Food Chemistry 147 (2014) 1–9

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Carotenoid composition of berries and leaves from six Romanian sea

buckthorn (Hippophae rhamnoides L.) varieties
Raluca Maria Pop a,b, Yannick Weesepoel b, Carmen Socaciu a,⇑, Adela Pintea a, Jean-Paul Vincken b,
Harry Gruppen b
a
University of Agricultural Sciences and Veterinary Medicine, Mănăsßtur Street, 3-5, 400372 Cluj-Napoca, Romania
b
Laboratory of Food Chemistry, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history: Berries and leaves from six varieties of Carpathians’ sea buckthorn (Hippophae rhamnoides L., ssp.
Received 18 May 2012 Carpatica) were analysed for their carotenoid composition (free and esterified) using a combination of
Received in revised form 23 May 2013 HPLC-PAD, GC–MS and UHPLC–PAD–ESI-MS techniques. GC–MS techniques revealed the fatty acid profile
Accepted 13 September 2013
specific for each berry variety, while targeted UHPLC–MS analysis identified the fatty acids involved in
Available online 27 September 2013
carotenoids esterification: palmitic (C16:0), myristic (C14:0) and stearic (C18:0). Total carotenoid content
varied between 53 and 97 mg/100 g dry weight in berries, and between 3.5 and 4.2 mg/100 g DW in
Keywords:
leaves. The carotenoid di-esters represented the main fraction among berry varieties having zeaxanthin
Carotenoid esters
Chlorophylls
di-palmitate as major compound, while leaves contained only free carotenoids like lutein, b-carotene,
Principal component analysis violaxanthin and neoxanthin. Principal component analysis identified the suitable carotenoid biomarkers
Electrospray ionization mass spectrometry characteristic for the Carpathians’ sea buckthorn from Romania with contribution to their taxonomic
Chemotaxonomic classification classification and authenticity recognition.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Alpha-Carotene, c-carotene, dihydroxy-b-carotene, lycopene, and

Sea buckthorn (Hippophae rhamnoides L.) is a hardy, fast grow-
ing, deciduous, spiny shrub, between 2 and 4 m tall, with yellow
or orange berries. It has brown or black rough bark and a thick
grayish-green crown. Leaves are alternate, narrow-lanceolate with
a silver–grey color on the bottom side but according to the species
and microclimate variation, morphological structure of sea buck-
thorn shows much variation. The berries are rich in bioactive com-
pounds including vitamins, steroids, tocopherols, carotenoids,
chlorophylls, flavonoids, and fatty acids (FAs), all with potentially
beneficial effects on human health (Singh, 2005; Suryakumar &
Gupta, 2011). Among biologically active substances, carotenoids
receive much interest, as they have multiple activities such
as anti-oxidant, anti-mutagenic and anti-tumour (Britton,
Liaaen-Jensen, & Pfander, 2009). Previous investigations on sea
buckthorn berries have shown differences in composition and con-
tent of carotenoids (Andersson, Olsson, Johansson, & Rumpunen,
2009; Bal, Meda, Naik, & Satya, 2011). Forty-one different carote-
noids have been reported in various cultivars, with zeaxanthin,
b-cryptoxanthin, and b-carotene as the predominant ones
(Andersson et al., 2009; Raffo, Paoletti, & Antonelli, 2004).
⇑ Corresponding author. Tel.: +40 264 596384/126; fax: +40 264 593792.
E-mail address: carmen.socaciu@usamvcluj.ro (C. Socaciu).
canthaxanthin represented the minor carotenoids (Yang & Kallio,
2005). The presence of carotenoid esters has been reported in sea
buckthorn berries (Giuffrida, Pintea, Dugo, Torre, Pop & Mondello,
2011; Pintea, Varga, Stepnowski, Socaciu, Culea & Diehl, 2005) but
the identification and quantification of the individual carotenoid
esters is poorly described, especially in Romanian varieties (ssp.
Carpatica). Therefore, the aim of this research was to identify and
quantify all carotenoids (including esters) in Romanian sea buck-
thorn varieties. Reversed phase high performance liquid chroma-
tography (RP-HPLC) was used for obtaining UV–Vis spectra of
individual carotenoids and to establish the fingerprint of each vari-
ety. In-line electrospray ionization mass spectrometry (ESI-MS) and
tandem fragmentation data were recorded for unambiguous iden-
tification of carotenoids, porphyrins and FA esterified carotenoids
(Frassanito, Cantonati, Flaim, Mancini, & Guella, 2008). To provide
additional information regarding the different carotenoid esters in
berries, the FAs profiles of the extracts were investigated using gas
chromatography–mass spectrometry (GC–MS). The results
obtained were integrated in development of a new method for
targeted carotenoid esters analysis using a combination of
GC–MS and UHPLC–ESI-MS. Furthermore, principal component
analysis (PCA) was applied to reveal whether free carotenoids
and carotenoid esters could be used as markers for taxonomic
classification and authenticity recognition.

