Profiling polyphenols in five brassica species microgreens by UHPLC-PDA-ESI/HRMSn

Abstract

Brassica vegetables are known to contain relatively high concentrations of bioactive compounds associated with human health. A comprehensive profiling of polyphenols from five Brassica species microgreens was conducted using ultra high-performance liquid chromatography photo diode array high-resolution multi-stage mass spectrometry (UHPLC-PDA-ESI/HRMS/MSn). A total of 161 polyphenols including 30 anthocyanins, 101 flavonol glycosides, and 30 hydroxycinnamic acid and hydroxybenzoic acid derivatives were putatively identified. The glycosylation patterns of the flavonols were assigned based on direct comparisons of their parent flavonoid glycosides reference compounds. The putative identifications were based on UHPLC-HRMSn analysis using retention times, elution orders, UV/Vis spectra and high resolution mass spectra, as well as an in-house polyphenol database, and literature comparisons. This study showed that these five Brassica species microgreens could be considered as good sources of food polyphenols.

Full text

Profiling Polyphenols in Five Brassica Species Microgreens by
UHPLC-PDA-ESI/HRMSn
Jianghao Sun,
†
Zhenlei Xiao,
§
Long-ze Lin,
†
Gene E. Lester,
‡
Qin Wang,
§
James M. Harnly,
†
and Pei Chen*
,†
†
Food Composition and Methods Development Laboratory, Beltsville Human Nutrition Research Center, Agricultural Research
Service, U.S. Department of Agriculture, 10300 Baltimore Avenue, Beltsville, Maryland 20705, United States
§
Department of Nutrition and Food Science, University of Maryland, College Park, Maryland 20742, United States
‡
Food Quality Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture,
10300 Baltimore Avenue, Beltsville, Maryland 20705, United States
ABSTRACT: Brassica vegetables are known to contain relatively high concentrations of bioactive compounds associated with
human health. A comprehensive profiling of polyphenols from five Brassica species microgreens was conducted using ultrahigh-
performance liquid chromatography photodiode array high-resolution multistage mass spectrometry (UHPLC-PDA-ESI/
HRMSn). A total of 164 polyphenols including 30 anthocyanins, 105 flavonol glycosides, and 29 hydroxycinnamic acid and
hydroxybenzoic acid derivatives were putatively identified.The putative identifications were based on UHPLC-HRMSnanalysis
using retention times, elution orders, UV−vis and high-resolution mass spectra, and an in-house polyphenol database as well as
literature comparisons. This study showed that these five Brassica species microgreens could be considered as good sources of
food polyphenols.
KEYWORDS: microgreens, Brassicaceae, acylated cyanidin 3-sophroside-5-mono- and diglucosides, acylated flavonol glycosides,
hydroxycinnamic acid derivatives, UHPLC-PDA-ESI/HRMSn
■INTRODUCTION
Microgreens are young edible greens produced from vegetables,
herbs, or other plants, ranging in size from 5 to 10 cm long
including stem and cotyledons (seed-leaves). They are popular
for their pretty colors, intense flavors, delicate textures, and
relatively high nutritional contents.
1
The entire plant (seedling)
is harvested at the ground level when cotyledon or seed-leaves
have fully expanded and before true leaves have fully emerged.
TheBrassicaceaeoffer some of the most commonly
consumed vegetables worldwide, which can be grown as
microgreens. Five Brassica vegetables commonly found in the
U.S. marketplace are red cabbage (Brassica oleracea var.
capitata), purple kohlrabi (B. oleracea var. gongylodes), red and
purple mustards (Brassica juncea), and mizuna (Brassica rapa
var. nipposinica or B. juncea var. japonica). Brassica vegetables
are known to be rich sources of ascorbic acid, carotenoids,
glucosinolates, polyphenols, and tocopherols,
2−4
which have
human-health beneficial attributes reportedly involved in
preventing cardiovascular diseases and some types of
cancers.
5−8
Previous studies have tentatively identified phenolic com-
pounds from 22 mature-leaf Brassica vegetables,
9−12
and
phenolic compounds have been found in tronchuda cabbage
(B. oleracea var. costata) seeds,
13
mature leaves,
14
and
internodal shoots and roots.
15,16
Twelve specific phenolic
compounds have been profiled in 2−12-day-old seedlings
possessing both seed-leaves and true leaves. The aim of the
present study was to characterize and quantify the naturally
occurring polyphenols in five commonly consumed Brassica
species (mizuna, red cabbage, purple kohlrabi, red mustard, and
purple mustard) at their microgreen growth stage. The analyses
of their native polyphenols and flavonol aglycones were
performed using state-of-the-art analytical tools: ultrahigh-
performance liquid chromatography photodiode array high-
resolution multistage mass spectrometry (UHPLC-PDA-ESI/
HRMS/MSn). Results showed that Brassica microgreens
contained notable levels of hydroxycinamic acids and may
contain different compounds from their true leaves. Totals of
30 anthocyanins, 105 flavonol glycosides, and 29 hydroxycin-
namic acid and hydroxylbenzoic acid derivatives were
tentatively identified. This is the first known reported study
of polyphenol compounds in vegetables at the cotyledonary leaf
(microgreen) stage of growth of an array of Brassica
microgreens.
■MATERIALS AND METHODS
Chemicals. Formic acid, HPLC grade methanol, and acetonitrile
were purchased from VWR International, Inc. (Clarksburg, MD,
USA). HPLC grade water was prepared from distilled water using a
Milli-Q system (Millipore Laboratory, Bedford, MA, USA).
Plant Materials and Sample Preparation. Five Brassica species,
at the microgreen growth stage, were obtained from Sun Growers
Organic Distributors, Inc. (San Diego, CA, USA). All of the fresh
samples were lyophilized and then powdered. Powdered samples (100
mg) were extracted with 5.00 mL of methanol/water (60:40, v/v)
using sonication for 60 min at room temperature and then centrifuged
Received: April 26, 2013
Revised: July 31, 2013
Accepted: October 21, 2013
Article
pubs.acs.org/JAFC
© XXXX American Chemical Society Adx.doi.org/10.1021/jf401802n |J. Agric. Food Chem. XXXX, XXX, XXX−XXX

at 1000gfor 15 min (IEC Clinical Centrifuge, Damon/IEC Division,
Needham, MA, USA). The supernatant was filtered through a 17 mm
(0.45 μm) PVDF syringe filter (VWR Scientific, Seattle, WA, USA),
and 10 μL of the extract was used for each HPLC injection.
UHPLC-PDA-ESI/HRMS/MSnConditions. The UHPLC-HRMS
system used consisted of an LTQ Orbitrap XL mass spectrometer with
an Accela 1250 binary pump, a PAL HTC Accela TMO autosampler, a
PDA detector (ThermoFisher Scientific, San Jose, CA, USA), and a
G1316A column compartment (Agilent, Palo Alto, CA, USA).
Separation was carried out on a Hypersil Gold AQ RP-C18 UHPLC
column (200 mm ×2.1 mm i.d., 1.9 μm, ThermoFisher Scientific)
with an UltraShield precolumn filter (Analytical Scientific Instruments,
Richmond, CA, USA) at a flow rate of 0.3 mL/min. The mobile phase
consisted of a combination of A (0.1% formic acid in water, v/v) and B
(0.1% formic acid in acetonitrile, v/v). The linear gradient was from 4
to 20% B (v/v) at 40 min, to 35% B at 60 min, and to 100% B at 61
min and held at 100% B to 65 min. The PDA was set at 520, 330, and
280 nm to record the peaks, and UV−vis spectra were recorded from
200 to 700 nm.
