Journal of Chromatography A, 1381 (2015) 229238

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Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Analysis of triterpenoids and phytosterols in vegetables by thin-layer

chromatography coupled to tandem mass spectrometry
Katerina Naumoska a , Irena Vovk a,b,
a
b

National Institute of Chemistry, Laboratory for Food Chemistry, Hajdrihova 19, 1001 Ljubljana, Slovenia
EN-FIST Centre of Excellence, Trg Osvobodilne fronte 13, 1000 Ljubljana, Slovenia

a r t i c l e

i n f o

Article history:
Received 14 October 2014
Received in revised form
28 December 2014
Accepted 1 January 2015
Available online 9 January 2015
Keywords:
HPTLC
TLCMS2
Mass spectral library
Triterpenoids
Phytosterols
Vegetables

a b s t r a c t
Three TLC methods were used for an initial screening of some common plant triterpenoids and phytosterols in cuticular wax extracts of different vegetables (zucchini, eggplant, tomato, red pepper, mangold,
spinach, lettuce, white-colored radicchio di Castelfranco, raddichio Leonardo, white cabbage, red cabbage and savoy cabbage). The preliminary experiments showed that the studied vegetables are potential
sources of triterpenoids and phytosterols. To identify the compounds present in the extracts with high certainty, the rst TLCMS2 method was developed for the analysis of eight triterpenoids (lupeol, -amyrin,
-amyrin, cycloartenol, cycloartenol acetate, lupeol acetate, lupenone and friedelin) and two phytosterols
(-sitosterol and stigmasterol). This method takes the advantages of: (1) a satisfactory separation of the
target compounds; (2) their differentiation according to the band colors; and (3) the potential of their
discrimination by the acquired rst-order mass (MS) and product ion (MS2 ) spectra. Since the closely
eluting compounds have complex and similar MS2 spectra, distinguishing between them was possible by
the proposed characteristic ions. Using a custom-built mass spectral library, the head to tail MS2 spectra
comparison of sample test solution zones and standard aided the compound identication. In addition to
the molecular mass information, the developed atmospheric pressure chemical ionization method (APCI)
in positive ion mode provided structural information, regarding the presence of functional group in the
molecule. This approach resulted in many positively assigned compounds in the investigated vegetable
waxes, from which more than a half are reported for the rst time.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Cuticular waxes constitute the waxy coverings of the plant
organs and serve as plant protectants against unfavorable environmental conditions and insects. They are composed of long-chain
aliphatic hydrocarbons, ketones, esters, fatty alcohols, fatty acids,
aldehydes, as well as triterpenoids (C30 ) and phytosterols (C18 C30 )
[1]. The last two groups of compounds present a very large
and structurally diverse family of secondary plant metabolites,
biosynthetically derived through the mevalonate pathway from six
isoprene units (C5 H8 ) [2]. They can exist as free compounds or in
the form of esters and saponins. Pharmacological studies of triterpenoids showed anticancer, anti-inammatory, anti-ulcerogenic,
anti-microbial, anti-viral (including anti-HIV), anti-fungal, analgesic, antioxidative, hepatoprotective and some other activities

Corresponding author at: National Institute of Chemistry, Hajdrihova 19, SI-1000

Ljubljana, Slovenia. Tel.: +386 1 4760 341; fax: +386 1 4760 300.
E-mail address: irena.vovk@ki.si (I. Vovk).
http://dx.doi.org/10.1016/j.chroma.2015.01.001
0021-9673/ 2015 Elsevier B.V. All rights reserved.

[310]. Phytosterols are well known for their cholesterol-lowering

properties, and also demonstrate anticancer, anti-inammatory
and immunoregulatory activities [11,12]. Due to the growing interest in triterpenoids and phytosterols, the development of suitable
modern analytical methods for the determination of these metabolites in natural products, is of paramount importance.
Determination of triterpenoids in plant extracts is rather
difcult, since many plants contain a vast amount of various triterpenoid compounds. Presence of isomeric triterpenoids in plant
cuticular waxes renders the determination of triterpenoids even
more difcult. Among the separation techniques, thin-layer (TLC)
[1316], supercritical uid (SFC) [17], gas (GC) [1824] and highperformance liquid (HPLC) [13,14,2333] chromatography and
capillary electrophoresis (CE) [34] have been used in their analysis. GC is favorable for the separation of positional triterpenoid
isomers, and coupled to ame ionizaton (FID) [18,19,2124] and
mass spectrometric (MS) detection [1820,22,24] has been widely
used for their qualitative [1820,22,24] and quantitative analysis
[18,19,2124]. A big disadvantage of this technique is the need of a
prechromatographic derivatization, due to the non-volatility of the