0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.09.083
2 R.M. Pop et al. / Food Chemistry 147 (2014) 1–9
2. Materials and methods
2.1. Sea buckthorn samples
Fruits and leaves of 6 varieties of Hippophae rhamnoides L., ssp.
Carpatica (Victoria, Tiberiu, Sf. Gheorghe, Serpenta, S ß erbănesßti 4,
and Ovidiu) were harvested fully matured from an experimental
field at the Fruit Research Station from Bacău (Romania), in the
first week of October 2008. Botanically, they can be considered
established cultivars having been cultivated for more than 10 years
in the Bacău region. The morphologic characteristics of both ber-
ries and leaves are presented in Table 1. The sea buckthorn pictures
included in the table were taken from the PhD Dissertation of Do-
garu (2011) (unpublished results). The berries and leaves were
kept in plastic pots, directly refrigerated at 4 °C, kept for 8 h, and
subsequently frozen and stored at 20 °C. Prior to extraction, seeds
were separated from the fresh berries.
2.2. Chemicals
ULC/MS grade acetonitrile and ethyl acetate were purchased
from Biosolve BV (Valkenswaard, The Netherlands). Water was
prepared using a Milli-Q water purification system (Millipore,
Billerica, MA, USA). All other chemicals were purchased from
Merck (Darmstadt, Germany).
2.3. Pigment extraction
The extraction procedure described by Pintea et al. (2005) was
followed with slight modifications. Total carotenoids were ex-
tracted from frozen sea buckthorn berries (SBB) and sea buckthorn
leaves (SBL) (1 g) using methanol: ethyl acetate: petroleum ether
(1:1:1, v/v/v). Repeated extractions were done under continuous
stirring for 4 h in the dark. The combined extracts were filtered
and then partitioned successively, using a separation funnel, with
water, diethyl ether and saturated NaCl solution. The remaining
water from the ether phase containing the carotenoids was re-
moved using anhydrous sodium sulphate. The organic phase was
collected and then evaporated using a rotavapor at 35 °C. The ex-
tract was dissolved in a known volume of ethyl acetate for HPLC-
PAD and UHPLC analysis, and used as such for GC–MS analysis.
All extractions were done in triplicate.
2.4. Analysis of fatty acids by GC–MS
Fatty acid composition of SBB was determined by mixing the
extract (100 lL) with 2 mL n-heptane. After homogenization, the
FAs were converted to methyl esters using 0.5 mL 0.5 mol/L NaOH
in methanol. The reaction mixture was stirred and left to stand for
15 min at room temperature. n-Heptane (1 mL) was collected from
the upper phase, dried with anhydrous sodium sulphate, filtered,
and analysed using a Thermo Trace GC Ultra (San Jose, CA,
USA).Samples were loaded (injection volume 1 lL) onto a Restek
Famewax column (30 m, 0.25 mm ID, 0.25 lm PEG film, Restek,
Bellefonte, PA, USA). Fatty acids were separated at a flow rate of
1.5 mL/min using helium as carrier gas. Injector and detector tem-
peratures were set at 250 °C. The oven thermal gradient started at
195 °C, linear gradient to 240 °C at 5 °C/min, held at 240 °C for
3 min, then linear gradient to 250 °C at 10 °C/min, and held at
250 °C for another 2 min. Data were acquired using a Thermo
DSQ II MS (San Jose, CA, USA) set at a mass range of 50–650 Da.
Fatty acids were identified based on their mass fragmentation pat-
terns by comparison with those of authentic standards (Supelco
F.A.M.E. Mix RM-3 (O7256-1AMP, St. Louis, MO, USA), using Xcal-
ibur (Thermo Finnigan 2.07, San Jose, CA, USA) software.
2.5. Pigment identification and quantification by RP-PAD-HPLC
Carotenoid pigments were identified and quantified using a Shi-
madzu HPLC system (Shimadzu Corporation, Kyoto, Japan), con-
sisting of LC-20AT solvent delivery module, a DGU-20A3 degasser
and a SPD-M20A UV–Vis photodiode array detector (PAD).
Samples were injected onto a LiChrosorb RP 18 (250  4.6 mm,
5 lm) column. Mobile phases consisted of acetonitrile: water 9:1
(v/v) (solvent A) and ethyl acetate (solvent B) both containing
0.25% (v/v) triethylamine, according to Pintea et al. (2005). The
addition of TEA as a modifier in the mobile phases was to improve
carotenoids recovery, separation and retention on the column
(Hart & Scott, 1995). The flow was maintained at 1000 lL/min.
The following elution profile was used: 0–16 min, isocratic on
15% B; 16–54 min, linear gradient from 15% to 62% B, 54–56 min,
isocratic on 62% B; 56–60 min, linear gradient from 62% to 15%
B; 60–70 min, isocratic on 15% B. Pigments were quantified by
in-line UV–Vis at 450 and 660 nm using lutein as an external stan-
dard. The calibration curve for pure lutein was prepared using five
different concentrations (0.04–0.2 mg/ml). Lutein standard was
dissolved in ethyl acetate and the absorption was measured at
450 nm. The linear regression factor of the calibration curve was
0.977. The quantification of free carotenoids was done using this
calibration curve, based on the average of triplicate injections. In
case of carotenoid esters, the accurate quantitative analysis was
achieved by multiplying the results, expressed as lutein equiva-
lents, with the specific correction factors applied for each ester.
The correction factor was calculated according to their specific
absorption coefficient A1% 1%
1 cm , comparative to lutein A1 cm value
(Britton, Liaaen-Jensen, & Pfander, 1995).
2.6. Pigment analysis by UHPLC-PAD–ESI-MS
Samples were analysed on a Thermo Accela UHPLC system (San
Jose, CA, USA) equipped with Acella 1250 Pump, autosampler and
photo-diode array (PAD) detector. Volumes of 5 lL were injected
on to a Waters Acquity UPLC BEH Shield RP18 column (2.1
 150 mm, 1.7 lm particle size). Ethyl acetate (solvent A), acetoni-
trile (solvent B), and acetonitrile: water 1:1 (v/v) (solvent C), each
with 0.1% (v/v) formic acid, were used as eluent. Because TEA
showed high proton affinity when in MS, it was excluded from
the mobile phase to avoid compromising the separation and pig-
ment identification. Therefore, formic acid, known to improve the
ionization by an optimum protonation process, was used only in
subsequent UHPLC–MS analysis (Guaratini et al., 2004).
The flow was maintained at 400 lL/min. The following elu-
tion profile was used: 0–3 min, isocratic on 5% A; 3–13 min, lin-
ear gradient from 5% to 30% A, 13–15 min, isocratic on 30% A;
15–17 min, linear gradient from 30% to 5% A; 17–27 min, iso-
cratic on 5% A. During elution a constant flow of 5% C was kept
in order to improve carotenoid ionization. PAD recorded full
spectra as well as at 450 and 660 nm for carotenoid and chloro-
phyll detection, respectively. In-line MS data were recorded by
directing the LC flow into a LTQ-XL ion trap mass spectrometer
(Thermo Scientific, San Jose, CA, USA) equipped with an ESI
probe. Helium was used as the sheath gas and nitrogen as the
auxiliary gas. Data dependent MSn analysis was performed with
a normalized collision energy of 35%. The MS3 fragmentation
was always performed on the most intense product ion in the
MS2. Most settings were optimized using ‘tune plus’ (Xcalibur
2.07, Thermo Scientific) via automatic tuning. The system was
tuned by direct injection using b-carotene in PI mode. The ion
transfer tube temperature was 450 °C and the source voltage
3.5 kV. When berries were analysed, data were collected using
a parent mass list containing all possible carotenoid ester masses
calculated based on the combination of free carotenoids present
 R.M. Pop et al. / Food Chemistry 147 (2014) 1–9 3

Table 1
The morphologic characteristics of sea buckthorn berries (SBB) and leaves (SBL) varieties.

