Article

pubs.acs.org/JAFC

Generic Characterization of Apolar Metabolites in Red Chili Peppers

(Capsicum frutescens L.) by Orbitrap Mass Spectrometry.
Sebastiaan Bijttebier,*,, Kaouther Zhani,, Els DHondt, Bart Noten, Nina Hermans, Sandra Apers,
and Stefan Voorspoels

Business Unit Separation and Conversion Technology (SCT), Flemish Institute for Technological Research (VITO), Boeretang 200,
2400 Mol, Belgium

Department of Horticulture and Landscape, Sousse University, Higher Institute of Agronomy, 4042 Chott Mariem, Tunisia

NatuRA, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium

*
S Supporting Information

ABSTRACT: The aim of the present study was to develop a generic analytical method for the identication and quantitation of
apolar plant metabolites in biomass using liquid chromatographyphotodiode arrayaccurate mass mass spectrometry (LC-
PDA-amMS). During this study, a single generic sample preparation protocol was applied to extract apolar plant metabolites.
Compound identication was performed using a single generic screening method for apolar compounds without the need for
dedicated fractionation. Such a generic approach renders vast amounts of information and is virtually limited by only the
solubility and detector response of the metabolites of interest. Method validation conrmed that this approach is applicable for
quantitative purposes. Furthermore, an identicationquantitation strategy based on amMS and molar extinction coecients was
used for carotenoids, eliminating the need for reference standards for each carotenoid. To challenge the validated method, chili
peppers (Capsicum frutescens L.) were analyzed to unravel their complex phytochemical composition (carotenoids, glycolipids,
glycerolipids, capsaicinoids, lipid-soluble vitamins).
KEYWORDS: LC-PDA-amMS, quantitation without reference standards, extinction coecients,
generic analysis of apolar plant metabolites, biomass composition, Capsicum frutescens L., carotenoids

INTRODUCTION
Biomass such as algae and vegetables often contains natural
components of high value including the so-called phytochem-
icals, such as antioxidants and essential oils, that are barely
utilized from these resources today.1,2 These natural products
are thought to provide benecial health eects and have an
increasing sales market in the pharmaceutical, cosmetics, and
food supplements industry.3 Knowing the composition of these
resources is thus an essential rst step in a potential valorization
process.
Because of the complexity of compound mixtures in
biological samples, the usage of simple analytical instrumenta-
tion such as liquid chromatographyultraviolet detection (LC-
UV) has shifted toward more selective and more complex
separation and detection systems such as ultrahigh-performance
liquid chromatographyphotodiode arrayaccurate mass mass
spectrometry (UHPLC-PDA-amMS) instrumentation to
achieve more denitive compound identication.4 Accurate
mass MS detectors allow the tentative identication of
compounds without the use of analytical standards.5 Because
of the limited availability of analytical standards for plant
metabolites, this utility has shown to be essential in natural
products exploration.6 Furthermore, modern high-end ana-
lytical instrumentation allows the detection of multiple
compound classes, such as carotenoids, capsaicinoids, and
glycolipids, in a single analysis, resulting in enormous time and
consumables savings. To avoid compound discrimination
during extraction, no cleanup steps should be used and the
number of sample preparation steps should be limited as much
as possible. Such a generic approach is virtually limited only by
the solubility (extractability, chromatographic retention) and
detector response (UV absorbance and ionization eciency) of
the compounds of interest. Furthermore, the use of a generic
analytical screening application allows the setup of a compound
database with specic chromatographic and spectrometric data.
This database enables an eective dereplication during structure
elucidation of the extract composition.7
The aim of the present study was to develop, optimize, and
validate a generic analytical method for the identication and
quantitation of apolar plant metabolites in biological matrices
using UHPLC-PDA-amMS. Authentic standards of multiple
phytochemical classes and various biological matrices were used
to conrm and emphasize the generic character of the method.
To challenge the validated method, the widely studied chili
pepper matrix (Capsicum frutescens L.) was selected as it is
known for its complex phytochemical composition (carote-
noids, glycolipids, glycerolipids, capsaicinoids, lipid-soluble
vitamins).813 Finally, an identicationquantitation strategy
based on the use of amMS data and extinction coecients was
applied for carotenoids, eliminating the need for reference
standards for each carotenoid.
Received: January 17, 2014
Revised: April 23, 2014
Accepted: April 24, 2014
Published: April 24, 2014