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K. Naumoska, I. Vovk / J. Chromatogr. A 1381 (2015) 229238

compounds, which additionally prolongs the analysis. On the other

hand, HPLC reduces the sample pretreatment step by avoiding the
derivatization. However, triterpenoids and phytosterols lack chromophores, which limits the mobile phase choice and reduces the
sensitivity of UV detection [13,14,23,25]. Therefore, coupling HPLC
with evaporative light scattering (ELSD) [24,26] or corona charged
aerosol (CAD) detector [27] can be suitable, since they showed
increased sensitivity compared to UV detectors for the compounds
of interest. In addition, triterpenoids can be tagged with uorescent groups and determined by uorescence detector (FLD), as well
[28,29]. However, UVvis, as a non-selective and universal detector,
is the most commonly used in HPLC. Furthermore, the mass spectrometry (MS) detector enables identication and quantication
of compounds in real samples, and aids the structural elucidation
of unknown compounds as well [13,14,26,3033]. When product
ion (MS2 ) analysis is used in the identication of the triterpenoid
positional isomers, the spectra differ only by the relative intensities
of some mass peaks, which indicates that a good chromatographic
separation prior to MS analysis is obligatory for unambiguous identication of the compounds [13,14]. Although, TLC offers lower
separation efciencies compared to GC and HPLC, it is a highly
applicable technique especially for fast screening of compounds in
various complex matrices simultaneously, since the sample purication step is usually avoided or is minimal. In addition, there are
no limitations in the selection of mobile phase solvents in comparison to HPLC. Moreover, an increase in the specicity and sensitivity
of the analysis can be achieved by its coupling to tandem MS [35].
To the best of our knowledge, there is a lack of methods for
simultaneous identication or determination of common plant
neutral triterpenoids and phytosterols. Moreover, there is scarce
information on their presence in the cuticular waxes of various vegetables. Therefore, the main objective of the present study was to
develop and apply a new TLCMS2 method for the analysis of the
triterpenoids and phytosterols in vegetable extracts.
2. Experimental
2.1. Chemicals
All the solvents used in the study were at least of analytical grade. Dichloromethane, chloroform, n-propanol, n-hexane,
ethyl acetate, acetic acid (glacial, 100%), sulfuric acid (9597%),
hydrochloric acid (fuming, 37%) sodium sulfate (anhydrous), potasium hydroxide and 4-methoxybenzaldehyde (anisaldehyde) were
obtained from Merck (Darmstadt, Germany), while HPLC grade
methanol and acetonitrile were produced by J.T. Baker (Deventer,
The Netherlands). Acetone, LCMS purity acetonitrile as well as reference standards for ursolic acid (90%) and -sitosterol (97%)
were purchased from SigmaAldrich (St. Louis, MO, USA). Other
reference standards such as lupeol (99%), -amyrin (98.5%),
-amyrin (98.5%), cycloartenol (90%), lupeol acetate (95%),
cycloartenol acetate (90%), lupenone (95%), friedelin (99%)
and betulinic acid (97%) were supplied by Extrasynthse (Genay,
France), while stigmasterol (99%) was obtained from Serva Feinbiohemica (Heidelberg, Germany) and oleanolic acid (97%) from
Carl Roth (Karlsruhe, Germany). Ultrapure water was supplied by a
Milli-Q water purication system (18 M cm) from Millipore (Bedford, MA, USA).
2.2. Preparation of standard solutions
Stock solutions of all standards (1 mg mL1 ; except those of
friedelin and betulinic acid with concentration of 0.1 mg mL1 )
were prepared in n-propanol and were further diluted with
the same solvent to obtain working solutions (25 g mL1 ). A