Sea buckthorn Sea buckthorn berries Sea buckthorn berries (SBB) Sea buckthorn leaves characteristics Sea buckthorn leaves
varieties characteristics (SBL)
VICTORIA Color: orange Color: white pearl on the back side and light
green color on top
Shape: oval–elongated Shape: wide elliptic
Average weight: 0.70 g Average length: 6–6.5 cm

TIBERIU Color: yellow–orange Color: light green on top and gray on the back
(Serbanesti 1) side
Shape: oval–flat Shape: narrow elliptic
Average weight: 0.49 g Average length: 5–6 cm

Sf GHEORGHE 5 Color: yellow–orange Color: light green on top and silvery white on
(Auras) the back side
Shape: round–oval Shape: elliptical
Average weight: 0.49 g Average length: 5.5 cm

SERPENTA Color: orange–yellowish Color: white silvery

(Serpeni 11) Shape: elliptical Shape: elliptical
Average weight: 0.38 g Average length: 6–7 cm

SERBANESTI 4 Color: orange Color: copper–silvery back side and light green
(Silvia) top side
Shape: round Shape: narrow elliptic
Average weight: 0.39 g Average length: 4–5 cm

OVIDIU (SF Color: orange–yellow Color: light color on the back side and pale
Gheorghe 9) green on the top
Shape: oval Shape: wide elliptic
Average weight: 0.44 g Average length: 5–6 cm

(lutein, zeaxanthin and b cryptoxanthin), esterified to one or two (http://www.sisweb.com/referenc/tools/exactmass.htm). When