Both positive and negative ionization modes were used, and the
conditions were set as follows: sheath gas at 70 (arbitrary units),
auxiliary and sweep gases at 15 (arbitrary units), spray voltage at 4.8
kV, capillary temperature at 300 °C, capillary voltage at 15 V, and tube
lens at 70 V. The mass range was from 100 to 2000 amu with a
resolution of 15000, FTMS AGC target at 2e5, FT-MS/MS AGC
target at 1e5, isolation width of 1.5 amu, and maximum ion injection
time of 500 ms. The most intense ion was selected for the data-
dependent scan to offer their MS2to MS5product ions, respectively,
with a normalization collision energy at 35%.
■RESULTS AND DISCUSSION
Strategies for Systematic Identification of Polyphe-
nols from Microgreen Brassica.Brassicaceae polyphenol
composition has been extensively investigated. The main
flavonols in Brassica vegetables are the O-glycosides of
quercetin, kaempferol, and isorhamnetin.
2,17−22
The sugar
moiety found in Brassica vegetables is glucose, occurring as
Table 1. Typical Substitutional Groups and Common Neutral Losses of Polyphenols in Five Brassica Species Microgreens
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf401802n |J. Agric. Food Chem. XXXX, XXX, XXX−XXXB

mono-, di-, tri-, tetra-, and pentaglucosides.
17−23
They are also
commonly found acylated by different hydroxycinnamic acids.
Anthocyanins are another main class of flavonoid found in
Brassica vegetables, and cyanidin is the most common
anthocyanidin in colored-leaf Brassica vegetables.
2,24
Hydrox-
ycinnamic acids (C6−C3) are phenolic acids characterized in
Brassica vegetables with the most common ones being p-
coumaric, caffeic, sinapic, and ferulic acids, often found in
conjugation with sugars or other hydroxycinnamic
acids.
2,17−19,21,22
The five Brassica species microgreen phenolic compounds
exhibit absorbance maxima at three wavelengths (280 nm for
flavonols and flavonol glycosides, 320 nm for hydroxycinnamic
acid derivatives, and 520 nm for anthocyanins).
2,17−19,21,22
HRMS was used for the determination of chemical formulas.
Neutral loss information from MS was used for identification of
sugar moiety and acyl groups. In MS analysis, cleavage of the
first glycosidic linkage is expected to take place at the O-
glycosidic bond at the 7-position of the flavonols and at the 5-
position of the anthocyanins, leading to the fragmentations [(M
−H) −162]−for monohexosides and [(M −H) −324]−for
dihexosides.
23,25,26
The remaining glucose moieties of the
flavonoid molecule are expected to be linked to the hydroxyl
group at the 3-position of the aglycone. The disaccharide
moieties of the flavonoids in Brassica species are mainly
sophorosides.
2
The MS fragmentation behavior can be used for
the determination of interglucoside linkage, and neutral losses
of 180, 162, and 120 amu indicate a sophoroside with a 1→2
interglucoside linkage, whereas loss of 324 amu, and in some
cases low abundance of 162 amu, corresponds to a diglucoside
with a 1→6 linkage such as gentiobioside.
27
The saccharides
(mono-, di-, trisaccharides) and acyl groups of flavonol
glycoside and their possible neutral losses in CID MS/MS
analysis are listed in Table 1, and the basic structures of the
phenolic compounds found in these five Brassica species
microgreens are shown in Figure 1.
Anthocyanins. Among the five Brassica species micro-
greens, red cabbage, red mustard, purple mustard, and purple
kohlrabi have red to purple seed-leaves. UHPLC chromato-
grams at 520 nm revealed 30 different anthocyanins are likely
responsible for this coloration (Figure 2). The retention times
(tR), HRMS masses [M]+, molecular formulas, errors (ppm)
between theoretical and measured values, and major MS2and
MS3product ions are summarized in Table 2.
In these five Brassica species microgreens, only cyanidin (Cy)
derivatives were found, which is in accordance with the other
studies on Brassica species.
24,28−30
The anthocyanins found in
red cabbage microgreens were Cy 3-diglucoside-5-glucoside
derivatives acylated with different hydroxycinnamic acids at the
diglucosyl moiety in the 3-position. High-resolution mass
spectroscopic analysis with multistage mass fragmentation was
used as an important tool for anthocyanin characterization.
Among the 30 Cy glycosides found in red cabbage, red
mustard, purple mustard, and purple kohlarabi microgreens,
Figure 1. Basic chemical structures identified from five Brassica species microgreens.
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf401802n |J. Agric. Food Chem. XXXX, XXX, XXX−XXXC

peak 1 at m/z773.2106 (C33H41O21,−1.36 mmu) was the
lowest molecular weight anthocyanin, and losses of three
hexosyl units were observed in MS2spectra, suggesting Cy 3-
diglucoside-5-glucoside, a typical compound reported in red
cabbage. The major acylated anthocyanins were Cy 3-
diglucoside-5-glucoside derivatives with various acylated
groups, for example, coumaroyl, feruloyl, and sinapoyl
connected to the diglucoside. The MS/MS of most of the
molecular ions of acylated anthocyanins gave the major product
ions at m/z449, a Cy 5-glucoside residue, and at m/z611, a Cy
3-diglucoside residue. The MS/MS fragments of the acylated
anthocyanins allow for a rough determination of the location of
the acylating groups. Peaks 12, 13, and 14 are the major
anthocyanins in microgreen red cabbage, and they were
identified as cyanidin 3-diferuloyl-sophoroside-5-glucoside, Cy
3-(sinapoyl)(sinapoyl)sophoroside-5-glucoside, and Cy 3-
(sinapoyl)(feruloyl)sophoroside-5-glucoside, respectively.
Using peak 12 as an example, HRMS gave the [M]+ion at
1125.3070, corresponding to the formula of C53H57O27.
Fragmentation of the ion at m/z1125 in positive mode
produced ions at m/z963 by loss of a glucosyl residue (162
amu) from the 5-position. The ion at m/z449 was produced by
a total loss of 676 amu, corresponding to a diferuloyl-diglucosyl
residue (176 + 176 + 324 amu), from the terminal 3-position.
In a previous study of purple kohlrabi, 12 anthocyanins have
been identified. The major ones are Cy 3-(feruloyl)(sinapoyl)
diglucoside-5-glucoside, Cy 3-(feruloyl) diglucoside-5-gluco-
side, and Cy 3-(sinapoyl)(sinapoyl) diglucoside-5-glucoside.
31
In our study, acylated anthocyanins with one malonyl group
attached to the hexose of C-5 and other aromatic groups
(caffeic, p-coumaric, sinapic, or ferulic acid) attached to the C-3
glycosidic substituent were found. In the MS2spectra, the
fragment ions at (m/z1023, 993, and 963), with the two acyl
groups attached to the dihexose of C-3, are usually observed as
the base peak. This fragmentation pattern was evidenced with
most anthocyanins analyzed and led to the tentative
identification of Cy 3-(feruloyl)(feruloyl)diglucoside-5-(malon-
yl)-glucoside (m/z1211, peak 19), Cy 3-O-(sinapoyl)-
(feruloyl)diglucoside-5-O-(malonyl)glucoside (m/z1241, peak
20), and Cy 3-O-(sinapoyl)(sinapoyl)diglucoside-5-O-
(malonyl)glucoside (m/z1271, peak 21). Peaks 11a−14awere
identified as Cy 3-p-(coumaroyl)sophoroside-5-(malonyl)-
glucoside, cy 3-O-(p-coumaroyl)(sinapoyl) diglucoside-5-O-
(malonyl) glucoside, Cy 3-O-(feruloyl)glucoside-5-O-(malon-
yl) glucoside, and Cy 3-O-(sinapoyl)glucoside-5-O-(malonyl)-
glucoside in red mustard microgreens.