2014 American Chemical Society 4812 dx.doi.org/10.1021/jf500285g | J. Agric. Food Chem. 2014, 62, 48124831
Journal of Agricultural and Food Chemistry
MATERIALS AND METHODS
Chemicals. LC-MS grade methanol, acetonitrile, and ethyl acetate
were purchased from Biosolve (Valkenswaard, The Netherlands).
Ultrapure water with a resistivity of 18.2 Mcm at 25 C was
generated with a Millipore system. Dichloromethane for gas
chromatography, n-hexane for gas chromatography, acetone for gas
chromatography, sodium hydrogen carbonate, sodium hydroxide, and
sodium chloride for analysis were purchased from Merck (Darmstadt,
Germany). Ammonium acetate, (D-Ala2)-leucine enkephalin, sand
(quartz), and butylated hydroxytoluene were purchased from Sigma-
Aldrich (Bornem, Belgium). Certied reference material BCR 485
consisting of freeze-dried mixed vegetables was bought from IRMM
(Geel, Belgium). Commercially available mixtures to calibrate the mass
spectrometer, that is, MSCAL5-1EA (caeine, tetrapeptide Met-Arg-
Phe-Ala, Ultramark) for positive ion mode and MSCAL6-1EA
(sodium dodecyl sulfate, taurocholic acid sodium salt, Ultramark)
for negative ion mode, were purchased from Supelco (Bellefonte, PA,
USA).
Lycopene, -carotene, phytoene, lutein, zeaxanthin, tunaxanthin,
astaxanthin dipalmitate, phytouene, antheraxanthin, violaxanthin,
canthaxanthin, and astaxanthin were purchased from Carotenature
(Ostermundigen, Switzerland). -Carotene, trans--apo-8-carotenal,
campesterol, stigmasterol, -sitosterol, glyceryl trioleate, glyceryl
dioleate, -tocopherol, -tocopherol, phylloquinone, ergocalciferol,
and cholecalciferol were purchased from Sigma-Aldrich. Glycerol
dioleate and glycerol trioleate were obtained from Chem Service
(West Chester, PA, USA). Monogalactosyldiacylglycerol with average
MW 752.369 and digalactosyldiacylglycerol with average MW 926.767
were purchased from Avanti (Alabaster, AL. USA).
Preparation of Standard Solutions. Standard stock solutions
and working solutions were prepared of each analyte separately at a
concentration of approximately 200 g/mL. Stock solutions of glyco-
and glycerolipids were prepared in methanol. The stock solutions of
sterols and lipid-soluble vitamins were prepared in methanol + 0.1%
butylated hydroxytoluene. Stock solutions of carotenoids were
prepared in dichloromethane + 0.1% butylated hydroxytoluene.
Standard stock and working solutions were stored at 25 C in the
dark under an inert atmosphere (nitrogen). Dilutions of the stock
solutions were prepared in dichloromethane + 0.1% butylated
hydroxytoluene for analysis.
Sample Preparation. Red bell peppers (Capsicum annuum) and
apples (Malus domestica) were bought in a local store. After removal of
the stems and kernels, they were cut into pieces, frozen with liquid
nitrogen, and immediately freeze-dried. After drying, they were milled
and homogenized with a Grindomix GM 200 from Retsch (Haan,
Germany) at 10000 rpm and stored in the dark under nitrogen at 25
C.
Fruits of ve chili pepper accessions (Capsicum frutescens L.),
namely, Tebourba, Korba, Somaa, Awlad Haouzz, and Souk
jedid, were collected from plants cultivated in the experimental station
of the Higher Institute of Agriculture, Chott Mariem, Tunisia. The
peduncles of the peppers were removed, and the fruits were freeze-
dried, milled, and stored at 25 C until analysis.
Before sample preparation, the samples were allowed to equilibrate
to room temperature. One gram of sample was mixed with 1 g of
sodium hydrogen carbonate, spiked with trans--apo-8-carotenal
(internal standard), and mixed with sand. Ultrapure water was
added until the sample was visually completely hydrated (approx-
imately 3 mL, depending on the matrix). The mixture was kept in the
dark under N2 to allow swelling of the matrix for better analyte
extraction. Afterward, the sample was mixed again with sand and
loaded into a 33 mL accelerated solvent extraction (ASE) cell
(Thermo Fisher Scientic, Bremen, Germany). The mixture was
extracted three times (5 min static extraction) with 70:30 acetone/
methanol + 0.1% butylated hydroxytoluene at 40 C and 1050 psi,
resulting in approximately 120 mL of sample extract. The three
extracts were combined in a separation funnel, and 100 mL of 10%
sodium chloride (aqueous) and 15 mL of hexane were added. The
hexane phase was retained, and the polar phase was extracted twice
Article
more with 15 mL of hexane. The pooled hexane fractions were
evaporated, dissolved in 10 mL of dichloromethane + 0.1% butylated
hydroxytoluene, an aliquot was used for analysis, and the remaining
extract was stored in the dark under nitrogen at 25 C until further
treatment.
For saponication 2 mL of 10% sodium hydroxide (in methanol)
was added to 2 mL of dichloromethane extract and was consecutively
shaken for 6 h at room temperature in the dark under an inert
atmosphere. Afterward, the solution was washed ve times with water
to remove the alkali.14 An aliquot of the saponied dichloromethane
extract was used for analysis.
Instrumental Analysis. The analytical method was previously
described4 and is briey summarized as follows. For analysis, 1.25 L
of extract was injected with a CTC PAL autosampler (CTC Analytics,
Zwingen, Switzerland) on a 100 mm 2.1 mm, 1.8 m, Acquity
UPLC HSS C18 SB column (Waters, Milford, MA, USA) and
thermostatically (35 C) eluted with an Accela quaternary solvent
manager and a Hot Pocket column oven (Thermo Fisher Scientic,
Bremen, Germany) with a chromatographic gradient. The mobile
phase solvents consisted of 50:22.5:22.5:5 (v/v/v/v) water + 5 mM
ammonium acetate/methanol/acetonitrile/ethyl acetate (A) and 50:50
(v/v) acetonitrile/ethyl acetate (B), and the gradient was set as follows
(min/% A): 0.0/90, 0.1/90, 0.8/70, 20.0/9, 20.1/0, 20.4/0, 20.5/90,
23.0/90. For detection, an Exactive amMS (Thermo Fisher Scientic)
was used in positive atmospheric pressure chemical ionization (APCI)
mode. Full scan and all-ion fragmentation data were acquired with a
scanning range of m/z 1001400 at a resolution of 50000 full width at
half-maximum (fwhm). The Accela PDA detector (Thermo Fisher
Scientic) was set to scan from 190 to 800 nm.
RESULTS AND DISCUSSION
Method Validation. Carotenoids were chosen as model
compounds for the optimization of a generic sample
preparation and screening method for apolar plant metabolites.
Carotenoids are apolar plant metabolites that can easily degrade
or isomerize under the inuence of light, oxygen, enzymes,
heat, oxidants, and acid or alkaline conditions.15 It can hence be
assumed that if no isomerization or degradation occurs for
carotenoids during sample preparation, most other apolar
compounds should also be extracted without any artifact
formation. The chromatographic gradient and MS settings were
optimized on an UHPLC-PDA-amMS for a set of carotenoids
representing the wide polarity range of carotenoids found in
nature.4 A validation study of the optimized method was
performed for a range of compounds from several apolar
metabolite classes (12 carotenoids, 5 lipid-soluble vitamins, 1
phytosterol) and dierent matrices. Furthermore, the extraction
eciency of other apolar plant metabolites (glycolipids,
glycerolipids, steryl derivatives, chlorophyll) for which no
commercial reference standards were available was investigated
to conrm its generic character.
Dynamic Range and Sensitivity. High dynamic ranges
(up to 4 orders of magnitude) with good linear ts were
obtained in MS for most compound calibration curves. Because
of the lower sensitivity of carotenoids in UV detection and the
high cost of carotenoid standards, the dynamic ranges of the
carotenoids were not tested to their full extent with PDA
detection. However, it could be observed that smaller dynamic
ranges were acquired for the very apolar carotenes in both MS
and PDA, suggesting lower solubility to be the cause. Detection
and quantitation limits are of less importance for valorization
objectives as predominantly compounds with high concen-
trations are targeted.
Precision. The repeatability (intraday RSD) was obtained
from the relative standard deviations (RSDs) of the recoveries
from the spiking experiments. Because of the high price of
4813 dx.doi.org/10.1021/jf500285g | J. Agric. Food Chem. 2014, 62, 48124831
Journal of Agricultural and Food Chemistry
Figure 1. Representative structures of the dierent compound groups detected in chili peppers during this study.
analytical standards, it was chosen to obtain the intermediate
precision (interday RSD) of the method with compounds
natively present in red bell pepper (Capsicum variety). Five
aliquots of freeze-dried pepper were extracted and analyzed the
same day. This experiment was repeated for 3 days in total.
Intraday RSDs ranged between 0.5 and 8.4% and interday
RSDs between 0.0 and 6.0%.
Trueness. Certied reference material (CRM) is commer-
cially available for only a very limited set of plant metabolites.
Furthermore, reference standards are often not commercially
available or very expensive for plant metabolites. Therefore, the
trueness could only be assessed for a select set of compounds. A
CRM of mixed vegetables, BCR 485 (consisting of sweet corn,
tomatoes, and carrots), with certied concentration values for
all-trans-- and -carotene, lutein, and lutein + zeaxanthin was
extracted in triplicate. Values of 110 8, 105 3, 100.5 2.7,
and 94.5 1.4% of the certied concentration values were
found with MS, respectively, and 110.5 2.5, 106.2 1.1, 105
4, and 101 5% with PDA, respectively, with the
uncertainties being the RSD of the three replicates. These
results show good accuracy of the method for the quantitative
determination of carotenoids natively present in biological
matrices.
Spiking experiments were performed in triplicate on aliquots
of freeze-dried apple (M. domestica) using an analytical standard
mixture with 11 carotenoids, 6 lipid-soluble vitamins, and 1
phytosterol (concentrations ranging between 5.3 and 140 g/g
dw). The recoveries (R (%)) for all spiked compounds except
violaxanthin ranged between 86 and 115% with MS detection,
showing good accuracy. The slightly lower recoveries (7487%
in MS) found for violaxanthin can be attributed to epoxide
rearrangements (which lead to large hypsochromic shifts in UV
4814
Article
absorbance), catalyzed by the acids naturally present in the
apple matrix.16 Because of the low UV absorbance of
phytosterols and lipid-soluble vitamins, only the recoveries of
most of the spiked carotenoids could be calculated with PDA
detection, showing similar values compared to the results
calculated with MS.
Extraction Eciency. Because of the lack of commercially
available CRMs and reference standards, the conventional
approach of method validation could be performed for only a
select set of apolar plant metabolites. To investigate the
extraction eciency for a larger range of apolar plant
metabolites, the two most critical steps in sample preparation
were tested by exhaustive extraction of compounds naturally
present in peppers. In a rst experiment, an aliquot of freeze-
dried red bell pepper was extracted ve times with ASE
followed by separate preparation of the obtained extracts.
Assuming 100% extraction, the relative abundances of the
compounds were calculated in the respective extracts. In a
second experiment, the transfer of compounds to the hexane
phase during liquidliquid extraction was investigated by back-
extracting the polar phase six times with hexane. After the rst
three hexane extracts had been pooled, an extra 240 mL of 10%
sodium chloride solution was added to the polar phase. The
polar phase was extracted three more times. The rst two
apolar extracts resulting from this second series of extractions
were pooled. Subsequently, the three remaining extracts
(consisting of three pooled, two pooled, and a single extract)
were prepared separately for analysis. Again assuming 100%
extraction, the relative abundances of the compounds were
calculated in the respective extracts. The results showed that
during the two most critical steps in the extraction procedure,
dx.doi.org/10.1021/jf500285g | J. Agric. Food Chem. 2014, 62, 48124831
 Table 1. Values for Maximum Absorbance, Precursor and Product Ions, and Retention Times of the Carotenoids Present in the Five Chili Pepper Accessions
retention time max absorbance MS accurate mass of mass deviation identification
compd identity mol formula (min) (nm) mode precursor ion (ppm) in-source fragments type
1 cucurbitaxanthin A C40H56O3 6.77 426, 449, 476 pos 585.43068 0.79 567.420 a, b
neg - - -

2 cycloviolaxanthin C40H56O4 7.29 418, 443, 472 pos 601.42497 0.28 a, b

neg - - -

3 cis-capsanthin C40H56O3 7.77 467 pos 585.43029 0.12 567.419 c

neg 584.42392 0.74 -

4 all-trans-capsanthin C40H56O3 7.93 474 pos 585.43027 0.09 567.419, 549.409, 493.352, 475.35735, 461.342 d
neg 584.42352 0.05 -

5 cis-antheraxanthin C40H56O3 8.67 450, 472 pos 585.43029 0.12 567.419, 549.409, 493.352, 475.357 35 a
Journal of Agricultural and Food Chemistry

neg 584.42385 0.62 -

6 all-trans-zeaxanthin C40H56O2 10.12 455, 481 pos 569.43546 0.26 551.425 d

neg 568.42884 0.46 -

7 all-trans-apo-carotenal C30H40O 10.38 465 pos 417.31602 1.99 - d

neg 416.30875 0.70 -

4815
8 nigroxanthin C40H54O2 12.62 448, 478 pos 567.41995 0.51 - a, b
neg - - -

9 -cryptoxanthin C40H56O 13.61 456, 483 pos 553.44074 0.63 535.43 a, b, c, e

neg 552.43436 1.27 -

10 capsorubin-laurate (C12:0) C52H78O5 13.85 479 pos 783.59123 1.24 583.414, 565.405 b,e
neg 782.58620 0.93 -

11 karpoxanthin-laurate (C12:0) C52H80O5 14.77 424, 448, 477 pos 785.60732 0.67 767.597, 585.431, 567.420, 549.410, 475.357 a, b,f
neg 784.60170 0.74 -

12 capsorubin-myristate (C14:0) C54H82O5 15.01 pos 811.62242 1.33 583.414, 565.405 b, e

neg 810.61682 0.06 -

13 all-trans-capsanthin-laurate (C12:0) C52H78O4 15.01 475 pos 767.59687 0.55 749.587, 567.420 c, e
neg 766.59080 0.31 -

14 - C52H76O4 15.38 435, 465, 498 pos 765.58206 0.55 565.404 -

neg 764.57505 0.18 672.513, 658.497, 564.397
Article

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 Table 1. continued
retention time max absorbance MS accurate mass of mass deviation identification
compd identity mol formula (min) (nm) mode precursor ion (ppm) in-source fragments type
15 all-trans-capsanthin-laurate (C12:0) C52H78O4 15.72 474 pos 767.59639 1.17 749.587, 567.420, 549.409 c, e
neg 766.59059 0.04 674.528, 660.513, 566.413

