064 mmol) was used to prepare
alkylmagnesium bromide by the procedure described with
compound 1. Sodium salt of acid 3 (14.5 mg; 0.058 mmol)
was obtained by using a procedure analogous to that used
with acid 2. Reaction of the sodium salt of 3 with the alkyl-
magnesium bromide of 6 gave acid 7 as a colorless, waxy
compound in a 86.1% yield (33.8 mg). LC–MS/APCI: m/
z 677 [M + H]+; HREIMS m/z 676.7100 C46H92O2 [M]+,
calculated for [M]+ 676.7097; 1H NMR (400 MHz, CDCl3)
d 0.92 (3H, t, J = 6.8, H-46), 1.22–1.32 (84H, m, H-4–H-
45), 1.59 (2H, m, H-3) and 2.26 (2H, m, H-2); 13C NMR
A suspension of 60 mg the sodium salt of 2 (0.2 mmol)
in 8 ml absolute THF at ꢀ10 ꢁC was supplied under con-
tinuous stirring and in argon atmosphere with Li2CuCl4
(4.38 mg, 0.02 mmol) and dropwise with the solution of
0.22 mmol of alkylmagnesium bromide of 1, see above.
The mixture was stirred at ꢀ10 ꢁC for 4 h, acidified with
10% sulfuric acid and extracted with 3 · 5 ml diethyl ether.
The extract was washed with water, dried, and evaporated
to dryness. The yield of 4 referred to compound 2 was
87.4%, i.e., 93.8 mg, colorless, waxy compound, LC–MS/
APCI: m/z 537 [M + H]+; HREIMS m/z 536.5530
1
C36H72O2 [M]+, calculated for [M]+ 536.5532; H NMR
(400 MHz, CDCl3) d 0.91 (3H, t, J = 6.8, H-36), 1.22-
1.32 (62H, m, H-4–H-35), 1.59 (2H, m, H-3) and 2.26
(2H, m, H-2); 13C NMR (100 MHz, CDCl3) d 13.1 (C-
36), 19.8 (C-35), 24.8 (C-3), 29.3-29.8 (C-4–C-33), 32.1
(C-34), 34.2 (C-2), 180.1 (C-1).
Br
COOH
(CH2)20
Br
(CH2)8
1
2
EtONa
Mg, Et2O
N
Li2CuCl4, THF
1) (CF3CO)2O
2)
O
(CH2)30
COOH
(CH2)30
OH
4
5
O
N
1) LiAlH4
2) PBr3
Br
COOH
(CH2)30
Br
(CH2)6
6
3
Mg, Et2O
EtONa
N
Li2CuCl4, THF
1) (CF3CO)2O
O
(CH2)40
COOH
(CH2)40
2)
OH
7
8
O
N
Fig. 4. Scheme of the preparation of hexatriacontanoic and hexatetracontanoic acids (4, 7) and corresponding picolinyl esters (5, 8).

ˇ
T. Rezanka, K. Sigler / Phytochemistry 67 (2006) 916–923
922
(100 MHz, CDCl3) d 13.2 (C-46), 19.9 (C-45), 24.7 (C-3),
29.3–29.8 (C-4–C-33), 32.2 (C-44), 34.2 (C-2), 181.0 (C-1).
The synthesis of both picolinyl esters (5 and 8) is
described below.
Picolinyl esters (5 and 8) were prepared from acids 4 and
7 (see Fig. 4) by the procedure described above.
3.2. Preparative chromatography
5 as colorless, waxy compound, LC–MS/APCI: m/z 628
[M + H]+; HREIMS m/z 627.5958 C42H77NO2 [M]+, cal-
A Chromatospac Prep 100 preparative chromatograph
(Jobin-Yvon, Longjumeau, France) with axial compression
of the chromatographic bed (61.2 · 8 cm I.D.) was used for
the isolation of the fraction enriched with C35–C50 picolinyl
esters. The column efficiency was 6510 theoretical plates,
V0 was 1625 ml and tR of the picolinyl ester of hexatriacon-
tanoic acid (36:0) was 37.5 min. The mobile phase flow rate
was 64 ml/min. The mean particle diameter of the Separon
C18 reversed-phase packing was 15 lm (formerly Labora-
1
culated for [M]+ 627.5954; H NMR (400 MHz, CDCl3)
d 0.91 (3H, t, J = 6.8, H-36), 1.22–1.32 (62H, m, H-4–H-
35), 1.48 (2H, m, H-3), 2.23 (2H, m, H-2), 5.10 (2H, m,
H-70), 8.40 (1H, m, H-20), 7.15 (1H, m, H-50), 8.37 (1H,
m, H-60), 7.50 (1H, m, H-40), 13C NMR (100 MHz, CDCl3)
d 13.2 (C-36), 19.8 (C-35), 24.8 (C-3), 28.9–29.7 (C-4–C-
33), 32.0 (C-44), 34.4 (C-2), 174.1 (C-1), 124.4 (C-50),
135.7 (C-40), 136.5 (C-30), 147.4 (C-20), 147.9 (C-60), 65.4
(C-70).