FAs, previously determined by GC–MS. The calculation was done leaves were analysed data were collected over an m/z-range of
using The Exact Mass Calculator, Single Isotope Version 150–1500.
4 R.M. Pop et al. / Food Chemistry 147 (2014) 1–9
Finally, data were used to identify and correlate previous HPLC
results with the UHPLC-MS results by comparison with elution or-
der on C18 HPLC and C18 UHPLC columns, UV–Vis absorption spec-
tra from both HPLC and UHPLC, and the mass spectrum compared
with literature values.
2.7. Statistical analysis
The chemotaxonomic classification and discrimination of SBB
and SBL samples using their carotenoid composition was achieved
by principal component analysis (PCA), with UnscramblerX Soft-
ware, version 10.1 (CAMO Software AS, Norway).
Principal component analysis (PCA) is a technique usually used
to describe the variability of the data set, reducing the dimension-
ality of a data set by finding a new set of variables, smaller than the
original set of variables, which retains most of the samples’ infor-
mation. These new variables, linear combinations of the original
variables, called principal components (PCs) are uncorrelated with
each other but they may be correlated with many of the variables.
Each principal component independently accounts for the variance
of the original variables, and they are ordered by the fraction of the
total information each retains. Principle component PC1 explains
most of the relationship, being in the direction along which there
is greatest variation, while principal component PC2, orthogonal
to PC1 is the direction with maximum remaining variation. Such
score plots offer information about the samples in the PCA model,
and is characterised by the loadings value (Brereton, 2007). Corre-
lation with loading values is made through the location of values in
the loading plot, which indicates the influence of that value on a
given PC. The further away from the plot origin a variable lies,
the stronger impact that variable has on the PCA model. It is there-
fore possible to explain the differences between the various SB
samples and to determine which variables contribute the most in
their differentiation.
3. Results and discussion
3.1. Fatty acid composition of sea buckthorn berries
FAs in SBB originate either from triacylglycerides, free FAs, or
carotenoid esters (Kallio, Yang, Peippo, Tahvonen, & Pan, 2002).
In order to establish which FAs were esterified with carotenoids,
FA methyl esters in the SBB extract were determined by GC–MS.
The major FA was palmitic (C16:0) ranging from 28% in Ovidiu to
44% in Tiberiu followed by oleic acid (C18:1), with values between
17% and 34% in Tiberiu and Ovidiu, respectively, and palmitoleic
(C16:1) with values ranging from 21% to 28% in Tiberiu and Victoria,
respectively. These values are in accordance with other studies
made on Romanian SBB. For example, pulp oil obtained from
SBB, grown in the wild of the Danube Delta, had the same major
FAs: palmitic (C16:0) – 37%, oleic (C18:1) – 30% and palmitoleic
(C16:1) – 20% (Vescan, Pamfil, Bele, Matea, & Sisea, 2010). Further-
more, an average of 3% linoleic (C18:2) and 1% linolenic acid (C18:3)
was found in our varieties, which have also been described by oth-
ers for SBB pulp oil (Gutiérrez, Ratti, & Belkacemi, 2008). Traces of
stearic (C18:0) and myristic acid (C14:0) were also present, consis-
tent with reports on SBB pulp oil of different origin (Cakir, 2004).
3.2. Carotenoid composition of sea buckthorn berries
Quantitative HPLC-PAD analysis of non-saponified carotenoid
extract was used to identify free carotenoids and carotenoid esters
among the six varieties.
Fig. 1A shows a representative HPLC chromatogram of pigments
in SBB Victoria variety. Berries did not contain chlorophylls,
indicating that they were fully maturated. Carotenoid identifica-
tion was made based on their retention times, UV–Vis spectra
(kmax, spectral fine structure, and cis peak intensity), MS spectrum
(see 3.4), and comparisons with literature data.
Twenty-seven compounds were identified. The free carotenoids
were lutein, zeaxanthin, b-cryptoxanthin, a-carotene, c-carotene,
b-carotene and lycopene (peaks 1–12) as reported elsewhere
(Pintea et al., 2005; Yang & Kallio, 2005). The carotenoid esters
fraction was mainly represented by the zeaxanthin esters (peaks
14, 21, 24 and 27) followed by the lutein esters (peaks 17, 19, 20,
22, 23, 25 and 26) and -cryptoxanthin esters (peaks 13 and 15).
Carotenoid esters were tentatively identified by comparison
with chromophores of free carotenoids (Britton et al., 1995).
Identification of the FA substituent was done using ESI-MS as
described in 3.4.
3.3. Carotenoid and chlorophylls composition of sea buckthorn leaves
Fig. 1B shows a representative HPLC chromatogram of pigments
from SBL. The pigment identification was done according to their
retention times, UV–Vis spectrum and MS spectrum (see further)
as in case of berries. HPLC profiles showed that leaves from all
six varieties had the same pigment fingerprints. In total 11 com-
pounds were identified. The main carotenoids were lutein (peak
4), b-carotene (peak 9), violaxanthin (peak 2), and neoxanthin
(peak 1) while the main chlorophylls were chlorophyll a (peak
7), pheophytin a (peak 8) and chlorophyll b (peak 6). Trace
amounts of zeaxanthin (peak 5) was found in two SB varieties (Sf
Gheorghe and Serpenta). Characteristic for SBL varieties was lutein
and the other carotenoids that were not esterified.
3.4. Structural assignment by ESI-MS
ESI-MS analysis in the positive modus was used for confirma-
tion of the previously identified free carotenoids and for determi-
nation of the FA substituent of esterified carotenoids.
According to literature data, other compounds such as chloro-
phylls and triglycerides may co-elute with carotenoids during MS
analysis, hindering carotenoid acquisition in the full scan mass
spectra (Frassanito et al., 2008). To overcome this problem, we
combined the qualitative results obtained from the GC–MS and
HPLC-PAD analysis to create a new method to perform targeted
MS analysis of carotenoids in SBB, without any extract clean-up
procedure. Molecular weights (MW) of all possible combinations
of FAs and carotenoids (mono- and di-esters) were calculated,
knowing that carotenoid esters are formed by the esterification
of the carotenoid hydroxyl group (Britton, 1998).
In the case of the geometrical isomers, lutein (b,e-carotene-3,
30 -diol) and zeaxanthin (b,b-carotene-3,30 -diol), which differ in
the position of a double bond in one of the ionone rings, can be
esterified with FAs in the 3 and 30 carbon of the ionone ring (Krin-
sky, Landrum, & Bone, 2003).
Carotenoids, represented by both free forms and esters, were
determined in the non-saponified extracts as presented in
Table 2A. All free carotenoids in SBB had the fragmentation pattern
characterised by the loss of toluene, [M+HC7H8]+, originating
from in-chain fragmentation, as previously reported in the litera-
ture (Crupi, Milella, & Antonacci, 2010). Because geometrical iso-
mers, lutein and zeaxanthin, presented similar relative intensities
of the dehydrated fragments m/z 551, [M+H-18]+ and m/z 533,
[M+H-36]+, the structural characteristics of the hydroxylated end
groups was not sufficient to distinguish between isomers. Thus,
identification was achieved based on their elution time and fine
structure of the UV–Vis spectra from both HPLC and UHPLC. The
same principle was applied in case of carotenoid compounds that
 R.M. Pop et al. / Food Chemistry 147 (2014) 1–9
had identical parent masses with the molecular ion at m/z 537,
[M+H]+, (peaks 4–9) or m/z 536, [M]+ (peaks 10–12).
Peaks 13 and 15 represented mono-esters of -cryptoxanthin,
peaks 14, 16, 18 mono-esters of zeaxanthin, peak 17 mono-esters
of lutein, peaks 19, 20, 22, 23, 25, 26 di-esters of lutein and peaks
21, 24, 27 di-esters of zeaxanthin, (Table 2A). The results con-
firmed our previous studies, in which the carotenoid esters were
only tentatively identified based on the absorption pattern
(Pârlog, Vodnar, Dulf, Leopold, & Socaciu, 2009). Generally, two
peaks could be assigned to each ester. According to Breithaupt
and Bamedi (2002), the twin peaks are probably due to diastere-
oisomers formed during nonspecific oxidation (Breithaupt &
Bamedi, 2002). The FAs attached to b-cryptoxanthin, lutein and
zeaxanthin were identified by the loss of the FA moiety from their
corresponding esters in MS2 fragmentation of the parent ion.
Most dominant fragments found were the loss of one
([M+H-FA]+) or two ([M+H-2FA]+) FAs (Kurz, Carle, & Schieber,
2008). Esters, like free carotenoids, displayed the characteristic
in-source fragmentation behaviour forming fragments in MS2
[M+H-92]+, after losing the toluene. Other important characteris-
tic of the esters is the formation of ‘‘backbone’’ with m/z 535, in
case of b-cryptoxanthin monoesters, m/z 551 in case of lutein
and zeaxanthin mono-esters, and m/z 533, in case of lutein and
zeaxanthin di-esters. Accordingly, zeaxanthin dipalmitate (C16:0,
C16:0) was identified as the major compound in all Romanian
Fig. 1. Representative HPLC – PAD fingerprint of pigments from Victoria SBB variety
(A), recorded at 450 nm, and from Victoria SBL variety (B), recorded at 450 and
660 nm. For peak assignment see Table 1 (SBB) and Table 2 (SBL).
5
SBB varieties. Lutein – palmitate – myristate (C16:0, C14:0),
zeaxanthin – palmitate – myristate (C16:0, C14:0) and lutein
di-palmitate (C16:0, C16:0) were also present in all varieties. Even
though the FA profile of the carotenoid extract contained signifi-
cant amounts of unsaturated FAs, especially oleic acid (average
27% w/w) and palmitoleic acid (average 23% w/w), the carotenoids
were mainly esterified with saturated FAs like palmitic and
myristic.
For leaf analysis, untargeted MS data acquisition was performed
since lipid content was lower than observed in berries (10% com-
pared to 29% w/w, respectively) (Yuldasheva, Ulchenko, Chernenko,
& Glushenkova, 2006).
Pigments, carotenoids and chlorophylls were determined as
presented in Table 2B. Mass spectra of chlorophylls were charac-
terised by the formation of one major fragment in MS2,
[M+H–C20H38], which corresponds to the loss of the phytyl moiety
of 278 Da.
Other characteristics of chlorophylls, observed in the case of
pheophytin a (peak 8), was the presence of another fragment at
m/z 533 [M+H-338], which could be explained by the consecutive
loss of the phytyl tail followed by an acetic acid group bound at
C132 (Roy, Llewellyn, Egeland, & Johnsen, 2011).
3.5. Quantification of individual carotenoids in berries and leaves by
RP-HPLC
The carotenoid composition of the different SBB varieties is
summarized in Table 3A. The carotenoid composition varied
largely between varieties, Sßerbănesßti having the highest carotenoid
content with almost double value (97 mg/100 g DW) when
compared to Serpenta (53 mg/100 g DW). Next, Sf Gheorghe and
Ovidiu varieties were in the same range with 75 and 74 mg/
100 g DW, respectively, followed by Victoria (67 mg/100 g DW)
and Tiberiu (60 mg/100 g DW) varieties. The large variation of
carotenoid content is in agreement with other reports, which
suggest the fluctuation of carotenoid content according to sea
buckthorn origin, growth condition and subspecies (27 mg/100 g
DW in Hippophae neurocarpa from China and 105 mg/100 g DW
in ssp. turkestanica from Kyrgyzstan) (Andersson et al., 2009; Yang
& Kallio, 2005). Among the carotenoids, b-carotene (peak 10) was
found, on average, at 5 mg/100 g DW whereas the average
zeaxanthin diester (peak 24), among varieties as major peak, was
11 mg/100 g DW. Free lutein and b cryptoxanthin were present
only in traces while free zeaxanthin was present in all SBB caroten-
oid extracts. On average, the esterified carotenoids were 71% of the
total carotenoids, higher than the average of 55% identified by
Andersson et al. (2009) in the Swedish cultivars. Among the
esterified fraction, the ratio between mono- and di-esters tended
to favour the di-esters, on average 2.8 (w/w). Regarding the FAs
attached to carotenoids in the esterified fraction, palmitic acid
(C16:0) was found (average 58% w/w), myristic acid (C14:0, 19%),
both palmitic and myristic acids (17%) and, finally, palmitic and
stearic acid (C18:0, 6%).
The carotenoid composition of SBL is shown in Table 3B.Lutein
was found in the highest concentrations (average 0.9 mg/100 g
DW) followed by the isomers all trans-b-carotene (average
0.7 mg/100 g DW). The oxidized forms neoxanthin and violaxan-
thin, originating from the photosynthetic xanthophylls cycle,
varied between 0.5 and 0.6 mg/100 g DW in all samples.
3.6. Biomarker identification using principal component analysis
The chemometric analysis technique PCA was used for data
interpretation and visualization of metabolic differences among
SB varieties. Therefore, pigments analysis in both berries and
leaves was used to provide relevant biomarkers to distinguish
6 R.M. Pop et al. / Food Chemistry 147 (2014) 1–9

