International Journal of

Molecular Sciences

Article
Sulfated Phenolic Substances: Preparation and Optimized
HPLC Analysis
Lucie Petrásková 1 , Kristýna Káňová 1,2 , Katerina Brodsky 1,2 , Anastasiia Hetman 1,3 , Barbora Petránková 1,4 ,
Helena Pelantová 1 , Vladimír Křen 1 and Kateřina Valentová 1, *

1 Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague, Czech Republic;
[email protected] (L.P.); [email protected] (K.K.); [email protected] (K.B.);
[email protected] (A.H.); [email protected] (B.P.);
[email protected] (H.P.); [email protected] (V.K.)
2 Department of Biochemistry and Microbiology, University of Chemistry and Technology Prague, Technická 3,
166 28 Prague, Czech Republic
3 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University,
Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
4 Department of Analytical Chemistry, Faculty of Science, Charles University, Albertov 6,
128 43 Prague, Czech Republic
* Correspondence: [email protected]

Abstract: Sulfation is an important reaction in nature, and sulfated phenolic compounds are of
interest as standards of mammalian phase II metabolites or pro-drugs. Such standards can be pre-
pared using chemoenzymatic methods with aryl sulfotransferases. The aim of the present work
was to obtain a large library of sulfated phenols, phenolic acids, flavonoids, and flavonolignans
and optimize their HPLC (high performance liquid chromatography) analysis. Four new sulfates of
2,3,4-trihydroxybenzoic acid, catechol, 4-methylcatechol, and phloroglucinol were prepared and fully
Citation: Petrásková, L.; Káňová, K.; characterized using MS (mass spectrometry), 1 H, and 13 C NMR. The separation was investigated
Brodsky, K.; Hetman, A.; Petránková, using HPLC with PDA (photodiode-array) detection and a total of 38 standards of phenolics and
B.; Pelantová, H.; Křen, V.; Valentová,
their sulfates. Different stationary (monolithic C18, C18 Polar, pentafluorophenyl, ZICpHILIC) and
K. Sulfated Phenolic Substances:
mobile phases with or without ammonium acetate buffer were compared. The separation results
Preparation and Optimized HPLC
were strongly dependent on the pH and buffer capacity of the mobile phase. The developed robust
Analysis. Int. J. Mol. Sci. 2022, 23,
HPLC method is suitable for the separation of enzymatic sulfation reaction mixtures of flavonoids,
5743. https://doi.org/10.3390/
ijms23105743
flavonolignans, 2,3-dehydroflavonolignans, phenolic acids, and phenols with PDA detection. More-
over, the method is directly applicable in conjunction with mass detection due to the low flow rate
Academic Editor:
and the absence of phosphate buffer and/or ion-pairing reagents in the mobile phase.
David Arráez-Román

Received: 2 May 2022 Keywords: aryl sulfotransferase; Desulfitobacterium hafniense; HPLC analysis; sulfates; flavonoids;
Accepted: 19 May 2022 polyphenols; phenolic acid
Published: 20 May 2022

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in
published maps and institutional affil-
1. Introduction
iations. Plant constituents such as flavonoids and phenolic acids are an essential part of a
so-called healthy diet. These polyphenolic substances, once considered antioxidants and
protective agents against ROS, act more at the receptor level [1]. However, their function
is still not fully understood, and it is necessary to thoroughly study their toxicology,
Copyright: © 2022 by the authors. metabolism, and possible interactions with dietary supplements and drugs.
Licensee MDPI, Basel, Switzerland. Flavonoids and phenolic acids are preferentially metabolized (sulfated, methylated,
This article is an open access article
or glucuronidated) via the II biotransformation phase [2,3]. Sulfated flavonoids and other
distributed under the terms and
polyphenols are therefore of great pharmacological interest. They can serve as potential
conditions of the Creative Commons
(pro)drugs and have antiviral, antitumor, anticoagulant, and anti-inflammatory activi-
Attribution (CC BY) license (https://
ties [4]. Sulfation is also associated with molecular recognition, cell signaling, and hormone
creativecommons.org/licenses/by/
regulation [5]. A detailed study of all these properties requires substantial amounts,
4.0/).