mixture of all 13 standard solutions (MIX13, 25 g mL1 ; lupeol,

-amyrin, -amyrin, cycloartenol, cycloartenol acetate, lupeol
acetate, lupenone, friedelin, ursolic acid, oleanolic acid, betulinic
acid, -sitosterol and stigmasterol) and a mixture with all the
standards except ursolic, oleanolic and betulinic acids (MIX10,
25 g mL1 ) were prepared by mixing 1 mL of each working
solution, evaporating the solvent under nitrogen and redissolving
the solid residue in 1 mL of n-propanol.
2.3. Preparation of vegetable extracts and sample test solutions
The extraction of the vegetable cuticular waxes and preparation of sample test solutions followed the procedure given in
Ref. [13]. Fresh vegetables were purchased from a local market.
Fruits from zucchini (Cucurbita pepo L., Cucurbitaceae; 1845 g), eggplant (Solanum melongena L., Solanaceae; 2775 g), tomato (Solanum
lycopersicum L., Solanaceae; 4289 g) and red pepper (Capsicum
annuum L., Solanaceae; 696 g) and leaves from mangold (Beta
vulgaris L. ssp. vulgaris var. cicla, Chenopodiaceae; 77 g), spinach
(Spinacia oleracea L., Chenopodiaceae; 194 g), lettuce (Lactuca sativa
L. var. capitata, Cichoriaceae; 350 g), white-colored radicchio di
Castelfranco (Cichorium intybus L. var foliosum, Cichoriaceae; 368 g),
radicchio Leonardo (Cichorium intybus L. var. foliosum, Cichoriaceae; 529 g), white cabbage (Brassica oleracea L. subsp. oleracea
convar. capitata L. var. capitata L. f. alba, Brassicaceae; 488 g),
red cabbage (Brassica oleracea L. subsp. oleracea convar. capitata
L. var. capitata L. f. rubra, Brassicaceae; 250 g), and savoy cabbage (Brassica oleracea L. subsp. oleracea convar. capitata (L.) Alef.
var. sabauda, Brassicaceae; 157 g) were separately immersed into
dichloromethane for 1 min. After addition of anhydrous sodium
sulfate (1 g) to the extract (to bind the residual water), it was
ltered through paper lter and the ltrate was concentrated
under reduced pressure (Rotavapor, Bchi, Switzerland). The concentrated extract was transferred to a pre-weighted plastic tube
(15 mL), and the solvent was evaporated to dryness by using a
gentle stream of nitrogen. Dry wax residues (61370 mg) were dissolved in chloroform to a concentration of 10 mg mL1 and 1 mL
of each vegetable extract was transferred in a separate autosampler vial. The extract solvent was evaporated to dryness and the
solid residue was redissolved in n-propanol (1 mL) to give the nal
sample test solution. A hair dryer was used to speed up the solvation process of the waxy residues. The sample test solutions were
cooled down to room temperature and ltered through a 0.45 m
Millipore Millex-HV hydrophilic poly(vinyldiene diuoride) (PVDF)
membrane lter (Billerica, MA, USA).
2.4. Thin-layer chromatography
TLC was performed on the Merck 20 cm 10 cm glass-backed
HPTLC silica gel 60 (Art. No. 1.05641) and HPTLC C18 RP (Art. No.
1.05914) plates predeveloped with chloroformmethanol (1:1, v/v)
and acetone, respectively, and dried in an oven at 110 C for 30 min.
Standard solutions, MIX10, MIX13 and sample test solutions were
applied on the plates as 8 mm (or 6 mm for TLCMS) bands, 10 mm
from the bottom of the plates, by use of Linomat 5 (Camag, Muttenz,
Switzerland). Plates used for compounds screening were developed to a distance of 8 cm (in 12 min) in a horizontal developing
chamber (for 20 cm 10 cm plates; Camag) using 6 mL of developing solvents n-hexaneethyl acetate (5:1, v/v) for silica gel plates
and acetoneacetonitrile (5:1, v/v) and ethyl acetateacetonitrile
(3:2, v/v) for C18 RP HPTLC plates [13]. For each case, 10 mL of
the corresponding solvent was put in a tank for preconditioning
(10 min).
As a part of the optimization of the separation for TLCMS
analysis, C18 RP HPTLC plates were developed by ethyl

K. Naumoska, I. Vovk / J. Chromatogr. A 1381 (2015) 229238

231

acetateacetonitrile (3:2, v/v) in different developing chambers (Camag): normal, twin trough [13], horizontal (sandwich and
tank conguration) and Automatic development chamber ADC2
(Camag). Different application positions (5 mm, 8 mm and 10 mm)
from the bottom were also tested.
Finally, TLC screening and TLCMS2 analysis were performed
on 10 cm 20 cm C18 RP HPTLC plates, developed to a distance
of 18 cm (in 1 h 20 min) in a normal developing chamber for
20 cm 20 cm plates using 20 mL of ethyl acetateacetonitrile (3:2,
v/v) as a developing solvent.
Anisaldehyde detection reagent was prepared by mixing glacial
acetic acid (20 mL) and methanol (170 mL). The solution was cooled
with ice and water while sulfuric acid (16 mL) was added in a dropwise manner. Subsequently, anisaldehyde (1 mL) was added to the
mixture [36].
After developing and drying of the plates, the postchromatographic derivatization was performed by dipping
the plates into the anisaldehyde detection reagent for 2 s by using
an immersion device III (Camag), followed by drying with a stream
of warm air and heating them at 110 C on a TLC plate heater
(Camag) for 2 min (silica gel plates) or 30 s (C18 RP plates). The
chromatographic plates were captured under UV (366 nm) and
white light by using DigiStore 2 Documentation System (Camag)
and winCATS software (version 1.4.3.6336).