Table 2A
LC-(ESI) MS data and identification of carotenoids found in sea buckthorn berries. The wavelengths written in italics are indicators of carotenoid cis compounds.

No UVmax (nm) [M+H]+ MS2 product ions (relative intensity) Structure assignment References
1 448, 474 569 569½M þ Hþ
ð18Þ , 551½M þ H-18þ
ð25Þ , 533½M þ H-18-18þ
ð2Þ , 476½M þ H-92þ
ð100Þ
Lutein de Rosso and Mercadante
(2007)
2 454, 480 569 551½M þ H-18þ
ð20Þ , 533½M þ H-18-18þ
ð5Þ , 476½M þ H-92þ
ð100Þ
Zeaxanthin de Rosso and Mercadante
(2007)
3 436, 461, 553 553½M þ Hþ þ þ þ
ð10Þ , 535½M þ H-18ð100Þ , 497½M þ H-56ð6Þ , ½M-92ð18Þ
b-Cryptoxanthin de Rosso and Mercadante
492 (2007)
4 363, 471, 537 444½M þ H-92þ
ð100Þ
c-Carotene Giuffrida et al. (2011)
502
5 345, 362, 537 – cis Lycopene de Rosso and Mercadante
443, 467,498 (2007)
6 448, 473, 537 – Lycopene Giuffrida et al. (2011)
505
7 436, 463, 537 – cis c-Carotene de Rosso and Mercadante
494 (2007)
8 430, 456, 537 – d-Carotene de Rosso and Mercadante
488 (2007)
9 423,449, 474 537 444½M þ H-92þ
ð100Þ
a-Carotene de Rosso and Mercadante
(2007)
⁄
10 455, 480 536 444½M-92þ
ð100Þ
b-Carotene Giuffrida et al. (2011)
11 337, 416, 536⁄ 444½M-92þ
ð100Þ
15,15-cis b-Carotene de Rosso and Mercadante
448, 474 (2007)
⁄
12 453 536 444½M-92þ
ð100Þ
cis b-Carotene de Rosso and Mercadante
(2007)
13 448, 474 791 791½M þ Hþ þ þ þ
ð16Þ , 698½M-92ð100Þ , 535½M þ H-256ð70Þ , 443½M þ H-92-256ð65Þ
b-Cryptoxanthin– Pintea et al. (2005)
palmitate (C16:0)
14 453, 480 779 779½M þ Hþ þ þ
ð54Þ , 687½M þ H-92ð100Þ , 551½M þ H-228ð50Þ
Zeaxanthin–myristate Weller and Breithaupt
(C14:0) (2003)
15 791 791½M þ Hþ
ð26Þ , 698½M-92þ þ
ð100Þ , 535½M þ H-92-256ð70Þ ,
b-Cryptoxanthin– Pintea et al., 2005
443½M þ þ
H-92-256ð68Þ palmitate (C16:0)

16 453, 479 779 779½M þ Hþ þ þ

ð98Þ , 687½M þ H-92ð100Þ , 551½M þ H-228ð51Þ
Zeaxanthin–myristate Weller and Breithaupt
(C14:0) (2003)
17 448, 475 807 807½M þ Hþ þ þ
ð18Þ , 715½M þ H-92ð100Þ , 551½M þ H-256ð36Þ
Lutein–palmitate (C16:0) Weller and Breithaupt
(2003)
18 453, 479 807 807½M þ Hþ þ þ
ð100Þ ,715½M þ H-92ð62Þ , 551½M þ H-256ð32Þ
Zeaxanthin–palmitate Weller and Breithaupt
(C16:0) (2003)
19 448, 475 989 989½M þ Hþ þ þ þ
ð12Þ , 896½M-92ð32Þ , 761½M þ H-228ð42Þ , 669½M þ H-92-228ð100Þ ,
Lutein di-myristate Young, Abdel-Aal, Rabalski,
533½M þ H-228-228þ þ (C14:0, C14:0) and Blackwell (2007)
ð12Þ , 441½M þ H-92-228-228ð12Þ
20 448, 475 1017 1017½M þ Hþ þ þ
ð5Þ , 925½M þ H-92ð70Þ , 761½M þ H-256ð68Þ ,
Lutein–palmitate– Pintea et al. (2005)
669½M þ H-92-256þ þ myristate(C14:0, C16:0)
ð100Þ , 533½M þ H-256-228ð32Þ ,