Int. J. Mol. Sci. 2022, 23, 5743. https://doi.org/10.3390/ijms23105743 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2022, 23, 5743 2 of 17

which usually cannot be isolated from the biological material. On the other hand, nature-
inspired sulfated conjugates can be synthesized chemically or enzymatically to provide
well-characterized standards.
The enzymatic sulfation of (poly)phenolic compounds has recently been preferred to
the chemical one [6–13]. Bacterial aryl sulfotransferases such as the one from Desulfitobac-
terium hafniense use p-nitrophenyl sulfate (p-NP-S) as a sulfate donor. When monitoring
the reaction progress, we solve the analytical separation of polar (phenols, p-nitrophenol,
p-NP) and highly polar substances (phenolic sulfates, p-NP-S). Besides TLC, the HPLC
method is the analytical method of the first choice for monitoring sulfation reactions due
to its robustness and general availability. Sulfates of various polyphenols carrying one or
more highly charged sulfate group(s) are inherently very polar compounds. If the sulfate
group is not modified, or if the effect of the modification on the overall polarity is small,
conventional reversed-phase liquid chromatography will not provide adequate retention.
This shows up on the chromatogram as peak fronting, tailing, or very broad peaks.
One way to solve this problem is to modify the sulfate group using ion-pairing liquid
chromatography (IPLC). IPLC uses a conventional reversed stationary phase in combination
with a mobile phase enriched in an ion-pairing reagent. The ion-pairing reagent (generally
an amine) forms a hydrophobic pair with the highly charged molecule of opposite charge.
This dramatically increases the retention of the analytes and thus the sharpness of the
peaks. IPLC has been used, for example, for the analysis of oligonucleotides [14], for
the separation of reaction mixtures with sulfates of 2-phenylethyl alcohol, p-NP-glycerol,
p-NP-glucose, phenyl-glucose, p-NP-N-acetyl-D-glucosamine (GlcNAc) [6], and for 3-
sulfo-17-beta-estradiol-4,40 -disulfate, and for monitoring the sulfation of resveratrol and
phloretin [7,15]. However, IPLC applications may also have disadvantages, such as artifacts
when using gradient elution, incompatibility with mass spectrometry (MS) and preparative
chromatography, or more complex mobile phase preparations.
Therefore, in some cases, a better solution is required. Resolution and sensitivity
can be greatly improved by a careful combination of stationary and mobile phases, flow
rate, temperature, and the type of detection. Hereunder we show the advantages and
disadvantages of analytical methods developed for the separation of sulfated substances.
C18 columns in combination with phosphate-containing mobile phases and an ion-
pairing reagent required over 20 min for the separation of indoxyl sulfate, sulfated pri-
mary aliphatic alcohols, estradiol, or bisphenol-A [6,7,16]. Significantly shorter times
(2.5–6 min) were reached when separating silybin or isosilybin sulfates. However, the
width of the peaks in 5% of their height was greater than one minute (1–4.5 min) in all
cases [9]. Polyamine II column with a linear gradient of phosphate buffers was used to
separate fluorescently labeled N-sulfated polysaccharides [17]. However, phosphate is
incompatible with the LC-MS interface.
For monitoring the metabolic fate of (poly)phenolic substances in complex exper-
iments on animals, MS/MS detection can preferably be used. However, these devices
are expensive and not generally available. The sulfated metabolites of naringin and hes-
peridin were monitored in in vivo experiments using the UHPLC-ESI-MS/MS (ultra-high
performance liquid chromatography coupled with electrospray ionization tandem mass
spectrometry) analysis (Waters Acquity BEH C18 column, gradient elution). The metabo-
lites were identified based on chemical composition, retention time, MS/MS fragmentation
pattern, and comparison with available standards and references [2]. The sulfated product
of chlorocatechol was identified using the NUCLEODUR HILIC (hydrophilic interaction
chromatography) column with an ion trap mass analyzer. The peak of chlorocatechol sul-
fate was broad and the other components of the reaction mixture (p-NP, 3-chlorocatechol)
were not separated from the baseline [18]. The biotransformation products of quercetin,
isoquercitrin, and taxifolin sulfates formed by HepG2 cells were monitored by UHPLC
(XDB phenyl column) and ESI MS/MS detection [19]. The sulfation reaction (catalyzed by
aryl sulfotransferase from Clostridium innocuum) of various phenols, flavonoids, quinones,
primary alcohols, and sugars was analyzed using a C18 column (mobile phase acetonitrile,
Int. J. Mol. Sci. 2022, 23, 5743 3 of 17