16 cis-capsanthin-laurate (C12:0) C52H78O4 15.85 463 pos 767.59704 0.33 749.587, 567.420 c
neg 766.59063 0.09 -

17 cis-antheraxanthin-laurate (C12:0) C52H78O4 16.03 448, 478 pos 767.59672 0.74 749.587, 567.420, 549.409 c
neg 766.59051 0.07 -

18 all-trans-capsanthin-myristate (C14:0) C54H82O4 16.15 474 pos 795.62818 0.52 777.617, 567.420, 549.409 c, e
neg 794.62201 0.19 566.413, 227.202

19 all-trans--carotene C40H56 16.34 455, 482 pos 537.44556 0.15 457.384, 445.384 d
Journal of Agricultural and Food Chemistry

neg 536.43894 0.35 -

20 mutatoxanthin-laurate (C12:0) C52H78O4 16.45 405, 428, 457 pos 767.59653 0.99 567.420 b
neg 766.59097 0.53 -

21 cucurbitaxanthin A-laurate (C12:0) C52H78O4 16.54 426, 451, 474 pos 767.59682 0.61 749.587, 567.420, 549.409 a, b
neg 766.59099 0.56 -

4816
22 all-trans-phytofluene C40H62 16.66 334, 349, 369 pos 543.49255 0.22 - d
neg - - -

23 all-trans-capsanthin-myristate (C14:0) C54H82O4 16.83 473 pos 795.62760 1.24 777.617, 567.420, 549.409 c, e
neg 794.62178 0.10 566.413, 227.202

24 cis-antheraxanthin-myristate (C14:0) C54H82O4 17.05 450, 477 pos 795.62827 0.40 777.617, 567.420, 549.409 c
neg 794.62168 0.23 -

25 all-trans-phytoene C40H64 17.08 276, 288, 297 pos 545.50774 0.62 463.430 d
neg - - -

26 all-trans-zeaxanthin-laurate (C12:0) C52H78O3 17.16 455, 483 pos 751.60171 0.88 551.425 c
neg 750.59539 0.33 -

27 cis-capsanthin-palmitate (C16:0) C56H86O4 17.21 463 pos 823.65929 0.73 805.650, 567.420, 549.409 -
neg 822.65320 0.05 566.413, 255.232

28 cis-zeaxanthin-laurate (C12:0) C52H78O3 17.52 424, 447, 474 pos 751.60229 0.11 - -
neg 750.59536 0.37 -
Article

dx.doi.org/10.1021/jf500285g | J. Agric. Food Chem. 2014, 62, 48124831

 Table 1. continued
retention time max absorbance MS accurate mass of mass deviation identification
compd identity mol formula (min) (nm) mode precursor ion (ppm) in-source fragments type
29 mutatoxanthin-myristate (C14:0) C54H82O4 17.54 405, 430, 456 pos - - 777.617, 567.420, 549.409 b
neg 794.62109 0.97 -

30 all-trans-capsanthin-palmitate (C16:0) C56H86O4 17.83 472 pos 823.65985 0.05 805.650, 567.420, 549.409 e
neg 822.65301 0.18 -

31 cis-zeaxanthin-myristate (C14:0) C54H82O3 18.00 423, 447, 477 pos 779.63226 1.81 551.425 c
neg 778.62652 0.54 -

32 all-trans-zeaxanthin-myristate (C14:0) C54H82O3 18.16 454, 481 pos 779.63263 1.33 761.622, 551.425 c, e
neg 778.62677 0.22 550.418

33 all-trans-zeaxanthin-myristate (C14:0) C54H82O3 18.37 455, 479 pos - - - c, e

Journal of Agricultural and Food Chemistry

neg 778.62666 0.36 -

34 - - 18.44 473 pos - - - -

neg - - 804.642 44

35 - C58H88O4 18.58 434, 456 pos - - - -

neg 848.66846 0.41 -

4817
36 -cryptoxanthin-laurate (C12:0) C52H78O2 18.8 456, 482 pos 735.60815 0.94 535.430 c, e
neg 734.60119 0.63 -

37 all-trans-capsanthin-dilaurate (C12:0, C64H100O5 19.12 476 pos 949.76483 0.51 749.587, 549.409 e
C12:0) neg 948.75682 0.84 -

38 cis-capsanthin-dilaurate (C12:0, C12:0) C64H100O5 19.32 463 pos - - 749.587, 549.409 -

neg 948.75618 1.52 -

39 - - 19.50 474 pos - - - -

neg - - -

40 -cryptoxanthin-myristate (C14:0) C54H82O2 19.56 455, 483 pos - - 535.430 c, e

neg 762.63197 0.08 -

41 all-trans-capsanthin-laurate-myristate C66H104O5 19.78 474 pos 977.79388 1.81 749.587, 549.409 c, e

(C12:0, C14:0) neg 976.78806 0.88 -

42 cis-capsanthin-laurate-myristate (C12:0, C66H104O5 19.97 463 pos - - 749.587, 549.409 -

C14:0) neg 976.78790 1.04 -
Article

dx.doi.org/10.1021/jf500285g | J. Agric. Food Chem. 2014, 62, 48124831

 Table 1. continued
retention time max absorbance MS accurate mass of mass deviation identification
compd identity mol formula (min) (nm) mode precursor ion (ppm) in-source fragments type
43 cis-capsanthin-laurate-myristate (C12:0, C66H104O5 20.13 464 pos - - 749.587, 549.409 -
C14:0) neg 976.78853 0.40 -

44 -cryptoxanthin-palmitate (C16:0) C56H86O2 20.25 457, 482 pos - - - c, e

neg 790.66218 1.45 -

45 all-trans-capsanthin-dimyristate (C14:0, C68H108O5 20.36 473 pos 1.005.82526 1.68 777.617 c, e

C14:0) neg 1.004.81983 0.39 776.611, 227.202

46 all-trans-capsanthin-laurate-palmitate C68H108O5 20.57 473 pos - - 805.650, 749.588, 549.409 c, e

(C12:0, C16:0) neg 1.004.81839 1.82 776.611

47 cis-capsanthin-dimyristate (C14:0, C14:0) C68H108O5 20.72 464 pos - - 777.617, 549.409 -

Journal of Agricultural and Food Chemistry

neg 1.004.81860 1.61 776.611

48 all-trans-capsanthin-myristate-palmitate C70H112O5 20.93 475 pos - - 805.648, 777.617, 549.409 -

(C14:0, C16:0) neg 1032.85050 0.99 776.611, 548.4

49 cis-capsanthin-myristate-palmitate (C14:0, C70H112O5 21.10 464 pos - - 805.648, 777.617 -

C16:0) neg 1032.84970 1.76 776.611

4818
50 cis-capsanthin-myristate-palmitate (C14:0, C70H112O5 21.19 463 pos - - 805.648, 777.617, 549.409 -
C16:0) neg 1032.85057 0.92 776.611, 548.4

51 all-trans-capsanthin-dipalmitate (C16:0, C72H116O5 21.33 473 pos - - 805.648, 549.409 c, e

C16:0) neg 1060.88213 0.65 804.640, 255.233

52 all-trans-zeaxanthin-laurate-myristate C66H104O4 21.37 453, 480 pos - - 761.622, 733.591, 533.414 c, e

(C12:0- C14:0) neg 960.79331 0.73 760.614, 732.584

53 all-trans-zeaxanthin-dimyristate (C14:0- C68H108O4 21.37 453, 480 pos - - 761.622, 533.414 c, e