10
Peaks 19 and 20 were
two major anthocyanins identified in red and purple mustard.
Peaks 16b,17
b, and 18bwere identified as Cy 3-(sinapoyl)-
(coumaroyl)triglucoside-5-(malonyl)glucoside, Cy 3-(caffeoyl)-
(sinapoyl)(xylosyl)glucoside-5-(malonyl)glucoside and Cy 3-
(coumaroyl)(sinapoyl)diglucoside-5-(malonyl)glucoside, re-
spectively.
O-Glycosylated Flavonols and Their Acylated Deriva-
tives. Acylated flavonoid glycosides were easily identified on
the basis of the increased mass of the parent ions and the
wavelength maxima (330−336 nm) of their UV spectra (Figure
3). According to the MSn(n=2−5) data, the aglycones of the
flavonol glycosides were quercetin (Qn), kaempferol (Km), and
Figure 2. UHPLC chromatogram from four Brassica species microgreens, red cabbage (A), purple kohlrabi (B), red mustard (C), and purple
mustard (D), under 520 nm.
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf401802n |J. Agric. Food Chem. XXXX, XXX, XXX−XXXD

isorhamnetin (Is). Using the strategy described previously, 105
flavonol glycosides were characterized in five microgreens
vegetables (Figure 3). Among them, 18 were nonacylated
flavonoid glycosides and 87 were acylated flavonoid glycosides.
The compound distribution in these five microgreens is shown
in Table 3. Qn 3-sophoroside-7-glucoside, Qn 3-hydroxyfer-
uloylsophoroside-7-glucoside, Km 3-hydroxyferuloylsophoro-
side-7-glucoside, Km 3-sinapoylsophoroside-7-glucoside, and
Is 3-caffeoylsophoroside-7-glucoside are common peaks in all
five Brassica species microgreens. Is 3-O-glucoside, Qn 3,7-di-
O-glucoside, Km 3-p-coumaroyldiglucoside, Qn 3-caffeoylso-
phoroside, Qn 3-feruloylsophoroside, Qn 3-feruloylsophoro-
side-7-glucoside, and Km 3-sinapoylsophoroside were found
only in mizuna microgreens, whereas Km 3-sinapoylsophoro-
side-7-glucoside and Qn 3-sinapoylsophorotrioside were found
only in purple kohlrabi. Red cabbage microgreens had Km 3-p-
coumaroylsophorotrioside, Km 3-p-coumaroylsophoroside-7-
diglucoside, Km 3-hydroxyferuloylsophorotrioside-7-glucoside,
Km 3-disinapoyldiglucoside-7-glucoside, Km 3-sinapoylferu-
loylsophoroside-7-glucoside, and Qn 3-disinapoylsophorotrio-
Table 2. UHPLC-HRMS Data of Anthocyanins from Five Brassica Species Microgreens: Red Cabbage, Red Mustard, Purple
Mustard, Mizuna, and Purple Kohlrabi
peak tR
(min) [M]+formula error
(mmu) major and important MS2ions major MS3
ion tentative identification
1 5.97 773.2106 C33H41O21 −1.36 611 (29), 449 (40), 287 (100) 287 (100) Cy 3-diglucoside-5-glucoside
a
2 12.34 965.2528 C43 H49 O25 −2.12 803 (100), 641 (20), 287 (60) 287 (100) Cy 3-hydroxyferuloyl-5-glucoside
a
3 14.98 979.2699 C44H51O25 −1.53 817 (71), 449 (46), 287 (100) 287 (100) Cy 3-(sinapoyl)-diglucoside-5-glucosides
a
4 15.29 979.2708 C44 H51 O25 −0.61 817 (82), 449 (52), 287 (100) 287 (100) Cy 3-(sinapoyl)-diglucoside-5-glucoside
a
5 17.21 1141.3246 C50H61O30 0.34 979 (100), 449 (54) 287 (100) Cy 3-(glucopyranosyl-sinapoyl)
diglucoside-5-glucoside
a
6 24.63 919.249 C42H47O23 −1.38 757 (100), 449 (19), 287 (50) 287 (100) Cy 3-(coumaroyl)sophoroside-5-
glucoside
a
7 25.23 1287.3597 C59H67O32 −1.01 1125 (100), 449 (6) 963 (100), Cy 3-(glucosyl)(sinapoyl)(p-coumaroyl)
sophorside-5-glucoside
a
8 26.31 1317.369 C60H69O33 −1.94 1185 (100), 1155 (35), 449 (2) 1023 (100),
449 (3) Cy 3-(glucosyl)(sinapoyl)(feruloyl)
sophorside-5-glucoside
a
9 26.97 919.249 C42H47O23 −1.38 757 (100), 449 (19), 287 (50) 287 (100) Cy 3-(coumaroyl)sophoroside-5-
glucoside
a
10 27.71 949.2602 C43H49O24 −0.66 787 (100), 449 (18), 287 (49) 287 (100) Cy 3-(feruloyl)sophoroside-5-glucoside
a
11 28.31 1141.3016 C53H57O28 −1.30 979 (100), 449 (11) 287 (100) Cy 3-(caffeoyl)(sinapoyl)diglucoside-5-
glucoside
11a 29.14 1005.2492 C45H49O26 −1.46 757 (22), 535 (100), 491 (10), 287 (73) 287 (100) Cy 3-(coumaroyl)sophoroside-5-
(malonyl)glucoside
12 33.37 1125.307 C53H57O27 −1.04 963 (100), 449 (13) 287 (100) Cy 3-diferuloylsophoroside-5-glucoside
a
12a 31.07 1211.3088 C56H59O30 0.23 963 (100), 535 (81), 521 (9) 287 (100) Cy 3-(coumaroyl)(sinapoyl)diglucoside-5-
(malonyl)glucoside
a
13 34.50 1125.307 C53H57O27 −1.04 963 (100), 449 (13) 287 (100) Cy 3-diferuloylsophoroside-5-glucoside
a
13a 32.56 1035.2599 C46H51O27 −1.32 992 (7), 787 (40), 780 (5), 535 (100),
492 (12), 449 (6), 287 (5) 287 (100) Cy 3-(feruloyl)glucoside-5-(malonyl)-
glucoside
a
14 35.07 1155.3192 C54H59O28 0.40 993 (100), 449 (9) 287 (100) Cy 3-sinapoylferuloylsophoroside-5-
glucoside
a
14a 33.21 1065.2702 C47H53O28 −1.59 817 (73), 535 (100), 492 (2), 449 (3) 287 (100) Cy 3-(sinapoyl)glucoside-5-(malonyl)-
glucoside
a
15 35.91 1155.3192 C54H59O28 0.40 993 (100), 449 (9) 287 (100) Cy 3-(sinapoyl)(feruloyl)sophoroside-5-
glucoside
a
16 37.14 1185.3298 C55H61O29 0.50 1023 (100), 449 (10) 287 (100) Cy 3-(sinapoyl)(sinapoyl)sophoroside-5-
glucoside
a
16b 36.34 1373.3585 C62 H69 O35 −2.89 963 (100), 697 (66), 653 (28) 287 (100) Cy 3-(sinapoyl)(coumaroyl)triglucoside-5-
(malonyl)-glucoside
a
17 37.55 1155.3192 C54H59O28 0.40 993 (100), 449 (9) 287 (100) Cy 3-(sinapoyl)(feruloyl)sophoroside-5-
glucoside
a
17b 37.08 1197.2902 C55 H57O30 −2.72 949 (18), 860 (3), 535 (100), 517 (3),
491 (9) 287 (100) Cy 3-(caffeoyl)(sinapoyl)(xylosyl)
glucoside-5-(malonyl)glucoside
a
18 37.99 1185.3298 C55H61O29 0.49 1023 (100), 449 (10) 287 (100) Cy 3-(sinapoyl)(sinapoyl)sophoroside-5-
glucoside
a
18b 37.37 1227.3008 C56H59O31 −2.68 979 (82), 535 (100), 491 (10) 287 (100) Cy 3-(p-coumaroyl)(sinapoyl)diglucoside-
5-O-(malonyl)glucoside
19 38.00 1211.3082 C56H59O30 −1.46 963 (91), 535 (100), 491 (3) 287 (100) Cy 3-(feruloyl)(feruloyl)diglucoside-5-
(malonyl)glucoside
a
20 38.56 1241.3192 C57H61O31 −1.03 1206 (15), 1198 (30), 993 (100), 535 (88),
449 (8) 287 (100) Cy 3-(sinapoyl)(feruloyl)diglucoside-5-
(malonyl)glucoside
a
21 38.85 1271.3296 C58H63O32 −0.10 1023 (100), 535 (51), 491 (7) 287 (100) Cy 3-(sinapoyl)(sinapoyl)diglucoside-5-
(malonyl)glucoside
a
22 39.35 1241.3190 C57H61O31 −0.13 993 (100), 535 (70), 492 (13) 287 (100) Cy 3-(sinapoyl)(feruloyl)diglucoside-5-
(malonyl)glucoside
a
23 39.81 1211.3078 C56H59O30 −0.77 963 (86), 535 (100) 287 (100) Cy 3-(p-coumaroyl)(sinapoyl)diglucoside-
5-(malonyl)glucoside
a
a
Compared with literature data; Cy, cyanidin.