water, trifluoroacetic acid) and the identity of the reaction products was confirmed by
MS [20].
The analyses of hydroxytyrosol and acetyltyrosol sulfates [21], luteolin, myricetin, and
ampelopsin sulfates [8], quercetin mono- and disulfates [12] or quercetin, isoquercitrin, and
taxifolin sulfates [11] on the pentafluorophenyl column (mobile phase water, methanol,
trifluoroacetic acid) suffered from tailing peaks. Tailing peaks were also observed during
the separation of sulfated polysaccharides (cellobiose and GlcNAc-linker-tBoc) on the Mul-
tospher APS-HP-5 µm HILIC column (mobile phase acetonitrile, ammonium acetate) [22].
HPLC chromatograms of 2,3-dehydrosilybin sulfation obtained by separation on a mono-
lithic Chromolith C18 column (mobile phase acetonitrile, water, formic acid) also showed
tailing [13].
Separation of a chemically prepared mixture of mono- and disulfates of quercetin on a
BDS -Hypersil C18 column (mobile phase ammonium acetate with methanol) gave good
analytical separation (sharp peaks, baseline separation); up to 20 min was achieved in the
separation [23]. The sulfation reagent sulfur trioxide N-triethylamine probably also served
as an ion-pairing reagent and allowed sharp peaks. Analyses of the sulfates of quercetin
and epicatechin on the AQUA reversed-phase C18 column and the Spherisorb S3OD-2 C18
column (mobile phase trifluoroacetic acid in water and methanol, respectively) took over
30 min [24].
The following parameters can be considered as disadvantages for the separation of
sulfated polyphenols: Phosphate in the mobile phase, use of ion-pairing reagent (both
incompatible with LC-MS), MS/MS detection (not a common detector), separation time
over 20 min, and tailing and/or coelution of the peaks (poorer separation of the peaks
in the reaction mixture). The disadvantages of the published methods with at least one
undesirable parameter are summarized in Table 1.

Table 1. An overview of HPLC methods for sulfate separation published to date and their parameters.

Column C18 C18 a HILIC b Phenyl a PFP c Polyamine

Phosphate in
+ + + - - - - + + - - - - - - - +
mobile phase
Ion-pairing - + + - - - - + + - - - - - - - -
reagent
MS/MS, QTof - - - - - + - + - + + - + - - - Fluorescence
detection
Long separation
time (≥ 20 min) ?d + + + + - - + - + + - - + + - -
Tailing peaks,
? - + ? - ? + - + - + + + + + + +
coelution
Reference [16] [6] [7] [24] [23] [20] [11] [15] [9] [2] [18] [22] [19] [21] [8] [12,13] [17]
aUltra-high performance liquid chromatography (UHPLC), b hydrophilic interaction chromatography, c pentaflu-
orophenyl, d information not provided in the reference.

The aim of this work was to obtain a large library of sulfated phenols, phenolic acids,
flavonoids, and (2,3-dehydro)flavonolignans and to develop a robust and reliable HPLC
analytical method suitable for the separation of enzymatic sulfation reaction mixtures
of these phenolics. The authentic standards of sulfates of 2,3,4-trihydroxybenzoic acid,
catechol, 4-methylcatechol, and phloroglucinol were prepared in the frame of this work.
Four types of stationary phases (pentafluorophenyl (PFP), C18, C18 Polar, and HILIC)
were compared using mobile phases without or with buffer and with PDA detection. The
optimal method is also suitable for mass detection because it does not use phosphate buffers
and ion-paired reagents in the mobile phase. The duration of separation was up to 20 min
in most cases without peak tailing and coelution.

2. Results and Discussion

2.1. Synthesis of Sulfated Phenolics
A large number of metabolic standards used in this work were prepared earlier
using the bacterial aryl sulfotransferase of Desulfitobacterium hafniense [8,10–13,19]. Besides
previously published products, new authentic standards were prepared in the present
 2. Results and Discussion
2.1.2.Synthesis
Results andof Sulfated Phenolics
Discussion
2.1.ASynthesis
large number of metabolic
of Sulfated Phenolics standards used in this work were prepared earlier using
Int. J. Mol. Sci. 2022, 23, 5743 the bacterial
A large number of metabolic of
aryl sulfotransferase Desulfitobacterium
standards used in thishafniense
work were [8,10–13,19]. Besides
prepared earlier
4 ofpre-
17
using
viously published
the bacterial aryl products, new authentic
sulfotransferase standards were
of Desulfitobacterium prepared
hafniense in the Besides
[8,10–13,19]. present pre-
work
with the same
viously very effective
published products,enzymatic method
new authentic for selective
standards sulfationin
were prepared ofthe
polyphenols (Fig-
present work
ure 1) and fully characterized with HPLC, MS, and NMR (see Supplementary
with the same very effective enzymatic method for selective sulfation of polyphenols (Fig-
work with the same very effective enzymatic method for selective sulfation of Material).
polyphenols
(Figure
ure 1) and1) and fully
fully characterized
characterized with
with HPLC,
HPLC, MS,MS, and
and NMR
NMR (see
(see Supplementary
Supplementary Material).
Material).
O3SO 1 HO 1