C14:0) neg 988.82436 0.96 760.614
a
Dictionary of Natural Products:18 present in Capsicum annuum. bCarotenoids Handbook: present in Capsicum annuum and match with UV reference spectrum. cIdentied by Giurida et al..13 dMatch with
analytical standard. eIdentied by Schweiggert et al.;10 fIdentied by Deli et al.8
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Journal of Agricultural and Food Chemistry
Figure 2. Chromatogram of the PDA signal at 470 nm depicts the elution proles of the carotenoids present in the Korba chili pepper accession.
the greater part of compounds is extracted during the rst three
extractions (>90%).
Measurement Uncertainty. The results of the spiking
experiments, CRM analysis, and intermediate precision were
used to estimate the total expanded measurement uncertainty
(U; k = 2). As not all compounds included during the spiking
experiments were available for intermediate precision experi-
ments, the intra- and interday RSDs of compounds of the same
compound group were used to calculate U (e.g., for PDA
analysis, the RSDs of -carotene were applied for the
calculation of U for -carotene and lycopene). The obtained
U values ranged between 10 and 26% (MS) and between 14
and 21% (PDA) for all compounds except violaxanthin, for
which the U values were higher (44 and 59% for MS and PDA,
respectively) as the bias of the lower R (%) obtained for
violaxanthin was included in the calculation.
Identication Strategy. Identication of compounds is
relatively easy when analytical standards are available. However,
in natural products research, analytical standards are often very
expensive or not commercially available. Therefore, identi-
cation is often based upon the available chromatographic and
spectrometric information.813 Carotenoids absorb UV light in
a high-wavelength area (typical absorbance maxima at 400500
nm) because of their conjugated system in the polyene
backbone (e.g., molecular structures 4 and 45 in Figure 1).17
PDA detectors are therefore very selective for the detection of
carotenoids. However, the applicability of PDA detectors is
limited as they are only useful for compounds that absorb UV
or visible light and provide only very little structural
information, which makes it impossible to identify unknown
compounds with PDA spectra.10 MS detectors are much more
selective detectors than PDA detectors and can therefore
facilitate the identication of plant metabolites. Orbitrap MS
detectors can routinely generate mass spectra with a resolving
power up to 140,000 at fwhm and obtain mass accuracies
within 2 ppm (ppm), which enables the calculation of the most
probable molecular formulas of the generated ions and
fragments.5 However, to date, even the most state-of-the-art
MS detectors are often not capable of distinguishing between
isomers, whereas PDA detectors in many cases are.4,15
Furthermore, chromatography can be used to separate isomers
before detection. The combination of orthogonal analytical
techniques therefore provides a powerful tool for the
identication of unknown compounds.
During this study carotenoids, steryl derivatives, glycolipids,
glycerolipids, capsaicinoids, and lipid-soluble vitamins were
4819
Article
tentatively identied in chili pepper samples. The information
provided by the analysis alone is, however, not always enough
for peak identication at an acceptable condence level.
Therefore, other sources of information such as in-house and
commercial compound databases and peer-reviewed publica-
tions can give additional information for successful dereplica-
tion.7 During this study, the Dictionary of Natural Products18
and the Carotenoids Handbook19 were consulted as commercial
databases. Both of these databases give information on the
occurrence of plant metabolites in nature. The Carotenoids
Handbook also lists the UV absorbance spectra of most of the
carotenoids known to exist in nature. These were used as
reference spectra for comparison of experimental UV spectra if
identication by amMS spectra resulted in several possible
structures.
In the present study structures were assigned to unknown
peaks only when both the mass/charge (m/z) ratios and
molecular formulas of the ions were in agreement. PDA spectra
often provided conrmation for the proposed structures when
the reference information was available. Furthermore, the
assigned identities often matched with their occurrence in chili
peppers as described in the literature and databases. Figure 1
depicts the molecular structures of the identied plant
metabolite classes. Tables 1 and 4 show the diagnostic amMS
and PDA data used for compound identication in the chili
pepper samples. These tables also specify the literature and
databases used for conrmation of compound identity.
Carotenoids in Chili Peppers. Identication of Car-
otenoids. A wavelength of 470 nm is very specic to measure
carotenoid absorbance. This wavelength was used to depict the
carotenoids present in the Awlad Haouzz accession (Figure
2). The PDA chromatograms of the other four accessions
appeared identical (not shown). All of the peaks in Figure 2
could be tentatively attributed to carotenoids as no chlorophyll
peaks were detected (derived from the PDA proles of the
peaks). The greater part of PDA peaks in the chromatograms
contained the same absorption proles. As described
previously,10 carotenoid esterication with fatty acids does
not aect the polyene backbone, and therefore esterication has
virtually no eect on the absorption spectrum. The character-
istic UV proles of the nonesteried carotenoids, such as
capsanthin, antheraxanthin, and zeaxanthin, can therefore also
be used to locate their esters. Complementary to PDA
detection, fatty acid esters of carotenoids easily fragment
during MS ionization, and therefore information on the type of
fatty acid moieties (number of carbons, number of double
dx.doi.org/10.1021/jf500285g | J. Agric. Food Chem. 2014, 62, 48124831
Journal of Agricultural and Food Chemistry Article

Figure 3. Chromatogram of the PDA signal at 470 nm depicts the elution proles of the carotenoids present in the Korba chili pepper accession
extract after 1 year of storage, after saponication. Peaks: 1, capsorubin; 2, cucurbitaxanthin A; 3, cis-capsanthin; 4, cis-capsanthin; 5, trans-capsanthin;
6, cis-antheraxanthin; 7, mutatoxanthin; 8, karpoxanthin; 9, cis-zeaxanthin; 10, trans-zeaxanthin; 11, cis--apo-8-carotenal; 12, trans--apo-8-
carotenal; 13, -cryptoxanthin; 14, trans--carotene; 15, cis--carotene.

Figure 4. Correlation of the retention times and m/z values of the compounds identied in the chili peppers with the analytical screening method.
The data labels match the compound numbers in Tables 1 and 4

bonds) can be obtained from full scan amMS spectra, as
described earlier.4,10,13 Most of the carotenoids in the chili
pepper samples were esteried with laurate, myristate, and
palmitate, and the major carotenoid present in the sample was
4820
capsanthin, in agreement with previuos ndings.10 Other
studies10,17,20 have shown that the cis isomers of carotenoids
can be identied on the basis of a small hypsochromic shift
(shorter wavelength) of the maximum absorbance values and a
dx.doi.org/10.1021/jf500285g | J. Agric. Food Chem. 2014, 62, 48124831
Journal of Agricultural and Food Chemistry Article

Table 2. Carotenoid Concentrations in the Five Chili Pepper Cultivars Calculated with Analytical Standards and Molar
Extinction Coecients
concentration (g/g dw)
retention time Awlad
compd identity (min) Haouzz Tebourba Korba Somaa Souk jedid
4 all-trans-capsanthina 7.93 130 20 70 10 100 20 100 20 110 20
5 cis-antheraxanthina,e 8.67 27 5 18 3 22 4 26 5 24 4
6 all-trans-zeaxanthina 10.12 25 5 13.0 2.5 25 5 9.0 1.8 12.0 2.3
19 -carotenea 16.34 130 20 120 20 80 10 100 20 110 20
22 all-trans-phytouenea 16.66 27 6 22 5 17 4 21 4 20 4
25 all-trans-phytoenea 17.08 90 10 68 9 48 7 70 10 58 8
3 cis-capsanthinf 7.77
12 + 13 capsorubin-myristate + capsanthin-laurateb,g,h,i 15.01 260 50 220 50 180 40 310 60 190 40
15 all-trans-capsanthin-laurateb,g 15.72 70 20 80 20 70 10 90 20 50 10
16 cis-capsanthin-lauratef 15.85
17 cis-antheraxanthin-laurateb,e,j 16.03 70 20 70 20 70 20 100 20 60 10
18 all-trans-capsanthin-myristateb,g 16.15 430 90 390 80 320 70 540 110 450 90
23 all-trans-capsanthin-myristateb,g 16.83 120 30 150 30 120 30 140 30 110 20
24 cis-antheraxanthin-myristateb,e,j 17.05 80 20 80 20 70 10 80 20 60 10
26 + 27 all-trans-zeaxanthin-laurate + cis-capsanthin- 17.16 + 17.21 160 30 190 40 160 30 190 40 200 40
palmitateb,g,h,i
30 all-trans-capsanthin-palmitateb,g 17.83 60 10 80 20 50 10 70 10 60 10
31 cis-zeaxanthin-myristateb,k 18 11.3 2.3 19 4 14 2.8 13.4 2.7 14.6 2.9
32 all-trans-zeaxanthin-myristateb,k 18.16 80 20 70 10 90 20 50 10 37 7
33 all-trans-zeaxanthin-myristateb,k 18.37 39 7 50 10 50 10 70 10 28 5
37 all-trans-capsanthin-dilaurateb,g 19.12 380 80 420 90 330 70 430 90 210 40
38 cis-capsanthin-dilaurateb,g 19.32 18 4 13.8 2.9 20 4 19 4 11.2 2.4
41 all-trans-capsanthin-laurate-myristateb,g 19.78 700 200 800 200 700 100 900 200 500 100
42 cis-capsanthin-laurate-myristateb,g 19.97 37 8 27 6 44 9 50 10 30 10
43 cis-capsanthin-laurate-myristateb,g 20.13 120 30 110 20 120 20 150 30 80 20
45 all-trans-capsanthin-dimyristateb,g 20.36 700 100 800 200 700 100 800 200 600 100
46 all-trans-capsanthin-laurate-palmitateb,g 20.57 30 6 25 5 36 8 42 9 39 8
47 cis-capsanthin-dimyristateb,g 20.72 120 20 140 30 130 30 160 30 110 20
48 all-trans-capsanthin-myristate-palmitateb,g 20.93 370 80 500 100 400 80 370 80 330 70
49 cis-capsanthin-myristate-palmitateb,g 21.1 17 4 11.1 2.3 12.6 2.7 16 3 21 4
50 cis-capsanthin-myristate-palmitateb,g 21.19 70 10 90 20 80 20 80 20 80 20
51 all-trans-capsanthin-dipalmitateb,g 21.33
52 + 53 all-trans-zeaxanthin-dimyristate + all-trans-zeaxanthin- 21.37 + 21.37 80 20 110 20 120 20 80 10 60 10
laurate-myristateb,k
9 -cryptoxanthinc,k 13.61 30 10 29 9 24 7 26 8 19 6
10 capsorubin-lauratec,g 13.85 23 7 22 7 15 5 30 10 14 4
36 -cryptoxanthin-lauratec,k 18.8 28 9 28 9 24 8 27 9 12 4
40 -cryptoxanthin-myristatec,k 19.56 20 6 28 9 23 7 18 6 8 2
44 -cryptoxanthin-palmitatec,k 20.25 24 8 36 11 28 9 31 9 29 9
1 cucurbitaxanthin Ad,k 6.77 14 5 8.4 2.8 11 4 14 5 11 4
2 cycloviolaxanthind,l 7.29 11 4 12 4 6.4 2.1 15 5 10 3
8 nigroxanthind,k 12.62 7.4 2.4 5.9 1.9 4.7 1.6 7.4 2.4 3 1
11 karpoxanthin-laurated,l 14.77 30 10 30 10 30 10 40 10 20 10
20 + 21 mutatoxanthin-laurate + cucurbitaxanthin A 16.45 + 16.54 21 7 20 7 20 7 22 7 15 5
-laurated,h,k
28 + 29 cis-zeaxanthin-laurate + mutatoxanthin-myristated,h,k 17.52 + 17.54 23 5 24 5 29 6 27 5 25 5
a
Carotenoid concentrations calculated with analytical standards and LC-amMS. bQuantitation with standards containing the same polyene backbone
(the U (%) of zeaxanthin and capsanthin estimated during validation were used as uncertainties on the measurements of zeaxanthin and capsanthin
esters, respectively). cQuantitation with the extinction coecients (mol) from the literature.17 dQuantitation with the mol from a compound with a
similar polyene backbone. eBecause antheraxanthin was not available as a reference standard at the time of validation, the average U (%) obtained
from validation data was used as uncertainty for the measurement of antheraxanthin derivatives. fToo low intensity for quantitation. gQuantitated
with capsanthin standard. hPeak overlap. iPredominantly capsanthin is present. jQuantitated with antheraxanthin standard. kQuantitated with
zeaxanthin standard. lQuantitated with violaxantin standard.