2.5. TLCMS2 and mass spectral library

A TLCMS interface with oval elution head (4 mm 2 mm)
(Camag) was employed for the elution of the compounds from
the C18 RP HPTLC plates into a LCQ ion trap (Thermo Finnigan,
San Jose, CA, USA) system. Acetonitrile was used as an eluent at
a ow rate of 0.3 mL min1 . Acetonitrile ow (0.3 mL min1 ) and
working standard solution ow (5 L min1 ) were joined by a Tunion and directed into the MS source for direct syringe infusion
of the standards. Atmospheric pressure chemical ionization (APCI)
source operated in positive ion mode was used for ionization of the
target compounds. Capillary and source heater temperatures were
maintained at 225 C and 400 C, respectively. Other parameters
were as follows: sheath gas (N2 ) 65 arbitrary units (a.u.), auxiliary gas (N2 ) 27 a.u., discharge current 3 A, capillary voltage 18 V,
tube lens offset 25 V. The mass spectra were acquired in the m/z
range 300500. Compromise MS2 conditions were set for all the
compounds, except for friedelin, lupenone, -sitosterol and stigmasterol (Table 1). Each sample test solution was analyzed on a
separate 10 cm 20 cm C18 RP HPTLC plate in seven equal applications (30 L), which followed the application of MIX10 (20 L).
After the development, only the MIX10 track and the closest sample
test solution track were derivatized with anisaldehyde detection
reagent, while the remaining six applications were used for TLCMS
analysis (Fig. 1). Each zone with RF equal to the RF of the standards (marked with a soft pencil) was eluted from the plate and
transferred to the MS detector followed by rst-order mass (MS)
and product ion (MS2 ) spectra acquisition. The interday repeatability of the obtained spectra was also checked. The mass spectra
of the zones from sample test solutions were compared with those
of the standards (25 g mL1 ) acquired by direct syringe infusion
or directly by elution from the undeveloped HPTLC plates (250 ng).
In addition, plate background spectra at different RF values were
acquired in order to avoid false positives.
The NIST Mass Spectral Search Program 2.0 enabled creation of
a mass spectral library from the MS2 spectra of the individual standards. The acquired MS2 spectra of the sample test solution zones
eluted from the plates were exported to the library and compared
with the standard spectra.

Fig. 1. (Left side) The images of a plate, captured under white light and at 366 nm
are showing separation of eight triterpenoids and two phytosterols on HPTLC C18
RP plate developed with ethyl acetateacetonitrile (3:2, v/v) to a distance of 18 cm.
(Right side) Schematic representation of a plate prepared for band elution by TLCMS
interface.

3. Results and discussion

3.1. TLC
The initial screening of 11 triterpenoids (triterpenols: lupeol,
-amyrin, -amyrin and cycloartenol; ketones: friedelin and
lupenone; acetates: cycloartenol acetate and lupeol acetate; and
acids: ursolic, oleanolic and betulinic acids) and two phytosterols (-sitosterol and stigmasterol) in the vegetable sample
test solutions was performed by three TLC methods (Fig. 2) [13],
which did not enable satisfactory separation between the acids.
The rst method, employing HPTLC silica gel plate developed
with n-hexaneethyl acetate (5:1, v/v), enabled separation of the
compounds by their functional groups. Positional isomers were
completely or partly separated by the other two methods, using
HPTLC C18 RP plates and two different developing solvents, i.e.
acetoneacetonitrile (5:1, v/v) and ethyl acetateacetonitrile (3:2,
v/v). The latter developing solvent showed to be the most appropriate, as it provided higher selectivity and resolution among isomers,
i.e. cycloartenol and positional isomers, -amyrin and -amyrin,
while lupeol, cycloartenol acetate and friedelin bands were overlapped, which was not the case when using acetoneacetonitrile
(5:1, v/v). The observed bands from the sample test solutions were
compared with those obtained from the standards by their RF and
color (given also in Ref. [13]) after derivatization with anisaldehyde
detection reagent. At this stage the compounds identity cannot be
inferred with certainty. However, these experiments showed that
the vegetable cuticular waxes can be potential sources of triterpenoids and phytosterols.
As expected, development of C18 RP HPTLC plates by ethyl
acetateacetonitrile (3:2, v/v) in different developing chambers
(normal, twin-trough, horizontal (sandwich and tank conguration), ADC2) and application position from the bottom of the plate
inuenced the RF values and the resolution between the closely

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K. Naumoska, I. Vovk / J. Chromatogr. A 1381 (2015) 229238

Table 1
Selected MS2 conditions for the studied compounds. Isolation width of m/z 1.5 and activation radio frequency of 0.25 were set for all the compounds.
Compound

Parent mass (m/z)

Normalized collision energy (%)

Lupeol
-Amyrin
-Amyrin
Cycloartenol
Cycloartenol acetate
Lupeol acetate

Activation time (ms)

Range (m/z)

409

35

60.000

150420

Friedelin
Lupenone
-Sitosterol
Stigmasterol

427
425
397
395

45
55
45
35

100.000
60.000
60.000
60.000

150440
300430
150410
150410

Fig. 2. Screening of triterpenoids and phytosterols in the sample test solutions obtained from the different vegetable wax extracts and documented at 366 nm. a: HPTLC
silica gel 60 plates developed in n-hexaneethyl acetate (5:1, v/v); b: HPTLC C18 RP plates developed in acetoneacetonitrile (5:1, v/v); c: HPTLC C18 RP plates developed
in ethyl acetateacetonitrile (3:2, v/v). Tracks: 1 = mangold (15 L), 2 = savoy cabbage (10 L), 3 = red cabbage (11 L), 4 = MIX13 (10 L), 5 = spinach (15 L), 6 = red pepper
(8 L), 7 = white-colored radicchio di Castelfranco (4 L), 8 = white cabbage (7 L), 9 = eggplant (18 L), 10 = lettuce (9 L), 11 = radicchio Leonardo (1 L), 12 = tomato (1 L),
13 = zucchini (16 L).