O3SO 1 HO 1
4 4
HO 1 -
HO 2
4 Me O 3 SO 2
4 Me
HO 1 4 -
HO 2 Me O3SO 2 Me
HO 2 Me
4 4-methylcatechol sulfates
(MeCAT-S, 1- and 2-O-sulfate 64:36)
4-methylcatechol ( Me )
HO MeCAT 4-methylcatechol sulfates
2
(MeCAT-S, 1- and 2-O-sulfate 64:36)
4-methylcatechol (MeCAT) -
HO 3 1 OSO-3
HO 3 1 OH OSO3
HO 3 1
HO 3 1 OH
p-nitrophenyl sulfate - 5
5
OSO3 -
p-nitrophenyl sulfate OH
5
OH 5 OSO3 OH
OH
phloroglucinol (PG) O 2N phloroglucinol 1-O-sulfate (PG-S)
phloroglucinol (PG) O 2N phloroglucinol 1-O-sulfate (PG-S)
OH O
OH O OH OH O
2OH O OH HO 3 2 2
HO 3 HO 3 OH
HO 3
2
OH OH
OH O2N p-nitrophenol -
O2N p-nitrophenol HO 4 OSO-
HO 4 HO 4 OSO3 3
HO 4

2,3,4-trihydroxybenzoic acid THB) 2,3,4-trihydroxybenzoicacid

acidsulfates
sulfates (THB-S
) )
2,3,4-trihydroxybenzoic acid( (THB) 2,3,4-trihydroxybenzoic (THB-S

--
HOHO1 1 O3SO
O SO 11
3

HOHO2 2 HO
HO 22

catechol (CAT
catechol (CAT
) ) catechol1-O-sulfate
catechol 1-O-sulfate(CAT-S
(CAT-S) )

Figure
Figure
Figure 1.
1. Sulfation
1. Sulfation ofof
Sulfation 4-methylcatechol
4-methylcatechol
of (MeCAT),
(MeCAT),
4-methylcatechol (MeCAT),phloroglucinol
phloroglucinol (PG),
(PG),
phloroglucinol 2,3,4-trihydroxybenzoic
2,3,4-trihydroxybenzoic
(PG), acid
2,3,4-trihydroxybenzoic acid
(THB),
(THB),
acid andand
(THB), catechol
catechol (CAT)using
(CAT)
and catechol usingaryl
(CAT) arylsulfotransferase
usingsulfotransferase from Desulfitobacterium
aryl sulfotransferaseDesulfitobacterium hafniense
hafniense
from Desulfitobacterium and
andp-nitro-
p-nitro-
hafniense and
phenyl
phenyl sulfate
sulfate
p-nitrophenyl as sulfate
assulfate
sulfate donor.donor.
asdonor.
sulfate