characteristic peak around 330350 nm. Several cis-carotenoids
could be detected in the chili pepper samples, although in lesser
amounts compared to their trans counterparts. It is generally
known that absorbance maxima of carotenoids can shift
4821
depending on the diluent.15 These shifts can be similar to or
even greater than the shifts in maximum absorbances that occur
during trans to cis isomerization of carotenoids.15 The correct
dx.doi.org/10.1021/jf500285g | J. Agric. Food Chem. 2014, 62, 48124831
Journal of Agricultural and Food Chemistry
isomer form, cis or all-trans, was therefore attributed only when
a reference standard of the compound of interest was available.
Table 1 shows the diagnostic amMS and PDA data used for
carotenoid identication in the chili pepper samples. During
positive APCI, carotenoids predominantly ionized to generate
protonated molecules [M + H]+, whereas predominantly
radical molecular ions (M) were formed during negative
APCI. A substantial amount of carotenoid product ions was
detected due to in-source fragmentation, thereby providing
additional structural information. Five of the 53 detected
carotenoid peaks in the chili pepper accessions could be
identied with reference standards. Forty-four unknown
carotenoids could be tentatively identied on the basis of
spectrophotometric and mass spectrometric data, chromatog-
raphy, databases, and the literature. As the carotenoid content
of dierent Capsicum varieties has been frequently reported,
conrmation of identity was often obtained from comparison
with the ndings of other studies.8,10,13 Four minor unknown
carotenoids could not be assigned a tentative structure. These
data show that the applied generic apolar screening method
performs in the separation and identication of carotenoids as
well as earlier reported methods that were optimized for the
dedicated identication of carotenoids in chili peppers.10,13
To conrm the identity of the esteried carotenoids, the
sample extracts were saponied. Due to circumstances, the
saponication procedure was performed after 1 year of storage
of the extracts in the dark, at 25 C. Analysis of the extract
before saponication revealed that no signicant change had
occurred in the carotenoid content during storage. The
chromatogram of the saponied extract (Figure 3) shows the
prole of the hydrolyzed carotenoids. The peaks of cucurbita-
xanthin A (6.77 min), two cis-capsanthins (7.38 and 7.62 min),
all-trans-capsanthin (7.78 min), cis-antheraxanthin (8.55 min),
all-trans-zeaxanthin (10.05 min), and -cryptoxanthin (13.61
min) augmented in the chromatogram of the saponied extract,
which conrmed the presence of their esteried derivatives in
the untreated extract (Figure 2). Furthermore, new peaks
appeared in the saponied extract at 5.67, 8.26, 9.25, 9.82,
10.23, 13.61, and 16.88 min that could be identied as
capsorubin, mutatoxanthin, karpoxanthin, cis-zeaxanthin, cis--
apo-8-carotenal, -cryptoxanthin, and cis--carotene, respec-
tively. Their free form was not detected before saponication,
which conrms their presence as carotenoid esters in the
untreated chili pepper extracts.
The three main maximum absorbance wavelengths of the
isomers all-trans-lutein and cis-zeaxanthin are in close agree-
ment and are only distinguishable in UV absorbance by a
characteristic cis peak of cis-zeaxanthin. A cis peak was clearly
detected in the PDA spectrum of peak 9 in the chromatograms
of the saponied extracts (Figure 3), indicating the presence of
cis-zeaxanthin. It has also been reported that zeaxanthin and
lutein are distinguishable by in-source fragmentation in positive
APCI mode by the relative heights of the [M + H]+ and [M +
H H2O]+ peaks.4 Comparison of the MS spectrum of peak 9
in Figure 3 showed close resemblance with that of a trans-
zeaxanthin reference standard and diered from the spectrum
of a trans-lutein reference standard. Peak 9 was therefore
designated cis-zeaxanthin.
An overview of the chromatographic separation versus
molecular mass of all the compounds identied in the chili
pepper extracts during this study is given in Figure 4. Although
most of the compounds were separated by chromatography,
orthogonal detector selectivity remains essential for compound
4822
Article
dierentiation. This can in many cases not be achieved by PDA
detection alone and highlights the added value of high-end
instrumentation in this research eld.
Quantitation of Carotenoids with Analytical Reference
Standards. The concentrations of carotenoids for which an
analytical standard was available were calculated with amMS
(Table 2). The concentrations calculated with reference
standards represent only a small fraction of the total number
of carotenoids in the samples. Most of the xanthophylls in the
samples appeared esteried, for which analytical standards are
not commercially available.
Quantitation of Carotenoids with Extinction Coecients.
(a) Quantitation of Carotenoids Using Standards with the
Same Polyene Backbone. In UV analysis, carotenoids can be
quantitated by using reference standards having the same
polyene backbone and thus extinction coecient.15 However,
molar extinction coecients (mol) can be used for the
calculation of the concentration expressed in weight units
(e.g., g/g) only when the molecular mass is known. To our
knowledge earlier publications never used this quantication
strategy in combination with amMS. Accurate mass MS enables
the tentative identication of unknown carotenoids and
therefore the designation of its polyene backbone and
molecular weight. An LC-PDA-amMS conguration combined
with the use of extinction coecients should therefore allow
unparalleled speed in the identication and quantication of
unknown carotenoids. During this study, the majority of
carotenoid esters, predominantly capsanthin esters, were
quantitated with this procedure, that is, with a reference
standard of free capsanthin (Table 2). The uncertainties
represent the U of capsanthin estimated during method
validation. Because UV absorbance proles of carotenoids
generally overlap, coeluting carotenoids could not be
quantitated separately. In the few cases that coelution
happened, the sum of both compounds was quantitated with
the standard linked to the carotenoid with the highest
contribution to the signal, as was judged from the PDA and
MS spectra (Table 2).
To check the validity of quantitating carotenoid esters with
standards containing the same polyene backbone, the total
capsanthin content in the extracts without saponication
(21003300 mg/kg dw) was compared to the free capsanthin
concentration in the saponied extracts (26003800 mg/kg
dw). The total capsanthin content in the extracts without
saponication was calculated by conversion of the ester
concentrations to free capsanthin concentrations followed by
summation. The recoveries of the total capsanthin content in
the extracts without saponication with respect to the
capsanthin concentrations in the saponied extracts ranged
between 83 and 87%, within the U of capsanthin (21%). These
results demonstrate the suitability of using standards having the
same polyene backbone as the analytes of interest for the
quantitation of compounds for which no reference standards
are available. The slightly lower concentrations obtained for the
samples without saponication can be explained by the high
density of peaks in the chromatograms, whereby the baseline is
articially elevated and integration is slightly biased. Care
should, however, be taken when all-trans-carotenoids are used
to quantitate cis-carotenoids, as extinction coecients of cis-
carotenoids are generally smaller.21 Only small amounts of cis
isomers were present in the chili pepper samples (4.56.0% for
capsanthin). Higher percentages of cis isomers were detected
(7.519% for capsanthin) in the saponied extracts. It is
dx.doi.org/10.1021/jf500285g | J. Agric. Food Chem. 2014, 62, 48124831
Journal of Agricultural and Food Chemistry Article

Table 3. Concentrations of Fat-Soluble Vitamins and Free Phytosterols in the Chili Pepper Accessions, Calculated with
Reference Standards
concentration (g/g dw)
compd identity retention time (min) Awlad Haouzz Tebourba Korba Somaa Souk jedid
54 -tocopherola 10.98 <100 <100 <100 <100 <100
55 -tocopherol 11.87 500 100 400 100 400 100 500 100 400 100
56 phylloquinone 13.37 1.55 0.34 <0.7 1.11 0.25 1.12 0.25 1.01 0.22
60 stigmasterol 14.55 80 10 90 10 90 10 110 10 100 10
61 campesterolb 14.59 800 100 500 70 560 70 580 80 530 70
62 -sitosterolb 15.19 1300 200 1100 100 1100 100 1100 200 1000 100
a
Present, however, not quantiable due to low intensity. bThe measurement uncertainty of stigmasterol obtained with amMS was used.