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf401802n |J. Agric. Food Chem. XXXX, XXX, XXX−XXXE

Figure 3. UHPLC chromatogram of five Brassica species microgreens, red cabbage (A), purple kohlrabi (B), red mustard (C), purple mustard (D),
and mizuna (E), under 330 nm.
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf401802n |J. Agric. Food Chem. XXXX, XXX, XXX−XXXF

Table 3. UHPLC-HRMS Data of flavonol Glycosides and Derivatives of Hydroxycinnamic Acids and Hydroxybenzoic Acids
from Five Brassica Species Microgreens: Red Cabbage, Red Mustard, Purple Mustard, Mizuna, and Purple Kohlrabi
peak tR(min) [M −H]−formula error
(mmu) major and important MS2ions MS3ion tentative identification
a
a1 1.66 133.0143 C4H5O50.40 24 (83), 153 (100) 115 (100) malic acid
b
a2 1.98 191.0192 C6H7O7−0.53 173 (22), 111 (100) 67 (100) citric acid
b
a3 3.37 205.0349 C7H9O7−0.48 173 (61), 159 (6), 143 (11), 111 (100) methyl citric acid
b1 5.56 503.1398 C21H27O14 −0.83 341 (100), 179 (9) 179 (100) courmaroyl-diglucoside
a4 5.71 355.1029 C16H19O9−1.56 217 (59), 193 (100), 175 (40) 134 (100) feruloyl-glucose
b2 5.93 353.0870 C16H17O9−0.80 191 (100), 179 (43), 135 (8) 173 (100) caffeoyl-quinic acid
5a 6.12 299.0768 C13H15O8−0.44 239 (90), 179 (71), 137 (100) salicyloyl-glucose
b
a6 6.21 547.1671 C23H31O15 0.47 223 (100) 208 (100) sinapoyl-gentiobiose
b
a7 7.18 447.0557 C20 H15O12 −1.12 357 (38), 275 (55), 259 (100) 139 (100) rhamnosyl-ellagic acid
a8 8.32 787.1942 C33H39O22 1.45 625 (100) 300 (100) Qn 3-diglucoside-7-glucoside
b
a9 9.20 787.1920 C33H39O22 −1.85 625 (100) 300 (100) Qn 3-diglucoside-7-glucoside
b
c1 9.68 933.2486 C39H49O26 −0.79 771 (100) 591 (100) Km 3-sophorotrioside-7-glucoside
b3 10.06 787.1916 C33H39O22 −2.25 625 (100) 300 (100) Qn 3-sophoroside-7-glucoside
b
d1 10.09 845.2113 C39H41O21 −3.28 683 (100), 477 (15), 315 (6) 353 (100) Is 3-sinapoylglucoside-7-glucoside
b
a10 10.11 771.1978 C33H39O21 −1.13 609 (100) 285 (100) Km 3-sophoroside-7-glucoside
b
d2 10.43 817.2015 C34H41O23 −2.91 609 (100), 447 (34) 447 (100) Km 3-diglucoside-7-glucoside with
HCOOH
c2 10.45 1141.2889 C49 H57O31 1.07 979 (100), 949 (93), 787 (72) 787 (100) Qn 3-hydroxyferuloylsophorotioside-7-
glucoside
b
a11 10.59 979.2349 C43H47O26 −1.23 817 (98), 787 (100), 625 (59) 625 (100) Qn 3 hydroxyferuloylsophoroside-7-
glucoside
b
c3 10.80 1111.2760 C48 H55O30 −2.13 949 (100), 787 (30) 787 (100) Qn 3-caffeoylsophorotrioside-7-
glucoside
b
a12 10.82 979.2333 C43H47O26 −2.80 817 (98), 787 (100), 625 (59) 625 (100) Qn 3 hydroxyferuloylsophoroside-7-
glucoside
b
b4 10.92 787.1906 C33H39O22 −3.25 625 (100) 300 (100) Qn 3-sophoroside-7-glucoside
b
d3 10.97 979.2359 C43H47O26 −0.20 817 (92), 787 (100), 625 (51) 625 (100) Qn 3 hydroxyferuloylsophoroside-7-
glucoside
b
a13 10.99 949.2256 C42H45O25 0.06 787 (100), 625 (22) 625 (100) Qn 3-caffeoylsophoroside-7-glucoside
b
c4 11.11 1111.2749 C48 H55O30 −3.46 949 (100), 787 (29) 787 (100) Qn 3-caffeoylsophorotrioside-7-
glucoside
b
a14 11.12 949.2231 C42H45O25 −2.44 787 (100), 625 (20) 625 (100) Qn 3-caffeoylsophoroside-7-glucoside
b
c5 11.31 1111.2762 C48 H55O30 −2.16 949 (100), 787 (29) 787 (100) Qn 3-caffeoylsophorotrioside-7-
glucoside
b
a15 11.36 1111.2780 C48H55O30 −0.36 949 (100), 787 (30) 787 (100) Qn 3-caffeoylsophorotrioside-7-
glucoside
b
d4 11.47 949.2234 C42H45O25 −2.14 787 (100), 625 (20) 625 (100) Qn 3-caffeoylsophoroside-7-glucoside
b
b5 11.53 609.1447 C27H29O16 −1.41 489 (7), 447 (100), 285 (10) 285 (100) Km 3-diglucoside
a16 11.65 1111.2761 C48H55O30 −2.26 949 (100), 788 (34), 625 (36) 625 (100) Qn 3-caffeoylsophorotrioside-7-
glucoside
b
b6 11.81 771.1976 C33H39O21 −1.33 609 (100) 285 (100) Km 3-sophoroside-7-glucoside
b
c6 11.92 787.1942 C33H39 O22 1.45 625 (100) 300 (100) Qn 3-sophoroside-7-glucoside
b
a17 12.05 963.2385 C43H47O25 −2.69 801 (100), 609 (2) 609 (100) Km 3-hydroxyferuloylsophoroside-7-
glucoside
b
d5 12.07 1111.2766 C48H55O30 −1.76 949 (100), 787 (38) Qn 3-caffeoylsophorotrioside-7-
glucoside
b
c7 12.08 979.2349 C43H47 O26 −1.23 817 (98), 787 (100), 625 (59) 625 (100) Qn 3 hydroxyferuloylsophoroside-7-
glucoside
b
b7 12.25 979.2329 C43H47O26 −3.21 817 (95), 787 (100), 625 (55) 625 (100) Qn 3 hydroxyferuloylsophoroside-7-
glucoside
b
c8 12.28 1125.2937 C49 H57O30 −0.28 963 (100) 771 (100) Km 3-hydroxyferuloylsophorotrioside-7-
glucoside
b
d6 12.37 949.2234 C42H45O25 −2.14 787 (100), 625 (20) 625 (100) Qn 3-caffeoylsophoroside-7-glucoside
b
a18 12.53 977.2541 C44H49O25 −2.80 831 (43), 771 (100), 625 (21) 301 (100) Qn 3 sophoroside-7-sinapoylrhamoside
c9 12.63 1095.2826 C48 H55O29 −0.28 975 (2), 933 (100), 809 (7) 771 (100) Km 3-caffeoylsophorotrioside-7-
glucoside
b
b8 12.69 949.2236 C42H45O25 −1.94 787 (100), 625 (20) 625 (100) Qn 3-caffeoylsophoroside-7-glucoside
b
a19 12.72 933.2289 C42H45O24 −1.73 771 (100) 609 (100) Km 3-caffeoyldiglucoside-7-glucoside
c10 12.91 547.1671 C23H31O15 0.47 223 (100) 208 (100) sinapoylgentiobiose
b
d7 12.94 963.2381 C43H47O25 −3.09 801 (100) 609 (100) Km 3-hydroxyferuloylsophoroside-7-
glucoside
b
b8 13.15 1111.2760 C48H55O30 −2.13 949 (100), 787 (30) 787 (100) Qn 3-caffeoylsophorotrioside-7-
glucoside
b