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233

width of the oval elution head (0.2 cm). This risk is especially pronounced when MS cannot aid in the differentiation of the possibly
coeluted compounds, as is the case with cycloartenol, -amyrin and
-amyrin and cycloartenol acetate and lupeol (further referred to as
critical compounds). To the contrary, the modied method resulting in wider bands for the critical compounds (0.3 cm), enabled
higher certainty that no coelution of the neighboring zone/s will
occur to the MS (especially when the central part of the band
is eluted). The obtained higher resolution between the bands in
comparison to the initial method, due to the longer developing distance and consequently the higher number of theoretical plates
(N), enabled more reliable visual identication, as well as optimal
separation of all 10 compounds for MS analysis (Fig. 1). Regardless of the non-selective elution, zigzag elution pattern could be
implemented for the TLCMS analysis of the tightly located zones
from the initial method to avoid leakage from the elution head. On
the contrary, the modied method enabled straight elution pattern (each zone could be eluted from the same track) without any
leakage originating from the elution head. The method was applied
for identication of eight triterpenoids and two phytosterols in
different vegetable sample test solutions. Triterpenoid acids were
further excluded from the study, since we already reported them
in the same vegetable waxes. For their unambiguous identication,
a TLCMS2 approach, based on the separation using HPTLC C18 RP
plate and n-hexaneethyl acetate (5:1, v/v) as a developing solvent
was used [15].
Fig. 3. C18 RP HPTLC plates developed with ethyl acetateacetonitrile (3:2, v/v)
in different developing chambers for 20 cm 20 cm (1), 20 cm 10 cm (49) and
10 cm 10 cm plates (2, 3, 1012): normal unsaturated (1, 2) and 10 min saturated
(3), ADC2 (4, 5, 6), twin trough (7, 8, 9), horizontal-tank conguration without (10)
and with (11) 10 min of preconditioning, horizontal-sandwich conguration (12).
Developing solvent volume: 20 mL (1), 6 mL (49), 5 mL (2, 3), 3 mL (1012). MIX13
(10 L) was applied 5 mm (4, 7), 8 mm (5, 8) or 10 mm (13, 6, 912) from the
bottom.

3.2. Stability of standards on the plate

The standards applied on the plate were stable for at least 14 h
after development, which enabled TLCMS analysis. On the other

eluting compounds (Fig. 3). However, none of them provided the

satisfactory resolution for TLCMS analysis.
To obtain more reliable qualitative results and discriminate
between the target compounds, we modied one of the methods,
by turning a 20 cm 10 cm HPTLC C18 RP plate for 90 and developing it with ethyl acetateacetonitrile (3:2, v/v) up to distance of
18 cm (Fig. 1). The RF values of the bands of each compound (Table 2)
were similar to those reported in Ref. [13] for the method employing the same stationary phase and developing solvent. However,
the bands of the standards of MIX10 were wider for the modied
method (cycloartenol, -amyrin, -amyrin, cycloartenol acetate
and lupeol 0.3 cm) in comparison to the initial one (cycloartenol,
-amyrin, -amyrin 0.2 cm, while the bands of cycloartenol
acetate and lupeol were overlapped). The danger to elute zones
in a non-selective manner (elution of the part of the neighboring
zone/s together with the target zone) was obvious for the initial
method where some bands were of even lower width than the
Table 2
Average RF values with standard deviations (SD) calculated for each studied compound from three HPTLC C18 RP plates developed in the normal developing chamber
with ethyl acetateacetonitrile (3:2, v/v) to a distance of 18 cm.
Compound

Average RF values SD (n = 3)

-Sitosterol
Stigmasterol
Cycloartenol
-Amyrin
-Amyrin
Cycloartenol acetate
Lupeol
Friedelin
Lupeol acetate
Lupenone

0.15
0.16
0.28
0.30
0.33
0.40
0.42
0.43
0.50
0.57

0.01
0.02
0.01
0.02
0.01
0.01
0.01
0.01
0.01
0.01

Fig. 4. Background mass spectra acquired at different RF values from two different batches of HPTLC C18 RP plates predeveloped with acetone and developed in
ethyl acetateacetonitrile (3:2, v/v) and mass spectrum of -amyrin eluted from
the corresponding plate.