generally known that saponication can induce the formation of
artifacts,15 although in this case cis isomer formation during 1
year of extract storage cannot be ruled out.
The shelf life of carotenoid esters in the pepper extracts was
examined over a period of 1 year by correlating the
concentrations of capsanthin esters in the extracts immediately
after extraction (x) with the concentrations in the extracts after
1 year of storage (y). The capsanthin ester concentrations in
solution did not decline over 1 year of storage (y = 1.1629x
21.895). Furthermore, a coecient of determination (R2) of
0.9639 states a good t between the measurements. These
results indicate a long storage life for carotenoid esters in the
extracts.
The above results demonstrate that the combination of
amMS and PDA allows the identication and quantitation of
esteried carotenoids without the need for individual standards
for each and every carotenoid or for a saponication procedure.
(b) Quantitation with Extinction Coecients from the
Literature. A group of carotenoids, for which no reference
standards with the same polyene backbone were available,
could not be quantitated as described above. This group of
carotenoids was quantitated with a carotenoid standard
containing a dierent polyene backbone. To correct for the
dierences in absorbance between the standard and the
carotenoid of interest, the calculated concentrations were
subsequently converted to the concentration of the carotenoid
of interest with the mol values as tabulated17 for 86 carotenoids.
The results are shown in Table 2. The extinction coecients
reported 17 were compared to in-house experimentally
determined extinction coecients for four carotenoids and
the deviations were 4.8, 22, 2.6, and 11% for -carotene, -
carotene, lycopene, and zeaxanthin, respectively. These
deviations were considered acceptable and conrmed that the
mol values from the literature can be used to estimate the
concentrations of carotenoids for which no standards are
available. The measurement uncertainty was estimated by
combining the average U value obtained for carotenoids during
method validation and the highest of the above-mentioned
deviations between the experimentally determined and
literature-described mol values, resulting in an uncertainty of
31%. Carotenoids for which no extinction coecients were
available from the literature were quantitated in a similar
manner, using the mol values of carotenoids with an analogous
polyene backbone (Table 2). The values of the listed extinction
coecients17 diered up to a factor of 4 over the whole range
of carotenoids, predominantly due to the very small and
atypical conjugated system of phytoene, which results in a very
low extinction coecient. Carotenoids with similar polyene
backbones have similar extinction coecients, for example,
137,900 for zeaxanthin, 126,600 for -carotene, 137,200 for
4823
antheraxanthin, 121,000 for capsanthin, and 132,000 for
capsorubin. An additional uncertainty in the concentration is
thus introduced by choosing an extinction coecient of an
analogous polyene backbone (estimated around 11%, corre-
sponding to 2 RSD of the above extinction coecients).
Combined with the average U value obtained for carotenoids
during method validation and the highest of the above-
mentioned deviations for the mol values, this allowed the
estimation of the measurement uncertainty at 33%. The
combination of LC, amMS, and PDA thus allows the
identication of unknown carotenoids and simultaneous
estimation of their concentrations without the need for
reference standards for each carotenoid.
(c) Comparison of the Carotenoids Content of the
Dierent Accessions. As only one analysis was performed per
chili pepper accession, the dierent peppers could not be
compared using ANOVA. Pair-wise comparison resulted in
good correlations (R2 0.90). Only slightly lower concen-
trations were found for the Souk jedid accession. The
carotenoid proles were thus very similar for the ve chili
pepper accessions.
Lipid-Soluble Vitamins and Phytosterol Derivatives in
Chili Peppers. The generic method that was developed for
carotenoids has a wider scope than rst envisaged. Other
compounds that are extracted and elute within the set
chromatographic window can be analyzed. The lipid-soluble
vitamins phylloquinone and -tocopherol and phytosterols
stigmasterol, campesterol, and -sitosterol were identied and
quantitated using MS and analytical standards (e.g., molecular
structures 55, 68, and 74 in Figure 1; Tables 3 and 4). The
concentrations of -tocopherol were in line with the
concentrations found previously.12 Although -tocopherol was
previously reported to be present in the seeds of chili peppers,
it was detected only in small signal intensities during this study
(most probably attributable to the dierence in studied tissue),
which prevented their quantitation.22 As was found for the
carotenoids, the concentrations for lipid-soluble vitamins and
free phytosterols were very similar for the ve studied cultivars.
It is known that phytosterols occur in plant tissues as the free
alcohol, as fatty acid esters, as steryl glycosides, and as acylated
steryl glycosides.23 The free phytosterols thus represent only a
fraction of the phytosterols in the chili peppers. Several steryl
glucosides, acylated steryl glucosides, and acylated sterols were
identied in the chili pepper extracts by amMS (Table 4 and
Figure 4). The locations of the fatty acid double bonds could
not be retrieved from the LC-MS data. However, the fatty acid
composition of phytosterol derivatives in Capsicum oil,
obtained by consecutive purications and dedicated analytical
methods, has been described in the literature and allowed for
the correct fatty acid moiety assignment.9,24 Most interestingly,
dx.doi.org/10.1021/jf500285g | J. Agric. Food Chem. 2014, 62, 48124831
 Table 4. Values for Maximum Absorbance, Precursor and Product Ions, and Retention Times of Other Plant Metabolites Next to Carotenoids Identied in the Chili Pepper
Accessions
mass
max absorbance MS accurate mass of deviation identification
compd identity mol formula retention time (min) (nm) mode precursor ion (ppm) in-source fragments and adducts type
54 -tocopherol C28H48O2 10.98 297 pos 416.365 47 1.42 151.075, 401.342 b
neg 150.068, 189.079

55 -tocopherol C29H50O2 11.87 293 pos 430.381 35 1.91 165.091, 431.388, 206.117, 182.117 b
neg 414.350, 203.095, 164.084

56 phylloquinone C31H46O2 13.37 263, 271, 330 pos 451.357 42 0.80 452.360 b
neg 450.350 13 0.44 451.353, 211.063

57 5- or 7-avenasterol C29H48O 13.36 pos 395.369 c

neg
Journal of Agricultural and Food Chemistry

58 5- or 7-avenasterol C29H48O 13.69 pos 395.369, 255.210 c

neg

59 cholesterol C27H46O 14.03 pos 369.352 c

neg

4824
60 stigmasterol C29H48O 14.55 pos 395.367, 311.273, 255.210 b
neg

61 campesterol C28H48O 14.59 pos 383.367 b

neg

62 -sitosterol C29H50O 15.19 pos 397.382 b

neg

63 5- or 7-avenasterol glucoside C35H58O6 6.77 pos 395.368

neg 633.43779a 0.95

64 5- or 7-avenasterol glucoside C35H58O6 6.96 pos 395.368

neg 633.43759a 0.63

65 cholesterol glucoside C33H56O6 6.99 pos 369.352

neg 607.42098a 0.92

66 campesteryl glucoside C34H58O6 7.42 pos 383.367 d

neg 561.415 34 1.28 621.437a

67 stigmasteryl glucoside C35H58O6 7.6 pos 395.368 d

neg 633.4376a 0.65
Article

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 Table 4. continued
mass
max absorbance MS accurate mass of deviation identification
compd identity mol formula retention time (min) (nm) mode precursor ion (ppm) in-source fragments and adducts type
68 -sitosteryl glucoside C35H60O6 8.08 pos 397.383 d
neg 575.432 25 0.94 635.453a

69 campesteryl (6-O-linolenoyl) glucoside C52H86O7 16.45 pos 383.367 d

(C18:3) neg 881.65187a 0.75

70 campesteryl (6-O-linoleoyl) glucoside C52H88O7 17.52 pos 383.367 d

(C18:2) neg 883.66726a 0.45

71 campesteryl (6-O-palmitoyl) glucoside C50H88O7 19.5 pos 383.367 d

(C16:0) neg 859.6671a 0.28

72 -sitosteryl (6-O-linolenoyl) glucoside C53H88O7 16.92 pos 397.383 d

Journal of Agricultural and Food Chemistry

(C18:3) neg 895.66669a 0.19

73 -sitosteryl (6-O-linoleoyl) glucoside C53H90O7 17.97 pos 397.383 d

(C18:2) neg 897.68193a 0.65

74 -sitosteryl (6-O-palmitoyl) glucoside C51H90O7 19.98 pos 397.383 d

(C16:0) neg 873.68167a

4825
0.96

75 campesteryl linoleate (C18:2) C46H78O2 20.6 pos 663.607 42 0.06 383.367

neg 279.232

76 -sitosteryl linoleate (C18:2) C47H80O2 20.88 pos 677.623 91 1.18 397.383

neg 279.232

77 monogalactosyldiacylglycerol (18:3/18:3) C45H74O10 8.49 pos 613.482, 595.471 d

neg 773.521 41 0.63 833.542,a 277.217

78 monogalactosyldiacylglycerol (18:3/18:2) C45H76O10 9.4 pos 615.498, 597.488 d

neg 775.536 83 0.34 835.557,a 277.217, 279.232
79 monogalactosyldiacylglycerol (18:2/18:2) C45H78O10 10.3 pos 617.513, 599.503 d
neg 777.552 77 0.71 837.573,a 279.232

80 monogalactosyldiacylglycerol (18:3/18:1) C45H78O10 10.49 pos 617.51397, 599.503 36 d

neg 777.552 67 0.15 837.573,a 277.217, 281.248

81 monogalactosyldiacylglycerol (18:2/18:1) C45H80O10 11.44 pos d

neg 779.568 67 1.03 839.589,a 279.232, 281.248

82 monogalactosyldiacylglycerol (18:3/18:0) C45H80O10 12.03 pos 619.529 d

Article

neg 779.568 16 0.37 839.589,a 277.217, 283.264

dx.doi.org/10.1021/jf500285g | J. Agric. Food Chem. 2014, 62, 48124831

 Table 4. continued
mass
max absorbance MS accurate mass of deviation identification
compd identity mol formula retention time (min) (nm) mode precursor ion (ppm) in-source fragments and adducts type