´
ˇ´
tornı prıstroje, Prague, Czechoslovakia). 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.
8 as colorless, waxy compound, LC–MS/APCI: m/z 768
[M + H]+; HREIMS m/z 767.7522 C52H97NO2 [M]+, cal-
1
culated for [M]+ 767.7519; H NMR (400 MHz, CDCl3)
d 0.91 (3H, t, J = 6.8, H-46), 1.22–1.32 (84H, m, H-4–H-
45), 1.48 (2H, m, H-3), 2.23 (2H, m, H-2), 5.10 (2H, m,
H-70), 8.40 (1H, m, H-20), 7.15 (1H, m, H-50), 8.37 (1H,
m, H-60), 7.50 (1H, m, H-40); 13C NMR (100 MHz, CDCl3)
d 13.2 (C-46), 19.8 (C-45), 24.8 (C-3), 28.9–29.7 (C-4–C-
43), 32.0 (C-44), 34.4 (C-2), 172.5 (C-1), 124.4 (C-50),
135.7 (C-40), 136.5 (C-30), 147.3 (C-20), 147.9 (C-60), 65.4
(C-70).
The amount of 754 mg picolinyl esters (see above) was
dissolved in 19 ml of the mobile phase (injection volume)
(methanol–isopropanol–triethylamine, 87:13:0.05). In the
time interval 0–37 min the column was eluted with mobile
phase that was later replaced with diethyl ether (76 min).
The column was again conditioned with mobile phase for
14 min. The fraction up to 37.5 min was discarded and that
obtained within the interval 37.5–90 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 34 carbon atoms was 11.8 mg. These were separated
and identified by LC–MS.
3.1.2. Fatty acids from natural source and picolinyl esters
An amount of 10 g of refined sugar cane wax was mixed
with 2 g of KOH dissolved in 15 ml of water. The saponi-
fication process was maintained for 30 min with periodical
stirring. The fatty acid was extracted from the solid
obtained in the process using acetone in a solid–liquid
extraction system. The ensuing residue was cooled to room
temperature, extracted with heptane, the pH was adjusted
to approximately 3 by dilute sulfuric acid and another
extraction with heptane was performed. The yield of VLC-
FAs was 2 g with a purity amounting to 94%.
These free fatty acids (2 g) were dissolved in diethyl
ether (100 ml) and converted to the mixed anhydride deriv-
atives by reaction with trifluoroacetic anhydride (15 ml) at
50 ꢁC for 2 h. The excess reagent was evaporated, a 10%
solution of nicotinyl alcohol in tetrahydrofurane (15 ml)
was added and the mixture was left at 50 ꢁC for 2 h.
Diethyl ether (100 ml) and hexane (20 ml) were added
and the mixture was washed with water (50 ml), 1 M HCl
(20 ml, three times) and water (20 ml, three times) and
dried by anhydrous sodium sulfate. The solvents were
evaporated under reduced pressure and the picolinyl esters
were purified by silica gel chromatography with the hex-
ane–diethyl ether (1:1 v/v) mixture as the eluting agent;
the yield was 754 mg (Christie et al., 1986).
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
– approximately ꢁ53,000-plates/50 cm. A quadruple 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 tempera-
ture 220 ꢁC, corona current 5 lA, sheath gas – high-purity
nitrogen, pressure 0.38 MPa, and auxiliary gas (also nitro-
gen) flow rate 15 ml/min. Positively charged ions with m/z
200–900 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 ace-
tonitrile (ACN), dichloromethane (DCM) and propionit-
rile (EtCN) as follows: initial ACN/EtCN/DCM (60:30:
10, vol/vol/vol); linear from 10 to 40 min ACN/EtCN/
DCM (30:40:30, vol/vol/vol); held until 60.5 min; the com-
position was returned to the initial conditions over 8 min.
A peak threshold of 0.3% intensity was applied to the mass
spectra. Data acquisition and analyses were performed
The sample of 754 mg of derivatized fatty acids (as
picolinyl esters) was submitted to preparative chromatog-
raphy to obtain 11.8 mg of picolinyl derivatives of
C35–C50 fatty acids. This fraction was then analyzed by
LC–MS.

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T. Rezanka, K. Sigler / Phytochemistry 67 (2006) 916–923
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