441½M þ H-92-256-228þ
ð48Þ
21 453, 480 1017 1017½M þ Hþ þ þ
ð5Þ , 925½M þ H-92ð52Þ , 789½M þ H-228ð42Þ ,
Zeaxanthin–palmitate Pintea et al. (2005)
697½M þ H-92-228þ þ þ myristate(C16:0, C14:0)
ð100Þ , 533½M þ H-256-228ð18Þ , 441½M þ H-92-256ð36Þ
22 448, 475 989 989½M þ Hþ þ þ
ð5Þ , 896½M þ H-92ð46Þ , 761½M þ H-228ð84Þ ,
Lutein di-myristate Young et al. (2007)
669½M þ H-92-228þ þ þ (C14:0, C14:0)
ð100Þ , 533½M þ H-228-228ð16Þ , 441½M þ H-92-228ð30Þ
23 448, 475 1045 1045½M þ Hþ þ þ
ð2Þ , 953½M þ H-92ð26Þ , 789½M þ H-256ð48Þ ,
Lutein di-palmitate Giuffrida et al. (2011)
697½M þ H-92-256þ þ þ (C16:0, C16:0)
ð100Þ , 533½M þ H-256-256ð7Þ , 441½M þ H-92-256ð18Þ
24 453, 480 1045 1045½M þ Hþ þ þ
ð2Þ , 953½M þ H-92ð30Þ , 789½M þ H-256ð100Þ ,
Zeaxanthin di-palmitate Weller & Breithaupt (2003)
697½M þ H-92-256þ þ þ (C16:0, C16:0)
ð94Þ , 533½M þ H-256-256ð16Þ , 441½M þ H-92-256ð28Þ
25 448 1045 1045½M þ Hþ þ þ
ð8Þ , 953½M þ H-92ð32Þ , 789½M þ H-256ð100Þ ,
Lutein di-palmitate Giuffrida et al. (2011)
697½M þ H-92-256þ þ þ (C16:0, C16:0)
ð90Þ , 533½M þ H-256-256ð18Þ , 441½M þ H-92-256ð24Þ
26 448, 476 1073 1073½M þ Hþ þ þ
ð100Þ , 817½M þ H-256ð50Þ , 789½M þ H-284ð40Þ ,
Lutein–palmitate– Young et al. (2007)
533½M þ H-256-284þ stearate (C16:0, C18:0)
ð40Þ
27 453, 479 1045 1045½M þ Hþ þ þ
ð62Þ , 953½M þ H-92ð24Þ , 789½M þ H-256ð100Þ ,
Zeaxanthin di-palmitate Weller & Breithaupt (2003)
697½M þ H-92-256þ þ þ (C16:0, C16:0)
ð68Þ , 533½M þ H-256-256ð14Þ , 441½M þ H-92-256ð16Þ

*
Compounds that formed radical molecular ions [M]+d.

between varieties using carotenoids as biomarkers of sample qual-
ity or for authenticity recognition. Thus, PCA was applied to quan-
titative data from Table 3A for SBB and Table 3B for SBL. Fig. 2A
represents score plots of carotenoids from SBB, and Fig. 2B repre-
sents score plots of pigments from SBL. Both score plots were gen-
erated from comparisons of the first two principal components
(PC): PC1–PC2.
In case of SBB (Fig. 2A), the first two components explained 86%
of the sample variance. PC1, which explained 73% of the total
variance, presented high loadings for compounds (24) zeaxanthin
di-palmitate (C16:0, C16:0), (18) zeaxanthin–palmitate (C16:0), (21)
zeaxanthin–palmitate myristate (C16:0, C14:0), (20) lutein–palmi-
tate–myristate (C14:0, C16:0), (17) lutein–palmitate (C16:0), (23) lu-
tein di-palmitate (C16:0, C16:0) and (19) lutein di-myristate (C14:0,
C14:0). Moderate loadings were found for compounds (14) zeaxan-
thin–myristate (C14:0) and (2) zeaxanthin. In case of PC2, which ex-
plained 13% of the total variance, high loadings were observed for
compounds (10) b-carotene and (11) 15,15-cis b-carotene and
moderate for compounds (7) cis c-carotene, (4) c-carotene and
(5) cis lycopene. In explanation, the higher the loading of a variable
 R.M. Pop et al. / Food Chemistry 147 (2014) 1–9 7

Table 2B
LC-(ESI) MS data and pigment identification in sea buckthorn leaves.

No UVmax (nm) [M+H]+ MS2 product ions (relative intensity) Structure assignment References
1 414, 438, 466 601 583½M þ H-18þ þ þ
ð80Þ , 565½M þ H-18-18ð20Þ , 509½M þ H-92ð100Þ
Neoxanthin de Rosso & Mercadante (2007)
2 417, 440, 471 601 583½M þ H-18þ ð80Þ , 565½M þ H-18-18þ
ð20Þ , 509½M þ H-92þ
ð100Þ
Violaxanthin de Rosso & Mercadante (2007)
3 410, 665 593 533½M þ H-60þ ð100Þ
Pheophorbide a Roy et al., 2011
4 447, 475 569 569½M þ Hþ þ þ þ
ð18Þ , 551½M þ H-18ð25Þ , 533½M þ H-18-18ð2Þ , 476½M-92ð100Þ
Lutein de Rosso & Mercadante (2007)
5 453, 476 569 þ þ þ Zeaxanthin de Rosso & Mercadante (2007)
551½M þ H-18ð20Þ , 533½M þ H-18-18ð5Þ , 476½M þ H-92ð100Þ
6a 456, 646 907 629½M-278þ
ð100Þ
Chlorophyll b Roy et al., 2011
6b 456, 646 907 629½M-278þ
ð100Þ
Chlorophyll b’ Roy et al., 2011
7a 413, 431, 663 893 615½M-278þ
ð100Þ
Chlorophyll a Roy et al., 2011
7b 413, 431, 663 893 615½M-278þ
ð100Þ
Chlorophyll a’ Roy et al., 2011
8a 411, 504, 666 871 593½M-278þ þ
ð100Þ , ½M-278-60ð50Þ
Pheophytin a Roy et al., 2011
8b 410, 505, 666 871 593½Mhyphen278ð100Þ , 533½M-278-60þ
þ
ð50Þ
Pheophytin a’ Roy et al., 2011
9 454, 481 536⁄ 444½M-92þ
ð100Þ
b-Carotene de Rosso & Mercadante (2007)
10 449, 475 536⁄ 444½M-92þ
ð100Þ
cis b-Carotene de Rosso & Mercadante (2007)
11 449, 475 536⁄ 444½M-92þ
ð100Þ
cis b-Carotene de Rosso & Mercadante (2007)