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf401802n |J. Agric. Food Chem. XXXX, XXX, XXX−XXXG

Table 3. continued
peak tR(min) [M −H]−formula error
(mmu) major and important MS2ions MS3ion tentative identification
a
a20 13.18 1095.2797 C48H55O29 −3.42 949 (84), 933 (100), 787 (45) 787 (100) Qn 3-p-coumaroyltriglucoside-7-
glucoside
a21 13.32 1095.2826 C48H55O29 −0.28 975 (2), 933 (100), 809 (7) 771 (100) Km 3-caffeoylsophorotrioside-7-
glucoside
b
c11 13.44 625.1410 C27H29O17 −0.04 463 (8), 343 (16), 301 (100) 179 (100) Qn diglucoside
b
a22 13.52 1155.3022 C50H59O31 −2.38 993 (100), 950 (41), 787 (39) Qn 3-sinapoyltriglucoside-7-glucoside
d8 13.58 961.2592 C44H49O24 −2.84 623 (72), 609 (100), 592 (27) 257 (100) Km 3-sophoroside-7-sinapoylrhamnoside
a23 13.69 993.2493 C44H49O26 −2.45 801 (13), 787 (100) 607 (100) Qn 3-sinapoylsophoroside-7-glucoside
b
d9 13.77 933.2275 C42H45O24 −3.13 771 (100) 609 (100) Km 3-caffeoyldiglucoside-7-glucoside
b10 13.78 963.2391 C43H47O25 −2.09 801 (100) 609 (100) Km 3-hydroxyferuloylsophoroside-7-
glucoside
b
c12 13.92 355.1029 C16H19O9−1.56 217 (59), 193 (100), 175 (40) 134 (100) feruloylglucose
b
a24 14.12 1125.2937 C49H57O30 −0.28 963 (100) 771 (100) Km 3-hydroxyferuloylsophorotrioside-7-
glucoside
b
b11 14.21 355.1029 C16H19O9−1.56 217 (59), 193 (100), 175 (40) 134 (100) feruloylglucose
b
d10 14.21 933.2283 C42H45O24 −2.24 787 (10), 771 (100), 625 (11) 625 (100) Km 3-caffeoyldiglucoside-7-glucoside
c13 14.21 1155.3028 C50H59O31 −1.78 993 (100), 950 (29), 788 (30) Qn 3-sinapoyltriglucoside-7-glucoside
c14 14.33 1155.3023 C50H59O31 −2.28 993 (100), 950 (29), 788 (30) Qn 3-sinapoyltriglucoside-7-glucoside
b12 14.39 933.2306 C42H45O24 −0.03 787 (14), 771 (100), 625 (11) 625 (100),
607 (8) Qn 3-p-coumaroyldiglucoside-7-
glucoside
b
a25 14.52 1095.2810 C48H55O29 −2.45 949 (100), 933 (38), 771 (62), 625 (40) Km 3-caffeoyl-triglucoside-7-glucoside
b13 14.61 933.2280 C42H45O24 −2.63 771 (100) 609 (100) Km 3-caffeoyl-diglucoside-7-glucoside
a26 14.67 1095.2811 C48H55O29 −2.35 949 (100), 933 (38), 932 (6), 787 (6),
771 (62) Km 3-caffeoyl-triglucoside-7-glucoside
d11 14.89 385.1137 C17H21O10 −0.32 247 (52), 223 (100), 205 (55) 164 (100) sinapic acid-glucose
c15 14.91 993.2486 C44H49O26 −3.15 831 (99), 787 (100), 769 (6), 625 (44) 625 (100) Qn 3-sinapoylsophorotrioside
b
a27 15.01 1139.3093 C50H59O30 −0.32 977 (100) 771 (100) Km 3-sinapoylsophorotrioside-7-
glucoside
b
d12 15.04 993.2481 C44H49O26 −3.65 831 (100), 787 (94), 769 (6), 625 (45) Qn 3-sinapoyldiglucoside-7-glucoside
a28 15.21 977.2535 C44H49O25 −2.24 815 (100), 609 (3) 609 (100) Km 3-sinapoylsophoroside-7-glucoside
b
b14 15.22 993.2496 C44H49O26 −2.15 831 (99), 787 (100), 769 (6), 625 (44) 625 (100) Qn 3-sinapoyltriglucoside
c16 15.27 963.2387 C43H47O25 −2.49 801 (100), 609 (2) 609 (100) Km 3-hydroxyferuloylsophoroside-7-
glucoside
b
b15 15.55 963.2381 C43H47O25 −3.09 801 (100), 787 (45), 625 (26) 625 (100) Km 3-hydroxyferuloylsophoroside-7-
glucoside
b
d13 15.55 963.2374 C43H47O25 −3.79 801 (100), 787 (47), 625 (25) Km 3-hydroxyferuloylsophoroside-7-
glucoside
b
c17 15.63 1139.3103 C50H59O30 0.56 977 (100), 771 (3) 771 (100) Km 3-sinapoylsophorotrioside-7-
glucoside
b
b16 15.72 963.2391 C43H47O25 −2.09 801 (100), 787 (45), 625 (26) 625 (100) Km 3-hydroxyferuloylsophoroside-7-
glucoside
b
d14 15.82 933.2283 C42H45O24 −2.33 787 (10), 771 (100), 625 (11) 625 (100) Km 3-caffeoyldiglucoside-7-glucoside
a29 15.87 947.2429 C43H47O24 −2.28 827 (2), 785 (100), 609 (2) 609 (100) Km 3-feruloylsophoroside-7-glucoside
b
b17 15.93 933.2280 C42H45O24 −2.63 788 (10), 771 (100), 625 (11) 625 (100) Km 3-caffeoyldiglucoside-7-glucoside
c18 15.93 1109.2946 C49H57O29 −4.06 947 (100) 771 (100) Km 3-feruloylsophorotrioside-7-
glucoside
a30 16.28 917.2318 C42H45O23 −4.26 755 (100) 609 (100) Km 3-p-coumaroylsophoroside-7-
glucoside
b
d15 16.36 1095.2805 C48H55O29 −2.95 933 (100), 787 (28) Km 3-caffeoyltriglucoside-7-glucoside
c19 16.39 977.2535 C44H49O25 −2.24 815 (100), 609 (3) 609 (100) Km 3-sinapoylsophoroside-7-glucoside
b
a31 16.47 1079.2852 C48H55O28 −3.33 755 (100), 609 (12) 609 (100) Km 3-p-coumaroylsophoroside-7-
diglucoside
b18 16.48 1139.3065 C50H59O30 −3.16 977 (100) 771 (100) Km 3-sinapoylsophorotrioside-7-
glucoside
b
b19 16.63 977.2542 C44H49O25 −2.64 815 (100) 609 (100) Km 3-sinapoylsophoroside-7-glucoside
b
d16 16.76 609.1441 C27H29O16 −2.01 489 (13), 447 (100), 285 (19) 284 (100) Km 3-glucoside-7-glucoside
c
c20 16.93 947.2429 C43H47O24 −2.28 827 (2), 785 (100), 609 (2) 609 (100) Km 3-feruloylsophoroside-7-glucoside
b
c21 17.17 639.1566 C28H31O17 2.08 519 (10), 477 (100), 315 (12) 314 (100) Is 3-glucoside-7-glucoside
b
b20 17.20 947.2439 C43H47O24 −2.38 785 (100) 609 (100) Km 3-feruloylsophoroside-7-glucoside
b
d17 17.25 977.2535 C44H49O25 −3.34 815 (100), 771 (10) 609 (100) Km 3-sinapoylsophoroside-7-glucoside
b
b21 17.55 917.2328 C42H45O23 −2.91 755 (100) 609 (100) Km 3-p-coumaroylsophoroside-7-
glucoside
d18 17.97 947.2429 C43H47O24 −3.38 785 (100) 609 (100) Km 3-feruloylsophoroside-7-glucoside
b
c22 18.03 551.1753 C26H31O13 −1.71 389 (100), 341 (6) 341 (100) ferulic acid-rhamnosylglucose with a 48
amu group
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf401802n |J. Agric. Food Chem. XXXX, XXX, XXX−XXXH