83 monogalactosyldiacylglycerol (18:1/18:1) C45H82O10 12.6 pos d

neg 781.584 19 0.86 841.604a

84 monogalactosyldiacylglycerol (18:2/18:0) C45H82O10 13.03 pos d

neg 781.584 07 0.70 841.604,a 279.233, 283.264

85 monogalactosyldiacylglycerol (18:3/16:3) C43H70O10 7.3 pos 585.451, 567.440 e

neg 745.489 99 0.50 805.510,a 277.217, 249.185

86 monogalactosyldiacylglycerol (18:3/16:2) C43H72O10 8.11 pos 587.467, 569.457 e

neg 747.506 02 1.00 807.526,a 277.217, 251.201
Journal of Agricultural and Food Chemistry

87 monogalactosyldiacylglycerol (18:3/16:1) C43H74O10 9.06 pos 589.482, 571.472 e

neg 749.520 88 0.05 809.542,a 277.217, 253.217

88 monogalactosyldiacylglycerol (18:3/16:0) C43H76O10 10.49 pos 591.498, 573.487 e

neg 751.536 98 0.55 811.557,a 277.217, 255.233

89 monogalactosyldiacylglycerol (18:2/16:0) 11.46 pos 575.503 e

4826
C43H78O10
neg 753.553 01 1.05 813.573,a 279.232, 255.233

90 monogalactosyldiacylglycerol (18:1/16:0) C43H80O10 12.65 pos e

neg 815.58935a 0.43 255.233

91 digalactosyldiacylglycerol (18:3/18:3) C51H84O15 6.75 pos 613.481, 595.471 e

neg 935.574 01 0.29 995.594,a 277.217

92 digalactosyldiacylglycerol (18:3/18:2) C51H86O15 7.57 pos 615.498, 597.488 e

neg 937.589 81 0.45 997.610,a 277.217, 279.232

93 digalactosyldiacylglycerol (18:2/18:2) C51H88O15 8.42 pos 617.513, 599.503 e

neg 939.605 51 0.49 999.626a

94 digalactosyldiacylglycerol (18:3/18:1) C51H88O15 8.59 pos 617.513, 599.503 e

neg 939.605 88 0.88 999.626a

95 digalactosyldiacylglycerol (18:3/18:0) C51H90O15 10.09 pos 619.529, 601.518 e

neg 941.620 55 0.16 1001.641,a 277.217, 283.264

96 digalactosyldiacylglycerol (18:2/18:0) C51H92O15 11.04 pos e

neg 943.635 47 0.93 1003.657,a 283.264
Article

dx.doi.org/10.1021/jf500285g | J. Agric. Food Chem. 2014, 62, 48124831

 Table 4. continued
mass
max absorbance MS accurate mass of deviation identification
compd identity mol formula retention time (min) (nm) mode precursor ion (ppm) in-source fragments and adducts type
97 digalactosyldiacylglycerol (18:3/16:3) C49H80O15 5.7 pos 585.451, 567.440 e
neg 907.542 86 0.46 967.563,a 277.217, 249.185

98 digalactosyldiacylglycerol (18:3/16:0) C49H86O15 8.52 pos 591.498, 573.487 e

neg 913.588 93 0.50 973.610,a 277.217, 255.233

99 digalactosyldiacylglycerol (18:2/16:0) C49H88O15 9.44 pos 575.503 e

neg 915.605 16 0.12 975.626,a 279.232, 255.233

100 N-2-hydroxypalmitoyl-1-O-- C40H77O10N 6.91, 7.04 pos 732.560 95 1.46 714.550, 696.540, 570.509, 552.497, 272.258, 344.279, 326.269, d
glucopyranosyl-4-hydroxy-8-sphingenine 316.284, 308.258, 298.273, 280.263, 262.252
neg 730.548 19 0.99 790.568,a 568.494, 550.484, 532.473, 271.227
Journal of Agricultural and Food Chemistry

101 N-2-hydroxypalmitoyl-1-O-- C40H75O9N 7.68, 7.90 pos 534.487, 516.477, 272.258, 280.263, 262.252 d
glucopyranosyl-4,8-sphingadienine neg 712.536 73 0.25 772.557,a 550.484, 532.473, 514.462, 271.227

102 N-2-hydroxypalmitoyl-1-O-- C40H77O9N 8.18, 8.40 pos 554.513, 536.503 d

glucopyranosyl-8-sphingenine neg 714.553 41 1.19 774.574,a 552.500, 534.489, 516.478, 271.227

103 N-2-hydroxydocosanoyl-1-O-- C46H89O10N 11.44, 11.56, 11.73 pos 816.6557 0.27 798.645, 654.602, 636.592, 618.581, 356.351, 298.273, 280.263, d

4827
glucopyranosyl-4-hydroxy-8-sphingenine 262.252
neg 814.641 94 0.70 874.662,a 652.589, 634.578, 616.567

104 N-2-hydroxytricosanoyl-1-O-- C47H91O10N 12.34, 12.53 pos 830.671 24 0.40 812.660, 668.618, 650.607, 632.597, 298.273, 280.263 d
glucopyranosyl-4-hydroxy-8-sphingenine neg 828.657 38 0.43 888.678,a 666.604, 630.583

105 N-2-hydroxytetracosanoyl-1-O-- C48H93O10N 13.15, 13.34 pos 844.686 51 0.84 826.676, 682.633, 664.623, 384.383, 646.613, 298.273, 280.263 d
glucopyranosyl-4-hydroxy-8-sphingenine neg 842.672 66 0.01 902.694,a 680.620, 662.609, 644.599

106 N-2-hydroxypentacosanoyl-1-O-- C49H95O10N 13.93, 14.15 pos 858.702 18 0.80 840.691, 696.649, 678.638, 660.628, 298.273, 280.263 d
glucopyranosyl-4-hydroxy-8-sphingenine neg 856.688 78 0.54 916.710,a 694.636, 676.625, 658.614

107 triacylglycerol (18:3,18:3,18:2) C57H94O6 18.34 pos 875.711 17 1.31 597.486, 595.471
neg 277.217, 279.232

108 triacylglycerol (18:3,18:2,18:2) C57H96O6 18.95 pos 877.726 41 1.78 599.501, 597.486
neg 277.217, 279.232

109 triacylglycerol (18:2,18:2,18:2) C57H98O6 19.5 pos 879.742 13 1.69 599.502

neg 279.232

110 triacylglycerol (18:2,18:2,18:1) C57H100O6 20.2 pos 881.758 19 1.22 599.502, 601.517
Article

neg 279.232, 281.248

dx.doi.org/10.1021/jf500285g | J. Agric. Food Chem. 2014, 62, 48124831

 Table 4. continued
mass
max absorbance MS accurate mass of deviation identification
compd identity mol formula retention time (min) (nm) mode precursor ion (ppm) in-source fragments and adducts type

111 triacylglycerol (18:2,18:2,16:0) C55H98O6 20.32 pos 855.742 07 1.81 599.502, 575.502
neg 279.232, 255.233

112 triacylglycerol (18:2,18:1,16:0) C55H100O6 20.93 pos 857.758 03 1.45 601.517, 577.518, 575.502
neg 279.232, 255.233, 281.248

113 triacylglycerol (18:2,18:1,18:1) C57H102O6 20.96 pos 883.773 59 1.50 601.518, 603.533
neg 279.232, 281.248

114 triacylglycerol (18:1,16:0,16:0) C53H98O6 21.03 pos 831.7436 0.02 575.503, 551.503
neg 255.233
Journal of Agricultural and Food Chemistry

115 triacylglycerol (18:1,18:1,18:1) C57H104O6 21.33 pos 885.790 47 0.11 603.534

neg 281.248

116 diacylglycerol (18:3,18:2) C39H66O5 12.21 pos 615.497 74 0.91 597.487, 335.258, 337.273
neg 277.217

117 diacylglycerol (18:2,18:2) 13.11 pos 617.513 55 599.503, 337.273

4828
C39H68O5 0.65
neg 279.233

118 diacylglycerol (18:2,16:0) C37H68O5 14.28 pos 593.513 67 0.47 575.502, 313.273, 337.273
neg 279.232, 255.233

f
119 nordihydrocapsaicin C17H27O3N 1.56 pos 294.2065 0.44
neg 292.1919 0.27

120 capsaicin C18H27O3N 1.61 280 pos 306.206 24 0.42 f

neg 304.191 85 0.10 290.175

121 dihydrocapsaicin C18H29O3N 1.82 279 pos 308.2219 0.39 f

neg 306.2075 0.10

122 nonivamide C17H27O3N 1.82 pos f

neg 292.1921 0.96

123 homodihydrocapsaicin C19H31O3N 2.11 279 pos 322.2378 0.40 f

neg 320.2233 0.56
a
Acetate adduct formed in negative APCI mode. bIdentication with analytical standard. cIdentied by Matthaus et al.24 dIdentied by Yamauchi et al.9 eDescribed by www.lipidmaps.org. fDictionary of
Natural Products:18 present in Capsicum annuum.
Article