The
The
The structuresofof
structures
structures ofall
allphenolic
all phenoliccompounds
phenolic compounds and
compounds and their
and their respective
theirrespective sulfated
respectivesulfated derivatives
sulfatedderivatives
derivatives
used in the present study are shown in Figures 2–4. Altogether, these
used in the present study are shown in Figures 2–4. Altogether, these compoundsform
used in the present study are shown in Figures 2–4. Altogether, these compounds
compounds form
formaa a
unique library of sulfated simple phenols (Figure 2a), phenolic acids (Figure
unique library of sulfated simple phenols (Figure 2a), phenolic acids (Figure 2b), flavo-
unique library of sulfated simple phenols (Figure 2a), phenolic acids (Figure 2b), 2b), flavo-
flavonoids
noids
noids (Figure
(Figure
(Figure 3),3),
3), and andflavonolignans
and flavonolignans
flavonolignans (Figure
(Figure
(Figure 4). 4).
4).
-
(a) HO 1 - O3SO 1 (b) O -
O O
(a) HO 1 O3SO 1 (b) HO 3 O 3 O
-3
SO O
OH HO 3 O
OH
OH O SO 3
HO 3
HO 3 3
OH
HO 2 HO 2 OH - OH
HO 4 HO 4
O3SO 4
HOcatechol
2 (CAT) HO 2
catechol-1-O-sufate (CAT-S) -
HO 4 HO 4 O3SO 4
catechol-1-O-sufate (CAT-S) protocatechuic acid (PRO) protocatechuic acid sufates
catechol (CAT) -
HO 1 protocatechuic acid (PRO) (PRO-S, 3- and 4-O-sulfate 70:30)
protocatechuic acid sufates
O3SO 1
HO 1 - OH O (PRO-S,
OH 3-O and 4-O-sulfate 70:30)
O3SO 1 HO 1 4 HO 2
HO 1 4
4 - 3
OH O OH 2
HO 3 OH O
HO 2 Me O3SO 2 4 Me
HO 3 2
OH
HO 2
4 Me 4 - HO 3 2
Me O3SO Me OH -
HO 2 Me
HO 4-methylcatechol
2 2
sulfates
HO 4 HO 4 OSO OH 3
-
4-methylcatechol (MeCAT) (MeCAT-S, 1- and 2-O-sulfate 64:36) HO 4
2,3,4-trihydroxybenzoic HO OSO3
2,3,4-trihydroxybenzoic
4-methylcatechol sulfates 4
- acid (TBH) acid sulfates (TBH-S)
4-methylcatechol
HO 3 (MeCAT
OH ) (MeCAT-S,
HO 3 1-OSO
and 2-O-sulfate 64:36)
3 2,3,4-trihydroxybenzoic 2,3,4-trihydroxybenzoic
1 1 - acid (TBH) acid sulfates (TBH-S
O ) O
HO 3 OH HO 3 OSO3 O -
O3SO 3 HO 3
5 1 5 1 HO 3 OOH O
OH
O OH -
OH OH O3SO 4 3 - HO 3
5 5 HO 3 HO OHO3SO 4 OH
HO 4 OH
phloroglucinol
OH (PG) phloroglucinol-1-O-sulfate
OH caffeic acid (CAF) caffeic acid sulfates
-
(PG-S) O3SO 69:31)
HO 4(CAF-S, 3- and 4-O-sulfate 4
HO 4
phloroglucinol (PG) phloroglucinol-1-O-sulfate caffeic acid (CAF) caffeic acid sulfates
(PG-S) (CAF-S, 3- and 4-O-sulfate 69:31)
Figure 2. Structures of simple phenols (a) and phenolic acids (b, both blue) and their sulfates (crim-
son) 2.
Figure used
Figure 2. in this study.
Structures
Structures The sulfate
ofofsimple
simple phenols
phenolsgroups
(a)and
(a) andare highlighted
phenolic
phenolic acids in (b,
acids
(b,red.both
both blue)
blue) andand
theirtheir sulfates
sulfates (crim-
(crimson)
son) used
used in this
in this study.
study. TheThe sulfate
sulfate groups
groups are highlighted
are highlighted in red.in red.
Int. J.
Int. J. Mol.
Mol. Sci. 2022, 23,
Sci. 2022, 23, 5743
5743 55of
of 18
17
Int. J. Mol. Sci. 2022, 23, 5743 5 of 18

OSO3 -
OH OSO3 -
OH 4´ OH 4´ OSO3 OH
4´ OH 4´ OSO3
3´
4´ HO O 3´
4´ 4´ OH
3´
4´ HO O - 3´ 3´
4´ HO O 3´
4´
HO O HO7 O 3´
OSO3 - HO7 O OH HO O OH3´
HO7 O 3´
OH OH HO7 O OH3´ HO7 O
7 OSO3 7 OH
7 OH 7 OH OH 7 OH
3 OH 3 OH 3 O O OH
3 OH 3 OH 3 OH 3 O O OH O
OH OH O O 3 OHO OH
3 OH O 3 OHO OH OH O
OH O OH O OH O OH O HO OH OH HO OH OH
OH O quercetin (QUE) quercetin sulfates (QUE-S,3´ and 4´-O-sulfate 75:25) OH O OH O OH
OH
quercetin (QUE-) quercetin sulfates (QUE-S,3´
- and 4´-O-sulfate 75:25) OH isoquercitrin (ISQ) isoquercitrin-4´-O-sulfate (ISQ-S)
OSO3 - OSO3 - isoquercitrin ( ISQ ) isoquercitrin-4´-O-sulfate ( ISQ-S )
OH
4´ OSO3 4´ OSO3-
3´
4´ - 3´ -O3SO O -
HO O - -O SO O 4´
HO7 O 3´
OSO3 - O33SO7 O 3´
OH O3SO O OSO3 - -
OSO3 OH OSO3 OH OSO3 -
4´ OH 4´ OSO3
7 7
3 OH 3 OH OH 3´
4´ 3´
4´
3 OH 3 OH OH HO O HO O
OH O OH O OH O HO7 O OH 3´
HO7 O OH 3´
OH O OH O OH O 7 OH OH 7 OH OH
quercetin-disulfates (QUE-SS, mixture of 3´,4´-, 7,3´- and 7, 4´-di-O-sulfate) 3 O O OH 3 O O OH
quercetin-disulfates (QUE-SS, mixture of 3´,4´-, 7,3´- and 7, 4´-di-O-sulfate) 3 OHO O 3 OHO O O
OH O O OH O
OH
- HO OH O OH O HO OH O
OH OSO3 - OH O OH OH
4´ OH H3C O H 3C O
4´ OH 4´ OSO3 H3C H 3C
HO O 3´
4´ 3´
4´ HO O HO O
O 3´
OH HO O 3´
4´ - HO O HO HO HO HO
HO7 O 3´
OSO3 - HO7 O 3´
OH
OH HO7 OH HO HO
7 3
7 3 OSO3 7 3 OH OH
3
OH 3 OH OH
OH
3
OH OH rutin (RUT) rutin-4´-O-sulfate (RUT-S)
OH O OH OH rutin (RUT) rutin-4´-O-sulfate (RUT-S)
OH O OH O OH O
OH O OH O
taxifolin (TAX) taxifolin sulfates (TAX-S,3´ and 4´-O-sulfate 20:80)
taxifolin (TAX) taxifolin sulfates (TAX-S,3´ and 4´-O-sulfate 20:80)