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K. Naumoska, I. Vovk / J. Chromatogr. A 1381 (2015) 229238

MS2 spectra:

MS spectra:
409

100

257 271
285

100

lupeol

215 229
191 203
243
0
100

409

lupeol actate

0
100

191
0
100

409

229
215
243
203

-amyin

409

100

-amyrin

408

339353366

245
257

231 245 257

217
203
191

408

271
285
299

177191 203217

353
367

313

299 313

0
100
231

0
100

299
271
285

257

271

339

313

367

408

285
299

339 353 367

313

409

0
0
100

409

cycloartenol

161

409

0
100

cycloartenol
actate
427

friedelin
468

0
100

Relative Abundance

Relative Abundance

427
0
100

217

100

0
100

191 203
177

191 203
177
163

257
231

lupenone

271 285299

231

200

271
285 299
245 257
250

300

177191 217
163
203
231

259 285

313

0
100

395

381

350

409 400

357 369
407
337

311
0

-sitosterol

353
367

327

100

466
397

408

367

100

407
0
100

353

325

217

0
425

245

100

355
369

200
250
350
300
243 257
161
203
287
185 189
301315
229
261
215
275

400

327341 355

stigmasterol

0
100

161
0
300

350

400

450

500

m/z

382

297

187 201 215

200

255
283
269
241
250

m/z

311
325 339
300

350

367
400

Fig. 5. MS and MS2 spectra of the standards eluted from the HPTLC C18 RP plates.

hand, derivatized friedelin (after 30 s of heating on the TLC plate

heater) was stable only 12 min, after which the pale yellow color
observed under illumination at 366 nm disappeared. Interestingly,
after reheating the plate for additional 30 s, the colored band of
friedelin appeared again, indicating a reversible reaction. When
heated the plate for longer time (1, 1.5 or 2 min), the colored product of friedelin does not disappear for at least 25 min. However, we
chose 30 s of heating for C18 RP plates on the TLC plate heater as
it was optimal time for obtaining a good (bright) background and
the stability of other standards was not an issue under these conditions. Therefore, the plates were captured immediately after the
derivatization (in 12 min).
3.3. Background mass signals in TLCMS
Serious difculties in the TLCMS analysis can arise from the
plate (adsorbent) background spectrum, which can be inuenced
by the plate alone (different adsorbent areas of the same plate), as
well as solvents and solvent mixtures used for the predevelopment,
development or even for the elution to the MS. Some background
mass signals can lead to suppression of the analyte signal/s. This
was reported for TLC coupled to electrospray ionization MS when
acidic solvents were used for predevelopment or/and development
of silica gel plates [37,38]. Therefore, differentiation of analyte signal/s from background mass signals is playing a crucial role in the
TLCMS analysis. We examined the inuence of acetone as a predeveloping solvent and ethyl acetateacetonitrile (3:2, v/v) as a

developing solvent on the HPTLC C18 RP plate background spectrum and its impact on the analyte mass spectrum from elutions
at different RF values. The (+)APCI-MS spectra obtained from the
plates predeveloped with acetone showed the mass peaks at m/z
354, 368, 437, 453 and 497. The predeveloped HPTLC C18 RP plates
(batch: HX229037), which were additionally developed with ethyl
acetateacetonitrile (3:2, v/v), resulted in MS background spectra containing four major mass peaks at m/z 354, 391, 419, 475.
The same signals were obtained from zones at different RF values
on different plates with the same batch used for the whole study,
suggesting that the same contamination was occurring each time.
However, a batch-to-batch repeatability was not observed. Namely,
when another batch (HX43689814) was analyzed for background
signals, other major peaks at m/z 355 and 429 were repeating at
different RF values and different plates. Fortunately, no background
mass signals that could interfere with the mass signals of the target compounds were observed (Fig. 4). Additionally, no difculties
regarding the analyte/s ionization were noticed, which made this
TLCMS study possible without any special demands.
3.4. First-order mass (MS) and product ion (MS2 ) spectra
MS and MS2 spectra of the standards are given in Fig. 5.
Lupeol, -amyrin, -amyrin and cycloartenol standards (C30 H50 O,
M = 426.72 g mol1 ) gave MS spectra with a base peak at m/z
409 which was assigned to a dehydrated protonated molecule
[M+HH2 O]+ . Additionally, in the MS spectrum of cycloartenol,

K. Naumoska, I. Vovk / J. Chromatogr. A 1381 (2015) 229238

235

Fig. 6. Histograms of the proposed characteristic ions (marked by *) observed in the MS2 spectra of the triterpenoid compounds. For the ordinate axis, the mean values for
relative intensities, calculated from the four replications, are taken into account.