dx.doi.org/10.1021/jf500285g | J. Agric. Food Chem. 2014, 62, 48124831

Journal of Agricultural and Food Chemistry
linoleic, linolenic, and palmitic acids were bound to the steryl
and steryl glucoside moieties, corresponding to the results
reported by Yamauchi et al.9 This is dierent from the
distribution of fatty acids bound to carotenoids (lauric, myristic,
and palmitic acids) (Table 1). The elution order of the acylated
steryl glucosides was predominantly inuenced by the level of
saturation of the fatty acid moieties, as the retention time
increased in the order C18:3 < C18:2 < C16:0 (Figure 4).
Cholesterol, 5- and 7-avenasterol, and derivatives thereof
were also tentatively identied by amMS, corresponding to the
ndings of Matthaus et al.24 (Table 4). Due to the lack of
reference standards for sterol derivatives and the complex
relationship between detector response and concentration, the
various molecular species could not be quantitated. Extinction
coecients could not be used as phytosterols were not detected
by using PDA detection. An alkaline saponication procedure
to assess the total concentration of phytosterols would require
the use of both the free sterols and steryl glucosides, as the
ether bonds of the steryl glucosides are stable during
saponication.25 Steryl glucoside bonds can otherwise be
cleaved by acid hydrolysis.26 During this study, it was chosen
to calculate the relative abundances per group of the sterol
derivatives (e.g., steryl glucosides) to enable quantitative
comparison. This was achieved with the product ions
corresponding to the m/z of the free sterol alcohols with the
loss of water, found for all derivatives in positive APCI.
Liengprayoon et al.27 have previously reported this calculation
strategy, with the hypothesis that the response to ionization is
similar within a group of similar structures. Campesterol, -
sitosterol, and their derivatives were found to be the main
phytosterols present in the ve chili pepper accessions. The
proles of sterol derivatives were very similar between the
samples; however, the total content did vary signicantly
between the samples for steryl glucosides and acylated steryl
glucosides, being most abundant in the Awlad Haouzz and
Souk jedid accessions, respectively.
Yamauchi et al.9 previously reported the detection of free
phytosterols and phytosterol glucoside derivatives in chili
pepper by performing consecutive prefractionations before
analysis. During this study, a single generic sample preparation
protocol was applied to extract all of the compounds reported
in this paper. Subsequently, compound identication was
performed using a single generic screening method for apolar
compounds without the need for dedicated fractionation
(Figure 4).
Monogalactosyldiacylglycerols and Digalactosyldia-
cylglycerols in Chili Peppers. In total, 23 monogalacto-
syldiacylglycerols and digalactosyldiacylglycerols were identied
in the chili pepper accessions (e.g., molecular structure 84 in
Figure 1; Table 4). Their identication was based on the
ionization and in-source fragmentation pattern of a reference
standard of monogalactosyldiacylglycerols and digalactosyldia-
cylglycerols. In positive APCI mode, predominantly product
ions with the loss of the galactosyl moiety (and water) were
found in the spectra. During negative APCI mode, predom-
inantly acetate adducts were found. Dierentiation between
isomeric lipids containing dierent fatty acid moieties (e.g.,
C18:2/C18:2 and C18:1/C18:3) could be made by the fatty
acid product ions obtained by in-source fragmentation in
negative APCI mode. Almost all detected monogalactosyldia-
cylglycerols and digalactosyldiacylglycerols were separated with
the generic chromatographic gradient. Faster elution was
obtained for galactosyldiacylglycerol lipids with fatty acids of
4829
Article
shorter chain length and/or a higher number of double bonds
(Figure 4). The fatty acid moieties that were found to be part of
the galactosyldiacylglycerol lipids consisted of C18:3, C18:2,
C18:1, C18:0, C16:3, C16:2, C16:1, and C16:0, which
corresponds with previous ndings.9
Relative abundances were calculated for the monogalacto-
syldiacylglycerol and digalactosyldiacylglycerol families with the
acetate adducts obtained in negative APCI mode. Monoga-
lactosyldiacylglycerol (18:3/18:3), digalactosyldiacylglycerol
(18:3/18:3), digalactosyldiacylglycerol (18:3/16:0), and diga-
lactosyldiacylglycerol (18:3/18:0) were the most abundant
galactosyl diacylglycerol lipids present in the chili pepper
accessions, as previously found.9 The proles were very similar
for the galactosyl diacylglycerol lipids between samples.
However, the total content of monogalactosyldiacylglycerol
and digalactosyldiacylglycerol per sample varied signicantly,
which showed to be highest in the Awlad Haouzz accession
and lowest in the Souk jedid and Tebourba accessions,
respectively.
Cerebrosides in Chili Peppers. Yamauchi et al.9 identied
seven molecular species of glucocerebrosides in C. annuum L.
after HPLC purication to single compounds and consecutive
NMR analysis. Owing to this work, these compounds were
easily identied during the current study (e.g., molecular
structure 101 in Figure 1; Table 4). The protonated molecules
of these sphingolipids were detected together with several
product ions such as the ceramide moiety, owing to the loss of
the sugar moiety, and the -hydroxy fatty acyl moieties, because
of extensive in-source fragmentation during APCI positive
mode.9 Ionization in negative APCI mode rendered acetate
adducts, deprotonated molecules, and ceramide product ions
due to in-source fragmentation. For each of the identied
compounds, two almost baseline separated peaks appeared in
the chromatograms (Figure 4). A study reported by Whitaker
et al.28 showed that glucose is the only sugar present in the bell
pepper cerebrosides and that both the 8-cis and 8-trans isomers
of 4,8-sphingadienine and 8-sphingenine and the cistrans
isomers of 4-hydroxy-8-sphingenine are present in the fruits.
The two isomeric peaks are explained by the respective cis- and
trans-cerebrosides.
Relative abundances in MS detection were calculated for the
cerebrosides by combining the chromatographic cistrans peaks
obtained for the acetate adducts in negative APCI mode. The
proles were very similar for the cerebrosides between the
samples. The content per sample did vary signicantly, which
showed to be highest in the Awlad Haouzz accession and
lowest in the Tebourba accession.
Glycerolipids in Chili Peppers. Nine triglycerides and
three diglycerides were identied in the chili pepper accessions
(e.g., molecular structure 109 in Figure 1; Table 4). Their
identication was based on the ionization and in-source
fragmentation pattern of a glyceryl trioleate and glyceryl
dioleate standard. As in most cases the order of the fatty acids
bound to glycerol (i.e.sn-1, sn-2, or sn-3 position of the glycerol
backbone) could not be deduced from the MS spectra, the
lipids were noted as triglyceride (C18:2, C18:2, C16:0)
instead of triglyceride (C18:2/C18:2/C16:0) (Table 4).
Predominantly triglycerides were present in the extracts. Faster
elution was obtained with fatty acids of shorter chain length
and/or a higher number of double bonds (Figure 4). The fatty
acid moieties that were found to be part of the glycerolipids
consisted of linolenic acid (C18:3), linoleic acid (C18:2), oleic
acid (C18:1), and palmitic acid (C16:0), which is in line with
dx.doi.org/10.1021/jf500285g | J. Agric. Food Chem. 2014, 62, 48124831
Journal of Agricultural and Food Chemistry
the fatty acid composition of the acylated steryl glucosides, the
acylated sterols, monogalactosyldiacylglycerols, and digalacto-
syldiacylglycerols that were identied during this study.
Relative abundances in MS detection were calculated for the
diglycerides and triglycerides with the protonated molecules
obtained in positive APCI mode. The triglyceride with three
linoleic acid moieties (C18:2, C18:2, C18:2) and diglyceride
with two linoleic acid moieties (C18:2, C18:2) were most
abundantly present in the chili pepper accessions. The proles
were very similar for the di- and triacylglycerides in the samples.
However, the content did vary signicantly and was highest in
the Somaa and Korba accessions and lowest in the Awlad
Haouzz and Souk jedid accession for triglycerides and
diglycerides, respectively.
Capsaicinoids in Chili Peppers. Five capsaicinoids, which
are known to be present in high amounts in chili peppers, were
identied with amMS. Because these compounds are relatively
small and polar compared to the other studied compounds
(molecular structure 120 in Figure 1), they partially coeluted
within the rst 3 min of the chromatographic gradient. The
identities of nordihydrocapsaicin, capsaicin, dihydrocapsaicin,
and nonivamide were later conrmed with their respective
analytical standards (unpublished results). The proles of the
capsaicinoids were similar in the samples; however, the content
did vary signicantly, being lowest for Souk jedid and highest
in the Tebourba and Korba accessions.
During this study, a single generic sample preparation
protocol was applied to extract all of the compounds reported
in this paper. Subsequently, compound identication was
performed using a single generic UHPLC-PDA-amMS screen-
ing method for apolar compounds without the need for
dedicated fractionation. Method validation conrmed that the
generic approach is applicable for quantitative purposes and is
virtually limited only by the solubility and detector response of
the metabolites of interest. It was shown that although most of
the compounds were separated by chromatography, orthogonal
detector selectivity remains essential for compound dier-
entiation. This can in many cases not be achieved by PDA
detection alone and highlights the added value of high-end
instrumentation in this research eld.
Second, an identicationquantitation strategy based on the
use of LC-PDA-amMS and extinction coecients was applied
for carotenoids, enabling unparalleled speed in the identi-
cation and quantication of unknown carotenoids and
eliminating the need for reference standards for each
carotenoid. Finally, a case study on chili pepper fruits (C.
f rutescens L.) showed that carotenoids, steryl derivatives,
glycolipids, glycerolipids, capsaicinoids, and lipid-soluble
vitamins could be characterized simultaneously with the
analytical platform. This system is thus a powerful tool to
unravel the complex apolar phytochemicals composition of
biomass, as it is useful not only as a ngerprinting method but
also for the detailed proling of individual components.
ASSOCIATED CONTENT
*
S Supporting Information
Supplementary Tables 13. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*(S.B.) Phone: + 32 14 33 50 54. Fax: + 32 14 31 94 72. E-
mail: sebastiaan.bijttebier@vito.be.
4830
Article
Notes
The authors declare no competing nancial interest.
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