side, which were also found in mature red cabbage. Km 3-
sophorotrioside-7-glucoside, Qn 3-caffeoylsophorotrioside-7-
glucoside, and Qn 3-hydroxyferuloylsophorotioside-7-glucoside
existed only in microgreens of red mustard and purple mustard.
UsingMSanalysisofpeaka19,asanexample,the
deprotonated molecular ion at m/z933 (C42H45O24) lost a
hexosyl group from position 7, giving the product ion at m/z
771. The MS3product ion revealed a loss of 162 amu,
corresponding to a caffeoyl group, and a loss of dihexoxyl group
at the 3-position (324 amu), leading to the Km aglycone (m/z
285). Thus, peak a19 was tentatively identified as Km 3-
caffeoyldiglucoside-7-glucoside. Peak b13 also exhibited the
deprotonated ion at m/z933 but showed different
fragmentation pathways. During the MS fragmentation of
peak 18a, loss of 162 amu, corresponding to a hexosyl moiety at
the terminal 7-position, was observed. Further fragmentation of
the acylated ion, m/z625, gave the loss of p-coumaroyl group
and the loss of a dihexosyl group, producing the Qn aglycone
ion (m/z301). Thus, peak b13 was assigned as Qn 3-p-
coumaroyldiglucoside-7-glucoside. Using this strategy, the
remaining flavonols were identified on the basis of HRMS,
MS fragmentation pattern, UV maxima, and retention times as
flavonols, previously characterized in the five Brassica species
microgreens.
Derivatives of Hydroxycinnamic Acids and Hydrox-
ybenzoic acids. Hydroxycinnamic acids and hydroxybenzoic
acids are considered nonflavonoid phenolics and are charac-
terized by their C6−C3 and C6−C structures, respectively.
Most of the hydroxycinnamic acids and hydroxybenzoic acid
derivatives detected in mature vegetables
17−19,21
were also
detected in our five Brassica species microgreens. However, our
five Brassica species microgreens contained a greater variety and
higher concentrations of cinnamic acids than their mature leaf
counterparts. The retention times, HRMS molecular ions [M −
H]−, diagnostic MS2and MS3product ions, UV λmax, and
identification of the hydroxycinnamates, arranged by molecular
Table 3. continued
peak tR(min) [M −H]−formula error
(mmu) major and important MS2ions MS3ion tentative identification
a
d19 18.12 947.2449 C43H47O24 −1.38 785 (100) 609 (100) Km 3-feruloylsophoroside-7-glucoside
b
c23 18.28 993.2473 C44H49O26 −4.49 801 (13), 787 (100) 607 (100) Qn 3-sinapoylsophoroside-7-glucoside
b
d20 18.36 639.1548 C28H31O17 −1.87 519 (11), 477 (100), 315 (12) 314 (100) Is 3-glucoside-7-glucoside
d21 18.63 917.2330 C42H45O23 −2.71 755 (100) 609 (100) Km 3-p-coumaroyldiglucoside-7-
glucoside
b22 19.13 625.1382 C27H29O17 −2.82 505 (21), 463 (37), 445 (55), 301 (60),
300 (100) Qn 3-diglucoside
a32 19.40 935.2444 C42H47O24 −1.88 773 (100), 755 (29), 663 (52), 285 (30) 285 (100) Km aglycone with 7 glucoside and 3 acyl
glucosyls
a33 20.43 625.1414 C27H29O17 0.60 505 (18), 463 (17), 445 (54), 300 (100) 271 (100) Qn 7-sophoroside
b
b23 21.28 639.1548 C28H31O17 −2.9 315 (100), 300 (16) Is 3-diglucoside
a34 21.40 965.2516 C49H41O21 1.02 803 (100), 785 (24), 693 (48), 667 (9),
285 (21) 285 (100) Km 3-caffeoyldiglucoside-7-glucoside
d22 21.48 933.2271 C42H45O24 −3.53 787 (10), 771 (100), 625 (11) 625 (100) Km 3-caffeoyldiglucoside-7-glucoside
a35 21.90 935.2436 C42H47O24 −2.68 773 (100), 756 (31), 663 (55), 637 (10),
285 (24) 285 (100) Km aglycone with 7 glucoside and 3 acyl
glucosyls
c24 22.16 831.1997 C38H39O21 0.93 625 (100) 300 (100) Qn 3-sinapoylsophoroside
b
b24 23.13 193.0506 C10H9O4−0.03 178 (19), 149 (50), 134 (100) 106 (100) ferulic acid
c
b25 24.01 223.0607 C11H11O5−2.23 208 (8), 179 (11), 164 (100) 149 (100) sinapic acid
c
d23 24.79 193.0502 C10H9O4−0.43 178 (25), 149 (55), 134 (100) 106 (100) ferulic acid
c
b26 24.92 593.1503 C27H29O15 −1.51 447 (100) 284 (100) Km 3-glucoside-7-rhanmoside
d24 25.86 223.0607 C11H11O5−0.50 208 (8), 179 (11), 164 (100) 149 (100) sinapic acid isomer
b27 26.17 977.2536 C44H49O25 −3.24 815 (100), 653 (14) 653 (100) Km 3-sinapoylsophoroside-7-glucoside
b
d25 26.69 223.0607 C11H11O5−2.23 208 (8), 179 (11), 164 (100) 149 (100) sinapic acid isomer
a36 32.77 753.2253 C34H41O19 0.73 529 (100) 205 (100) disinapoylgentiobiose
b
a37 33.68 1123.2886 C53H55O27 −4.47 961 (100), 755 (20) 755 (100) Km 3-hydroxyferuloylsophorotrioside-7-
glucoside
b
a38 34.63 1153.2981 C54H57O28 1.32 991 (100), 785 (20) 785 (100) Km 3-sinapoylferuloylsophoroside-7-
glucoside
b
a39 35.20 1183.3081 C55H59O29 −5.62 1021 (100), 816 (19) 815 (100) Km 3-disinapoyldiglucoside-7-glucoside
a40 35.52 1183.3086 C55H59O29 −5.20 977 (22), 959 (7), 815 (100), 609 (14),
591 (7) 609 (100) Km 3-sinapoyldiglucoside-7-
sinapoylglucoside
b28 37.34 753.2253 C34H41O19 0.73 529 (100) 205 (100) disinapoylgentiobiose
b
a41 37.86 753.2253 C34H41O19 0.73 529 (100) 205 (100) disinapoylgentiobiose
b
d26 38.81 753.2258 C34H41O19 1.39 529 (100) 223 (100) disinapoylgentiobiose
b
a42 39.11 723.2144 C33H39O18 0.29 529 (100), 499 (21) 223 (100) sinapoyl-feruloylgentiobiose
b
a43 39.35 723.2125 C33H39O18 −1.69 529 (100), 499 (21) 223 (100) sinapoyl-feruloylgentiobiose
b
a44 39.99 1199.3057 C55H59O30 −3.96 993 (100, −206), 787 (12) 787 (100) Qn 3-disinapoylsophorotrioside