a mass peak at m/z 427 was observed corresponding to the protonated molecule [M+H]+ . Along with the base peak at m/z 427
[M+H]+ , the friedelin standard (C30 H50 O, M = 426.72 g mol1 ) spectrum showed also a mass peak of lower intensity at m/z 468
corresponding to a protonated cluster ion [M+H+CH3 CN]+ . The
standards of cycloartenol acetate and lupeol acetate (C30 H52 O2 ,
M = 468.75 g mol1 ) showed similar and identical MS spectra as
their free forms, respectively, revealing a base peak at m/z 409,
assigned as [MCH3 COO ]+ . The mass spectrum of lupenone
(C30 H48 O, M = 424.7 g mol1 ) showed a base peak at m/z 425,
corresponding to the protonated molecule [M+H]+ , as well as
dehydrated protonated molecule [M+HH2 O]+ at m/z 407 and a
protonated cluster ion [M+H+CH3 CN]+ at m/z 466. Standard MS
spectra of -sitosterol (C29 H50 O, M = 414.71 g mol1 ) and stigmasterol (C29 H48 O, M = 412.69 g mol1 ) revealed base peaks of the
dehydrated protonated molecules [M+HH2 O]+ at m/z 397 and
m/z 395, respectively. In addition to the molecular mass information, the developed APCI-MS method in positive ion mode provides
information with regard to the presence of functional group in
the compound structure. Thus, mass spectra of triterpenols and
phytosterols (containing hydroxyl functional groups), triterpenoid
ketones and triterpenoid acetates showed base peaks corresponding to ions assigned as [M+HH2 O]+ , [M+H]+ and [MCH3 COO ]+ ,
respectively, which is in line with the conclusions drawn by Van
Der Doelen and coworkers [31].
MS2 spectra did not show any signicant interday variation
in the fragmentation patterns, when acquired in four different
days. Under the MS2 conditions, the protonated molecules [M+H]+
of friedelin (m/z 427) and lupenone (m/z 425) were fragmented

to product ions with m/z values of 409 ([M+HH2 O]+ ) and 407
([M+HH2 O]+ ), respectively. The other compounds gave MS2 spectra of more complex nature, which cannot be interpreted in a
straightforward manner. The MS2 analysis of lupeol, -amyrin,
-amyrin, cycloartenol, cycloartenol acetate and lupeol acetate
resulted in similar MS2 spectra containing a group of intensive
product ions with m/z values of 257, 271 and 285, except for
cycloartenol and cycloartenol acetate. Moreover, for the MS2 spectra of cycloartenol, cycloartenol acetate, -amyrin and -amyrin,
additional group of product ions is observed with m/z values of
191, 203 and 217. For cycloartenol and cycloartenol acetate, this
group is dominant, while for -amyrin and -amyrin it is of lower
intensity. Lupeol and lupeol acetate have the additional common
product ion at m/z 215 which is of higher intensity than that at
m/z 217. In comparison to the other compounds, -sitosterol and
stigmasterol showed different fragmentation patterns. Histograms
of the proposed discrimination markers, denoted as characteristic
ions, observed in the MS2 spectra are shown in Fig. 4. Similar data
are published for the fragmentation of triterpenols [13,14,32,39]
and phytosterols [32].
3.5. TLCMS2
TLCMS2 was shown to be suitable for qualitative analysis of
the eight triterpenoids and two phytosterols. Their characteristically colored bands [13] were satisfactory separated and were
divided in four groups by their migration on the TLC plate (Fig. 1).
The compounds from a certain RF range gave characteristic ion(s)
in the MS or MS2 spectra, by which they could be differentiated

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K. Naumoska, I. Vovk / J. Chromatogr. A 1381 (2015) 229238

Fig. 7. Assignation of some compounds extracted from red pepper cuticular wax. (a) Parallel mass spectra comparison: the upper two spectra in each MS and MS2 comparison
section belong to the eluted sample test solution zones from the developed HPTLC C18 RP plate, whilst the third is the spectrum of the corresponding standard and (b) head
to tail comparison of the MS2 spectra obtained from the eluted sample test solution zones from the plate and the corresponding standards, represented by the upward and
downward pointed mass spectra, respectively.

from the closely eluting compounds (Figs. 5 and 6). -sitosterol

and stigmasterol from the rst group (RF range: 0.120.21) had the
lowest RF on the plate. The discrimination between them could be
done relatively easy, due to their good chromatographic separation, different band colors and different MS and MS2 spectra. The
second group (RF range: 0.240.35) was consisted of cycloartenol,
-amyrin and -amyrin. Despite their relatively close elution, they
were distinguished by different colors of their bands. Unfortunately, they could not be unambiguously identied by MS, since
their MS spectra showed the same base peak at m/z 409. However,
cycloartenol could be differentiated from the other two triterpenols
by the presence of an additional mass peak at m/z at 427 in its spectrum. Their MS2 spectra showed similar fragmentation patterns.
As previously explained for the cycloartenol standard, the spectrum showed dominant group of ions with m/z values of 191, 203
and 217, and a minor one with m/z values of 257, 271 and 285.
In contrast, the MS2 spectra of -amyrin and -amyrin showed a
dominant group of ions peaking at m/z 257, 271 and 285. However,
the characteristic group of ions with m/z values of 191, 203 and 217
was more intensive in the spectrum of -amyrin than that of its isomer, -amyrin. The third group of compounds (RF range: 0.370.45)
was comprised of closely eluting cycloartenol acetate, lupeol and
friedelin. However, their discrimination by mass spectrometry was