OH OSO3 OH
OH 4´ OSO-3 OH
4´ OH
4´ OH 3´
4´
- 3´
4´
3´
4´ HO O - -O3SO O -O SO O -
HO O O 3´
OSO3 - O3SO7 O 3´
OH O33SO O OSO3 -
HO7 O 3´
OH HO77 OSO3 7 OH OSO3
7 OH 3 3
3 3 3
3
OH O OH O OH O
OH O O
OH 3´-O-sulfate OH O OH O
OH O luteolin (LUT) luteolin (LUT-S) luteolin disulfates (LUT-SS, 7,3´- and 7, 4´-di-O-sulfate, 82:12)
luteolin (LUT) luteolin 3´-O-sulfate (LUT-S) luteolin disulfates (LUT-SS, 7,3´- and 7, 4´-di-O-sulfate, 82:12)
OH OH OH
OH OH 5´ OH 5´ OH -
5´ OH -
5´ OH 5´ OH -
OSO3 - 5´ OH 5´ OSO3 -
OH OSO - 5´
OH 4´ OSO3
5´ 5´
OSO3 4´ OSO3 4´
4´ OH 4´ 3 - 3´
4´ O 3´
4´
HO O 3´
4´
3´ O 3´
4´ -O3SO O HO 3´ 3´
HO O 4´ HO 3´ O3SO7 O 3´
OH HO7 O OH HO7 O OH
HO7 O 3´
OH HO7 O OH OH 7 3 OH 7 3 OH
7 OH 7 OH 7
3 3
3 OH OH OH
3 OH 3 OH 3 OH OH OH
3 OH 3 OH OH O OH O OH O
OH O OH O OH O OH O OH O
OH O OH O MYR-SS AMP
MYR
myricetin ( MYR) MYR-S
myricetin-4´-O-sulfate ( MYR-S) myricetin-7,4´-di-O-sulfate ( ) ampelopsin ( ) ampelopsin-4´-O-sulfate (AMP-S)
myricetin ( ) myricetin-4´-O-sulfate ( ) myricetin-7,4´-di-O-sulfate (MYR-SS) ampelopsin (AMP) ampelopsin-4´-O-sulfate (AMP-S)

Figure
Figure 3.
3. Structures
Figure 3. Structures of
Structures of flavonoids
of flavonoids (blue)
(blue) and
flavonoids (blue) and their
and their sulfates
their sulfates (crimson)
sulfates (crimson) used
(crimson) used in
used in this
in this study.
this study. The
study. The sulfate
The sulfate
sulfate
groups are highlighted in red.
groups are highlighted in red.
groups are highlighted in red.

O OH O
O OH OH O
OH O
HO O O O 19
19 OR
HO7 O2 O O
7 32 O HO O OR
20 HO7 O2
3
OH A 20
OR
A 7 32
OH OR 3 OH OH
OH O O OH OH OH O
OH O O OH OH O
OH OH O A O
HO O O OH O A O 19
19 OR
HO7 O2 O O HO O OR
7 32 O 20 HO7 O2
3 20
OH OR 7 32
B OH OR 3 OH
OH O B OH OH
OH O B OH
R=H: Silybin (SB, A:B 50:50) OH O B
- R=H: Silybin (SB, A:B 50:50) OH -O Silychristin ( SCH, A:B 89:11)
R=SO3 -: Silybin-20-O-sulfate (SB-S, A:B 50:50) Silychristin (SCH, A:B(89:11)
R=SO3 -: Silychristin-19-O-sulfate SCH-S, A:B 89:11)
R=SO3 : Silybin-20-O-sulfate (SB-S, A:B 50:50) R=SO3 : Silychristin-19-O-sulfate (SCH-S, A:B 89:11)