d27 40.41 723.2120 C33H39O18 −2.19 529 (100), 499 (21) 223 (100) sinapoyl-feruloylgentiobiose
a45 43.75 959.2830 C45H51O23 0.35 735 (100), 529 (7), 511 (11) 529 (100) trisinapoylgentionbiose
b
a47 44.67 959.2798 C45H51O23 −2.87 735 (100), 529 (10), 511 (13) 223 (100) trisinapoylgentionbiose
b
b30 45.98 929.2695 C44H49O22 −2.63 705 (100), 511 (6) 499 (100) feruloyl-disinapoyl-gentionbiose
a
Km, kaempferol; Qn, quercetin; Is, isorhamnetin.
b
Identified with literature data.
c
Identified with reference standards.
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf401802n |J. Agric. Food Chem. XXXX, XXX, XXX−XXXI

weight, are listed in Table 3. Their peaks are eluted with the
flavanol glycoside peaks, as shown in Figure 3. The
hydroxycinnamic acids, hydoxycinnamoylquinic acids, hydrox-
ycinnamoylmalic acids, and hydroxycinnamoyl saccharides with
one to three glucosides were identified using reference
compounds (designated by c) or from the literature (designated
by b). Sixteen of the hydroxycinnamoylsaccharides were formed
from di- or triglucoses, mainly gentiobiose, with one to three
hydroxycinnamoyl units. By direct comparison with reference
compounds in mustard greens, peaks a36, a41, b28, and d26
(Figure 3) were identified as disinapoylgentiobioses. Peaks a4,
b11, and c12 were identified as feruloyl-glucosides. Peaks a42,
d27, and a43 were identified as sinapoyl-feuloylgentiobioses.
Peaks a47 and b30, identified as trisinapoylgentionbiose and
feruloyl-disinapoyl-gentionbiose, are peaks common to micro-
greens of mizuna, purple kohlrabi, red mustard, and purple
mustard. Peak d11 is found only in mizuna and was tentatively
identified as sinapic acid-glucose.
Other organic acids, such as caffeoylquinic acid, ferulic acid,
sinapic acid, citric acid, malic acid, and caffeoylquinic acid, are
organic acids common in these five microgreens. There were a
number of organic acid isomers found in the five Brassisa
microgreens, and identification was based on their similar MS2
and MS3spectra. However, they exhibited different retention
times based on species. For example, peaks a42, a43, and d27
all had the same [M −H]−at m/z723. HRMS measurements
suggested the formula C33H39O18, with the main MS2product
ion at m/z529 (M −194, neutral loss of ferulic acid) and the
main MS3product ion at m/z223 (sinapic acid). These
compounds were identified as sinapoyl-ferulic acid and its
isomers. Similarly, peaks a36, b28, and a41 ([M −H]−at m/z
753, with a main MS2product ion at 529 and main MS3
product ions at 205) were identified as disinapoylgentiobiose
and its isomers.
In summary, this is the first study characterizing phenolic
profiles specifically in Brassica species microgreens. A total of
165 phenolic compounds were tentatively identified using
complementary information from UHPLC-PDA-HRMSnin
negative and positive modes, revealing a large number of highly
glycosylated and acylated quercetin, kaempferol, and cyanidin
aglycones and complex hydroxycinnamic and benzoic acids.
The results showed that the Brassica species microgreens
tended to have more complex polyphenol profiles and to
contain more varieties of polyphenols compared to their mature
plant counterparts. Thus, Brassica species microgreens could be
considered a good source for polyphenols. This compositional
study should serve as reference base for these five Brassica
species microgreens and enhance their value to health agencies
and consumers.
■AUTHOR INFORMATION
Corresponding Author
*(P.C.) Phone: (301) 504-8144. Fax: (301) 504-8314. E-mail:
Pei.Chen@ars.usda.gov.
Funding
This research is supported by the Agricultural Research Service
of the U.S. Department of Agriculture and an Interagency
Agreement with the Office of Dietary Supplements of the
National Institutes of Health.
Notes
The authors declare no competing financial interest.
■REFERENCES
(1) Xiao, Z.; Lester, G. E.; Luo, Y.; Wang, Q. Assessment of vitamin
and carotenoid concentrations of emerging food products: edible
microgreens. J. Agric. Food Chem. 2012,60, 7644−7651.
(2) Cartea, M. E.; Francisco, M.; Soengas, P.; Velasco, P. Phenolic
compounds in Brassica vegetables. Molecules 2011,16, 251−280.
(3) Guzman, I.; Yousef, G. G.; Brown, A. F. Simultaneous extraction
and quantitation of carotenoids, chlorophylls, and tocopherols in
Brassica vegetables. J. Agric. Food Chem. 2012,60, 7238−7244.
(4) Verkerk, R.; Schreiner, M.; Krumbein, A.; Ciska, E.; Holst, B.;
Rowland, I.; De Schrijver, R.; Hansen, M.; Gerhauser, C.; Mithen, R.;
Dekker, M. Glucosinolates in Brassica vegetables: the influence of the
food supply chain on intake, bioavailability and human health. Mol.
Nutr. Food Res. 2009,53 (Suppl. 2), S219.
(5) Terry, P.; Wolk, A.; Persson, I.; Magnusson, C. Brassica
vegetables and breast cancer risk. JAMA, J. Am. Med. Assoc. 2001,
285, 2975−2977.
(6) Verhoeven, D. T.; Goldbohm, R. A.; van Poppel, G.; Verhagen,
H.; van den Brandt, P. A. Epidemiological studies on Brassica
vegetables and cancer risk. Cancer Epidemiol. Biomarkers Prev. 1996,5,
733−748.
(7) Lampe, J. W.; Peterson, S. Brassica, biotransformation and cancer
risk: genetic polymorphisms alter the preventive effects of cruciferous
vegetables. J. Nutr. 2002,132, 2991−2994.