* 
Compounds that formed radical molecular ions [M ].

implies a larger contribution to the sample variation. The superpo-
sition of the loading and score plots (data not shown) indicated
that the discriminating compounds between samples along PC1
axis were 24, 18, 21, 20, 17, 23 and 19. Therefore, it would be rea-
sonable to expect higher values for these biomarkers in case of Ser-
banesti variety, placed on the right of the score plot (Fig. 2A) and
lower concentrations in opposing (negatively correlated) varieties
like Ovidiu, Tiberiu and Serpenta. The position of SBB groups along
PC2 axis, showed that Victoria, which lies at the top of the axis,
very close to Sf Gheorghe in the opposite direction to Ovidiu, Ser-
penta and Tiberiu varieties, situated at the bottom and negatively
correlated. In this case, the discriminating compounds were 10
and 11. Thus, major and minor compounds determined clustering
of SBB varieties as six groups. Among them, Tiberiu variety showed
large variations between individual sample repetitions.
Samples position along the PC1 and PC2 axis, together with the
carotenoids composition as related to their biosynthetic pathway,
allowed us to observe some berry varieties (e.g. Victoria and Sf
Gheorghe) were richer in carotenoid hydrocarbons such as lyco-
pene while Sßerbănesßti was rich in zeaxanthin esters and poor in
hydrocarbons. Serpenta variety proved to be poor in both caroten-
oid categories.

Table 3A
Carotenoid content in different sea buckthorn berry varieties.

No Identified Carotenoids Concentration (mg ± SD/100 g DW)b in sea buckthorn varieties

Victoria Tiberiu Sf Gheorghe Serpenta S
ß erbănesßti Ovidiu
1 Lutein NDa NDa 2.1 ± 0.2 1.4 ± 0.1 NDa NDa
2 Zeaxanthin 2.0 ± 0.0 2.0 ± 0.1 2.3 ± 0.2 1.8 ± 0.1 2.5 ± 0.1 2.3 ± 0.0
3 b-Cryptoxanthin 1.3 ± 0.0 NDa NDa NDa 1.3 ± 0.0 1.6 ± 0.0
4 c-Carotene 1.6 ± 0.1 NDa 1.8 ± 0.1 NDa NDa 1.8 ± 0.0
5 cis Lycopene 1.9 ± 0.2 1.6 ± 0.3 2.7 ± 0.0 1.4 ± 0.0 1.6 ± 0.1 1.9 ± 0.0
6 Lycopene 1.6 ± 0.0 NDa 2.3 ± 0.0 1.4 ± 0.0 1.4 ± 0.0 1.7 ± 0.0
7 cis c-Carotene 2.3 ± 0.0 2.0 ± 0.8 3.7 ± 0.1 NDa 2.1 ± 0.1 2.1 ± 0.0
8 dCarotene 1.4 ± 0.0 NDa 1.9 ± 0.0 1.5 ± 0.0 1.4 ± 0.0 NDa
9 a-Carotene 1.3 ± 0.0 1.3 ± 0.0 traces 0.9 ± 0.8 1.4 ± 0.0 1.6 ± 0.0
10 b-Carotene 7.4 ± 0.2 4.0 ± 0.6 6.1 ± 0.3 1.9 ± 0.1 7.5 ± 0.7 4.6 ± 0.0
11 15,15-cis b-Carotene 1.9 ± 0.0 1.6 ± 0.1 2.0 ± 0.1 1.4 ± 0.0 1.9 ± 0.1 1.9 ± 0.0
12 cis b-Carotene 1.3 ± 0.0 1.5 ± 0.3 2.1 ± 0.0 1.4 ± 0.0 1.5 ± 0.0 1.6 ± 0.0
13 b-Cryptoxanthin–palmitate (C16:0) 1.3 ± 0.0 1.4 ± 0.0 1.4 ± 0.0 1.4 ± 0.0 1.5 ± 0.0 1.6 ± 0.0
14 Zeaxanthin–myristate (C14:0) 1.4 ± 0.0 1.5 ± 0.1 1.6 ± 0.0 1.4 ± 0.0 1.8 ± 0.0 1.7 ± 0.0
15 b-Cryptoxanthin–palmitate (C16:0) 1.8 ± 0.0 NDa 2.2 ± 0.0 1.9 ± 0.0 2.6 ± 0.1 2.2 ± 0.0
16 Zeaxanthin–myristate (C14:0) 1.8 ± 0.2 2.1 ± 0.1 2.3 ± 0.0 1.9 ± 0.0 2.9 ± 0.1 3.1 ± 1.3
17 Lutein–palmitate (C16:0) 2.8 ± 0.0 2.9 ± 0.5 2.7 ± 0.0 2.3 ± 0.1 4.1 ± 0.1 3.2 ± 0.1
18 Zeaxanthin–palmitate (C16:0) 2.5 ± 0.0 2.9 ± 0.3 2.8 ± 0.0 2.1 ± 0.1 5.8 ± 0.6 2.9 ± 0.0
19 Lutein di-myristate (C14:0, C14:0) 2.7 ± 0.0 3.1 ± 0.2 3.3 ± 0.1 3.1 ± 0.2 5.1 ± 0.3 3.6 ± 0.2
20 Lutein–palmitate–myristate (C14:0, C16:0) 3.8 ± 0.0 3.3 ± 0.4 3.1 ± 0.0 3.0 ± 0.1 6.4 ± 0.5 4.0 ± 0.1
21 Zeaxanthin–palmitate myristate (C16:0, C14:0) 4.3 ± 0.1 3.9 ± 0.5 4.0 ± 0.1 3.2 ± 0.2 8.3 ± 0.7 4.2 ± 0.0
22 Lutein di-myristate (C14:0, C14:0) 2.3 ± 0.0 2.3 ± 0.0 2.5 ± 0.0 2.4 ± 0.0 2.5 ± 0.1 2.8 ± 0.0
23 Lutein di-palmitate (C16:0, C16:0) 3.5 ± 0.0 3.3 ± 0.7 3.6 ± 0.1 3.2 ± 0.2 6.3 ± 0.8 4.6 ± 0.0
24 Zeaxanthin di-palmitate (C16:0, C16:0) 8.2 ± 0.1 10.7 ± 2.5 9.9 ± 0.4 6.4 ± 1.0 18.3 ± 1.7 10.2 ± 0.1
25 Lutein di-palmitate (C16:0, C16:0) 2.4 ± 0.0 2.5 ± 0.0 2.7 ± 0.0 2.5 ± 0.0 2.5 ± 0.1 3.0 ± 0.1
26 Lutein–palmitate–stearate (C16:0, C18:0) 2.4 ± 0.0 2.8 ± 0.0 2.9 ± 0.0 2.6 ± 0.0 3.1 ± 0.1 3.1 ± 0.0
27 Zeaxanthin di-palmitate (C16:0, C16:0) 2.3 ± 0.0 2.7 ± 0.1 2.8 ± 0.0 2.5 ± 0.1 2.8 ± 0.1 3.1 ± 0.0
Total 67.4 ± 1.0 59.5 ± 7.6 74.9 ± 2.0 53.1 ± 3.3 96.7 ± 6.5 74.3 ± 1.9
Carotenoid monoesters 11.4 ± 0.2 10.7 ± 1.0 13.1 ± 0.1 11.1 ± 0.3 18.8 ± 1.0 14.8 ± 1.4
Carotenoid diesters 32.0 ± 0.3 34.7 ± 4.5 34.8 ± 0.7 28.9 ± 1.8 55.3 ± 4.4 38.5 ± 0.4
a
Not detected.
b
Average of triplicate samples.
8 R.M. Pop et al. / Food Chemistry 147 (2014) 1–9