O OH O
O OH OH O
OH O
R22O O 2 O O 19
19 OR
R O7 O 2 O O OR
O HO O 2
7 20 HO7 O 2
20
3 OH OR11 7
OH
3 OH OR 3 OH OH
OH O 3 OH
OH O
R11-,R22=H: 2,3-Dehydrosilybin (DHSB) OH O
R -,R2=H: 2,3-Dehydrosilybin (DHSB) OH O
R11=SO
=SO ,R 2=H: 2,3-Dehydrosilybin-20-O-sulfate (DHSB-S 2,3-Dehydrosilychristin (DHSCH )
R 1 332=SO - 2,3-Dehydrosilybin-20-O-sulfate (DHSB-S))
,R =H: -
2,3-Dehydrosilychristin (DHSCH)
R 1,R 2=SO3 -: 2,3-Dehydrosilybin-7,20-di-O-sulfate R=SO -: 2,3-Dehydrosilychristin-19-O-sulfate
R ,R 3 2,3-Dehydrosilybin-7,20-di-O-sulfate
: R=SO33 : 2,3-Dehydrosilychristin-19-O-sulfate
(DHSB-SS ) (DHSCH-S )
(DHSB-SS) (DHSCH-S)

Figure 4.
4. Structures
Figure 4. Structures of flavonolignans
flavonolignans (blue)
of flavonolignans (blue) and
and their sulfates
their sulfates (crimson)
(crimson) used
sulfates (crimson) in
used in this
in this study.
this study. The
study. The
The
Figure Structures of (blue) and their used
sulfate groups
sulfate groups are
groups are highlighted
are highlighted in
highlighted in red.
in red.
red.
sulfate
2.1.1.
2.1.1. Sulfation
2.1.1. Sulfation of
Sulfation of 4-Methylcatechol
of (MeCAT)
4-Methylcatechol (MeCAT)
4-Methylcatechol (MeCAT)
Sulfation
Sulfation of
of 4-methylcatechol
4-methylcatechol yielded
Sulfation of 4-methylcatechol yielded
yielded aa product
product
a product (MeCAT-S)
(MeCAT-S)
(MeCAT-S) thatthat
that appeared
appeared
appeared as one
as spot
as one one
spot on TLC and one peak on HPLC using Method M2. However,
spot on TLC and one peak on HPLC using Method M2. However, NMR analysis revealed
on TLC and one peak on HPLC using Method M2. However, NMRNMR analysis
analysis revealed
revealed a
aa mixture
mixture ofof two
two isomers:
isomers: 4-methylcatechol-1-O-sulfate
4-methylcatechol-1-O-sulfate and
and 4-methylcatechol-2-O-sulfate
4-methylcatechol-2-O-sulfate
mixture of two isomers: 4-methylcatechol-1-O-sulfate and 4-methylcatechol-2-O-sulfate
in
in aaa ratio
in ratio
ratio of
of 64:36
of 64:36 (Table
64:36 (Table S8a,b).
(Table S8a,b). The
The reaction
S8a,b). The reaction (Figure
reaction (Figure 1)
(Figure 1) was
1) was rapid,
was rapid, with
rapid, with the
with the entire
the entire amount
entire amount
amount
of
of MeCAT-S
MeCAT-S synthesized
synthesized within
within the
the first
first 20
20 min.
min. After
After purification
purification
of MeCAT-S synthesized within the first 20 min. After purification by gel chromatog- by
by gel
gel chromatog-
chromatog-
raphy,
raphy, 180
raphy, 180 mg
180 mgof
mg ofthe
of theproduct
the product was
productwas obtained
was obtained
obtained from 200
from
from mg
200200
mg mgof
of starting material
of starting
starting (isolated
material
material yield
(isolated
(isolated yield
Int. J. Mol. Sci. 2022, 23, 5743 6 of 17

yield 58 mol%) as a white powder with an overall purity of 99% (HPLC, Figure S1) and
corresponding m/z (Figure S2).

2.1.2. Sulfation of Phloroglucinol (PG)

In the case of PG, the formation of at least two products was observed (TLC, HPLC-
Method M2, Figure 1), but only one of them was successfully isolated and characterized.
According to NMR and MS analyses, it was confirmed to be a monosulfate. The product was
isolated as a brownish powder (73 mg, 44% yield, 90% purity, Figures S11 and S12, Table S13).

2.1.3. Sulfation of Protocatechuic Acid (PRO)

When PRO was sulfated, only one sulfated product was observed (TLC, HPLC-Method
M2). According to the NMR analysis, it was a mixture of two regioisomers: protocatechuic
acid-3-O-sulfate and protocatechuic acid-4-O-sulfate in the ratio of 7:3. The product tended
to associate with solvents and other chemicals used during the purification process (H2 O,
MeOH, HCOOH); however, after repeated purification, it was isolated as a white powder
(12 mg, 4% yield, 99% HPLC purity, Figure S3, Tables S9a,b).