(8) Cox, D. N.; Melo, L.; Zabaras, D.; Delahunty, C. M. Acceptance
of health-promoting Brassica vegetables: the influence of taste
perception, information and attitudes. Public Health Nutr. 2012,15,
1474−1482.
(9) Lin, L. Z.; Harnly, J. M. Phenolic component profiles of mustard
greens, yu choy, and 15 other Brassica vegetables. J. Agric. Food Chem.
2010,58, 6850−6857.
(10) Lin, L.; Sun, J.; Chen, P.; Harnly, J. UHPLC-PDA-ESI/HRMS/
MSnanalysis of anthocyanins, flavonol glycosides and hydroxycin-
namic acid derivatives in red mustard greens (Brassica juncea Coss
variety). J. Agric. Food Chem. 2011,59, 12059−12072.
(11) Lin, L.; Sun, J.; Chen, P.; Harnly, J. M. LC-PDA-ESI/MSn
identification of new anthocyanins in purple Bordeaux radish
(Raphanus sativus L. variety). J. Agric. Food Chem. 2011,59, 6616−
6627.
(12) Lin, L. Z.; Harnly, J. M. Identification of the phenolic
components of collard greens, kale, and Chinese broccoli. J. Agric. Food
Chem. 2009,57, 7401−7408.
(13) Ferreres, F.; Fernandes, F.; Sousa, C.; Valentao, P.; Pereira, J. A.;
Andrade, P. B. Metabolic and bioactivity insights into Brassica oleracea
var. acephala.J. Agric. Food Chem. 2009,57, 8884−8892.
(14) Ferreres, F.; Valentao, P.; Llorach, R.; Pinheiro, C.; Cardoso, L.;
Pereira, J. A.; Sousa, C.; Seabra, R. M.; Andrade, P. B. Phenolic
compounds in external leaves of tronchuda cabbage (Brassica oleracea
L. var. costata DC). J. Agric. Food Chem. 2005,53, 2901−2907.
(15) Sousa, C.; Lopes, G.; Pereira, D. M.; Taveira, M.; Valentao, P.;
Seabra, R. M.; Pereira, J. A.; Baptista, P.; Ferreres, F.; Andrade, P. B.
Screening of antioxidant compounds during sprouting of Brassica
oleracea L. var. costata DC. Comb. Chem. High Throughput Screening
2007,10, 377−386.
(16) Taveira, M.; Pereira, D. M.; Sousa, C.; Ferreres, F.; Andrade, P.
B.; Martins, A.; Pereira, J. A.; Valentao, P. In vitro cultures of Brassica
oleracea L. var. costata DC: potential plant bioreactor for antioxidant
phenolic compounds. J. Agric. Food Chem. 2009,57, 1247−1252.
(17) Olsen, H.; Aaby, K.; Borge, G. I. Characterization and
quantification of flavonoids and hydroxycinnamic acids in curly kale
(Brassica oleracea L. convar. acephala var. sabellica) by HPLC-DAD-
ESI-MSn.J. Agric. Food Chem. 2009,57, 2816−2825.
(18) Ferreres, F.; Valentao, P.; Pereira, J. A.; Bento, A.; Noites, A.;
Seabra, R. M.; Andrade, P. B. HPLC-DAD-MS/MS-ESI screening of
phenolic compounds in Pieris brassicae L. reared on Brassica rapa var.
rapa L. J. Agric. Food Chem. 2008,56, 844−853.
(19) Harbaum, B.; Hubbermann, E. M.; Wolff, C.; Herges, R.; Zhu,
Z.; Schwarz, K. Identification of flavonoids and hydroxycinnamic acids
in pak choi varieties (Brassica campestris L. ssp. chinensis var. communis)
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf401802n |J. Agric. Food Chem. XXXX, XXX, XXX−XXXJ

by HPLC-ESI-MSnand NMR and their quantification by HPLC-DAD.
J. Agric. Food Chem. 2007,55, 8251−8260.
(20) Rochfort, S. J.; Imsic, M.; Jones, R.; Trenerry, V. C.; Tomkins,
B. Characterization of flavonol conjugates in immature leaves of pak
choi [Brassica rapa L. ssp. chinensis L. (Hanelt.)] by HPLC-DAD and
LC-MS/MS. J. Agric. Food Chem. 2006,54, 4855−4860.
(21) Romani, A.; Vignolini, P.; Isolani, L.; Ieri, F.; Heimler, D.
HPLC-DAD/MS characterization of flavonoids and hydroxycinnamic
derivatives in turnip tops (Brassica rapa L. subsp. sylvestris L.). J. Agric.
Food Chem. 2006,54, 1342−1346.
(22) Llorach, R.; Gil-Izquierdo, A.; Ferreres, F.; Tomas-Barberan, F.
A. HPLC-DAD-MS/MS ESI characterization of unusual highly
glycosylated acylated flavonoids from cauliflower (Brassica oleracea L.
var. botrytis) agroindustrial byproducts. J. Agric. Food Chem. 2003,51,
3895−3899.
(23) Vallejo, F.; Tomas-Barberan, F. A.; Ferreres, F. Characterisation
of flavonols in broccoli (Brassica oleracea L. var. italica) by liquid
chromatography-UV diode-array detection-electrospray ionisation
mass spectrometry. J. Chromatogr., A 2004,1054, 181−193.
(24) Lin, L. Z.; Sun, J.; Chen, P.; Harnly, J. UHPLC-PDA-ESI/
HRMS/MS(n) analysis of anthocyanins, flavonol glycosides, and
hydroxycinnamic acid derivatives in red mustard greens (Brassica
juncea Coss variety). J. Agric. Food Chem. 2011,59, 12059−12072.
(25) Wu, X.; Prior, R. L. Identification and characterization of
anthocyanins by high-performance liquid chromatography-electrospray
ionization-tandem mass spectrometry in common foods in the United
States: vegetables, nuts, and grains. J. Agric. Food Chem. 2005,53,
3101−3113.
(26) Wu, X.; Prior, R. L. Systematic identification and character-
ization of anthocyanins by HPLC-ESI-MS/MS in common foods in
the United States: fruits and berries. J. Agric. Food Chem. 2005,53,
2589−2599.
(27) Ferreres, F.; Llorach, R.; Gil-Izquierdo, A. Characterization of
the interglycosidic linkage in di-, tri-, tetra- and pentaglycosylated
flavonoids and differentiation of positional isomers by liquid
chromatography/electrospray ionization tandem mass spectrometry.
J. Mass Spectrom. 2004,39, 312−321.
(28) Arapitsas, P.; Sjoberg, P. J. R.; Turner, C. Characterisation of
anthocyanins in red cabbage using high resolution liquid chromatog-
raphy coupled with photodiode array detection and electrospray
ionization-linear ion trap mass spectrometry. Food Chem. 2008,109,
219−226.
(29) Arapitsas, P.; Turner, C. Pressurized solvent extraction and
monolithic column-HPLC/DAD analysis of anthocyanins in red
cabbage. Talanta 2008,74, 1218−1223.
(30) McDougall, G. J.; Fyffe, S.; Dobson, P.; Stewart, D.
Anthocyanins from red cabbage −stability to simulated gastro-
intestinal digestion. Phytochemistry 2007,68, 1285−1294.
(31) Park, W. T.; Kim, J. K.; Park, S.; Lee, S. W.; Li, X.; Kim, Y. B.;
Uddin, M. R.; Park, N. I.; Kim, S. J.; Park, S. U. Metabolic profiling of
glucosinolates, anthocyanins, carotenoids, and other secondary
metabolites in kohlrabi (Brassica oleracea var. gongylodes). J. Agric.
Food Chem. 2012,60, 8111−8116.
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf401802n |J. Agric. Food Chem. XXXX, XXX, XXX−XXXK