Table 3B
Carotenoids content in different sea buckthorn leaf varieties.

No Identified carotenoids Concentration (mg ± SD/100 g DW)b in sea buckthorn varieties

Victoria Tiberiu Sf Gheorghe Serpenta S
ß erbănesßti Ovidiu
1 Neoxanthin 0.5 ± 0.0 0.5 ± 0.0 0.5 ± 0.0 0.5 ± 0.0 0.6 ± 0.0 0.6 ± 0.0
2 Violaxanthin 0.6 ± 0.0 0.5 ± 0.0 0.5 ± 0.0 0.5 ± 0.0 0.6 ± 0.0 0.6 ± 0.0
4 Lutein 1.0 ± 0.2 0.8 ± 0.1 0.8 ± 0.1 0.8 ± 0.2 1.0 ± 0.1 1.1 ± 0.0
5 Zeaxanthin NDa NDa 0.5 ± 0.0 0.5 ± 0.0 NDa 0.6 ± 0.0
9 b-Carotene 0.7 ± 0.1 0.7 ± 0.0 0.7 ± 0.1 0.7 ± 0.1 0.8 ± 0.1 0.9 ± 0.0
10 cis b-Carotene 0.5 ± 0.0 0.5 ± 0.0 0.5 ± 0.0 0.5 ± 0.0 0.6 ± 0.0 NDa
11 cis b-Carotene 0.5 ± 0.0 0.5 ± 0.0 0.5 ± 0.0 0.5 ± 0.0 0.6 ± 0.0 0.6 ± 0.0
Total Carotenoids 3.8 ± 0.2 3.5 ± 0.1 3.9 ± 0.2 4.2 ± 0.3 4.2 ± 0.2 4.2 ± 0.1
a
Not detected.
b
Average of triplicate samples.

When carotenoid concentrations from SBL were used, five ma-
jor groups on the score plot (Fig. 2B) could be observed. In this case,
the first two components explained 94% of the sample variance.
The groups were formed almost exclusively along PC1 axis, which
presented high loadings for compounds (9) b-carotene, (4) lutein
and (5) zeaxanthin. In the case of leaves, carotenoid did not give
reliable results due to the greater variability of leaf samples.
4. Conclusion
MS target analysis was used successfully to identify free carote-
noids, carotenoid esters and chlorophyll from both sea buckthorn
berries and leaves, without prior clean up procedures. In total, 27
compounds were identified in berries and 11 compounds in leaves.
Among berries, S ß erbănesßti had the highest total carotenoid con-
tent, mainly zeaxanthin diester (55 mg/100 g DW), and could be
the most suitable variety for intensive cultivation and industrial
application. In case of leaves the total carotenoid content was low-
er, on average 4 mg/100 g DW. The esterified carotenoids were
present only in berries. The results obtained from both berries
and leaves indicate that berries are suitable for sample classifica-
tion and variety recognition because the leaves were much more
variable among samples. Thus, PCA analysis identified the contri-
bution of both minor and major pigments in sea buckthorn culti-
vars that could be used in chemotaxonomic classification and

Fig. 2. Principal component analysis (PCA) represented as scores, for different SBB varieties (A) and SBL varieties (B).
 R.M. Pop et al. / Food Chemistry 147 (2014) 1–9
confirmation of authenticity. Therefore, carotenoid biomarkers for
the Carpathians sea buckthorn from Romania are major com-
pounds such as zeaxanthin di-palmitate (C16:0, C16:0), zeaxan-
thin–palmitate (C16:0), zeaxanthin–palmitate myristate (C16:0,
C14:0), lutein–palmitate–myristate (C14:0, C16:0), lutein–palmitate
(C16:0), lutein di-palmitate (C16:0, C16:0), lutein di-myristate (C14:0,
C14:0), b-carotene and 15,15-cis b-carotene. Among minor com-
pounds, zeaxanthin–myristate (C14:0), zeaxanthin, cis c-carotene,
c-carotene and cis lycopene were characteristic for sea buckthorn
varieties.
Acknowledgements
This work was financially supported by the 2010 POSDRU/89/
1.5 /S 52432 project, ‘‘National Postdoctoral School of Applied
Biotechnologies with Impact on Romanian Bioeconomy’’, project
co-financed by the European Social Fund through the Sectoral
Operational Programme Human Resources Development 2007–
2013. We gratefully acknowledge the partial support from a Grant
of the Romanian National Authority for Scientific Research, CNCS –
UEFISCDI, project number PN-II-ID-PCE-2011-3-0721’’. We thank
Prof. Dr. Viorel Rati from FRUCTEX Bacau, for providing the sea
buckthorn samples.
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