more straightforward. Friedelin gave different MS and MS2 spectra

than the closely eluting compounds. Although cycloartenol acetate
and lupeol had identical MS spectra, their MS2 spectra showed
differences between the intensities of the characteristic groups of
ions, allowing compound discrimination. Cycloartenol acetate and
lupeol MS2 spectra showed the same dominant group of ions as
cycloartenol and amyrins, respectively. The MS2 spectrum of lupeol
showed a characteristic product ion peak at m/z 215, which can
be observed also in the MS2 spectrum of lupeol acetate. Lupeol
acetate and lupenone from the forth group (RF : 0.470.59) were
well separated from each other and from the other compounds on
the plate. Lupeol acetate MS and MS2 spectra are practically identical to those of lupeol. A similar case was observed for cycloartenol
and cycloartenol acetate MS2 spectra, as well. Lupenones distinctive MS and MS2 spectra enabled straightforward identication.
3.6. Analysis of the sample test solution compounds MS2 spectra
by mass spectral library
Although the modied TLC method facilitated the discrimination between the target compounds compared to the initial
method, MS2 analysis was required for identication of the compounds in the sample test solutions. However, without the created

K. Naumoska, I. Vovk / J. Chromatogr. A 1381 (2015) 229238

237

Table 3
Triterpenoids and phytosterols identied in different sample test solutions veried by the mass spectral library. Shaded elds correspond to positively assigned compounds
in the vegetable test solutions. The assignments that were previously reported in the literature are marked with asterisk.
Vegetable

-Sitosterol

Stigmasterol

Zucchini
Eggplant
Tomato
Red pepper
Mangold
Spinach
Lettuce

*
*
*
*

*
*
*
*

White-colored
radicchio di
Castelfranco
Radicchio Leonardo
White cabbage
Red cabbage
Savoy cabbage

Cycloartenol

-Amyrin

-Amyrin

*
*

*
*

*
*

mass spectral library, built from the MS2 spectra of the standards,
the identication of the compounds in the sample test solutions
could not be done clearly. As the closely eluting positional isomers
gave complex and similar MS2 spectra, positive assignments based
solely on the library statistical results were not made. Therefore,
head to tail comparison between the acquired sample test solution
zones MS2 spectra and those of the corresponding standards was
done. This comparison mode plots the relative intensities against
m/z, where the rst spectrum points upward, and the second one
points downward from a common axis. Therefore, similarities and
differences in the fragmentation patterns could be seen clearer than
in the parallel mass spectra comparing. The axis symmetry in the
head to tail output indicates positive assignment. Example of how
the qualitative analysis of cycloartenol, -amyrin and -amyrin in
red pepper test solution was performed is shown in Fig. 7.
To the best of our knowledge, this is the rst report of the
presence of certain triterpenoids and phytosterols in the selected
vegetable waxes (Table 3). Table 3 summarizes the positive assignments veried by mass spectral library (head to tail comparison).
A great part of the previously published data (Table 3, marked
by *) was in accordance with that reported in the literature
[13,14,2022,4043]. The compounds that were not positively
assigned were not present in the studied vegetable types, not
detected due to the low concentrations in the sample test solutions
or not detected due to spectra averaging between the coeluting target and matrix compounds (false negative). Moreover, this is the
rst simultaneous qualitative TLCMS2 study of the common neutral triterpenoids and phytosterols in plant materials. A qualitative
TLCMS2 study of triterpenoid acids was also reported for the rst
time by our group [15].

4. Conclusions
Three TLC methods were employed for the initial screening
of triterpenoids and phytosterols in vegetable cuticular waxes.
The preliminary experiments showed that the vegetable cuticular
waxes could be potential sources of triterpenoids and phytosterols.
For the purpose of their identication, a new TLCMS2 method
was proposed. It combined the advantages of the satisfactory
separation of the target compounds, their different band colors,
as well as the possibility of their discrimination based on MS
and MS2 spectra. Since the MS2 spectra were similar among the
closely eluting compounds, the characteristic ions were proposed.
For unambiguous identication, a mass spectral library was built
from the MS2 spectra of the target compounds. The head to tail
mode of comparison between the sample test solution zones and

Cycloartenol
acetate

Lupeol

Friedelin

Lupeol
acetate

Lupenone

corresponding standard MS2 spectra have increased the clarity

of the results obtained. This study reports the rst simultaneous
TLCMS2 method for the identication of the common neutral
triterpenoids and phytosterols in vegetable extracts. In addition
to the molecular mass information, the developed (+)APCI-MS2
method provided information regarding to the nature of the
molecular functional group, as well. This approach enabled positive assignment of different triterpenoids and phytosterols in the
studied vegetable cuticular waxes, of which many are identied
for the rst time.
Acknowledgements
The study was carried out with the nancial support from the
Slovenian Research Agency (P1-0005) and EN-FIST Centre of Excellence. The authors want to express their most sincere gratitude
to Mateja Puklavec, Andreja Starc, Dr. Vesna Glavnik and Dr. Alen
Albreht for the generous help during the experimental work, as well
to Dr. Zoran Kitanovski for the help during the preparation of the
manuscript.
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