2.1.4. Sulfation of 2,3,4-Trihydroxybenzoic Acid (THB)

The gradual formation of one product was observed (TLC, HPLC-Method M2, Figure 1).
Furthermore, this product tended to associate with solvents. The purification on gel chro-
matography gave a brown oil product (34 mg, 8% yield, 96% HPLC purity, Figures S5 and S6,
Tables S10a,b).

2.1.5. Sulfation of Caffeic Acid (CAF)

The sulfation of caffeic acid yielded a product that behaved as one spot on TLC and
one peak on HPLC. Again, NMR analysis revealed a mixture of 3- and 4-O-sulfate in the
ratio of 69:31. After purification, we obtained 301 mg of white-brownish oily solid (100%
yield, 99% HPLC purity, Figures S7 and S8, Table S11a,b).

2.1.6. Sulfation of Catechol (CAT)

The sulfation of catechol (Figure 1) led to surprisingly only one regioisomer, catechol-
1-O-sulfate, as confirmed by MS and NMR. After purification, 116 mg of the product was
obtained (34% yield, 98% HPLC purity, Figures S9 and S10, Table S12) as a white powder.

2.2. Development and Optimization of Analytical Methods for the Separation of Sulfated Polyphenols
The typical composition of the reaction mixture to be analyzed was as follows: p-NP,
p-NP-S, the parent compound, and its sulfate (see Figure 1; in some cases, isomers and/or
disulfate(s) were also present). The main focus was on monitoring the separation of all
components in the mixture, the duration of the separation, and the width of the peaks at
five percent of their height. The following limits were established for evaluating the width
of the peaks: w0.05 < 0.300, very good; 0.300 < w0.05 < 0.500, good; and w0.05 > 0.500, poor.

2.3. Separations without Buffer in the Mobile Phase

Based on our previous experience and the articles published so far (Table 1), we
decided to test these four columns: Kinetex PFP, ZICpHILIC, Chromolith RP 18e, and Luna
Omega Polar C18. Initially, the Kinetex PFP, Chromolith RP 18e, and Luna Omega Polar
C18 columns were each tested without the use of buffers in the mobile phase (Table 2). The
use of buffers in the mobile phase is always associated with an increased washing effort of
the analytical system and thus a higher time requirement, so we tried to avoid this in the
first step. Since the samples contain ionizable compounds, formic acid or TFA (0.1%) was
added to all mobile phases to achieve a pH of about 3. The non-ionized form is less polar
and therefore more strongly retained in a reverse phase system.
Int. J. Mol. Sci. 2022, 23, 5743 7 of 17

Table 2. An overview of the columns used and separation conditions.

Stationary Method Flow Rate

Mobile Phase A Mobile Phase B T [◦ C] Gradient
Phase Number [mL/min]
10 mM 0 min 40% B,
CH3 COONH4 / 0–20 min 40–72% B,
M1 MeOH 0.6 45
HCOOH (100/0.1, 20–21 min 72–40%
PFP
v/v) B, 21–24 min 40%
10 mM 0 min 40% B,
CH3 COONH4 / 0–20 min 20–50% B,
M2 MeOH 0.6 45
HCOOH (100/0.1, 20–21 min 50–20%
v/v) B, 21–24 min 20%
0 min 40% B,
H2 O/CH3 COOF3 0–25 min 40–80% B,
M3 MeOH 0.6 45
(100/0.1, v/v) 25–26 min 80–40%
B, 26–28 min 40%
0 min 5% B,
10 mM 0–7.5 min 5–20% B,
AcCN/HCOOH CH3 COONH4 / 7.5–10 min 20% B,
ZICpHILIC M4 0.4 25
(100/0.1, v/v) HCOOH 10–12 min 20–5% B,
(100/0.1, v/v) 12–15 min 5% B,
15–17 5% B
0–2 min 5% B,
10 mM
2–7 min 5–90% B,
CH3 COONH4 /
M5 MeOH 1 25 7–8 min 90%B,
C18 HCOOH (100/0.1,
8–11 min 90–5% B,
v/v)
11–14 min 5% B
AcCN/H2 O/ AcCN/H2 O/ 0–5 min 0–30% B,
M6 HCOOH(5/95/0.1, HCOOH 1 25 5–7 min 30–0% B,
v/v ) (80/20/0.1) 7–9 min 0% B
0–7 min 0–90% B,
AcCN/H2 O/
H2 O, HCOOH 7–8 min 90% B,
C18 Polar M7 HCOOH 0.4 25
(100/0.1, v/v) 8–11 min 90–0% B,
(80/20/0.1)
11–14 min 0% B

The pentafluorophenyl column offers unique polar and aromatic selectivity thanks
to the fluorine atoms at the periphery of the phenyl units anchored to the core-sell silica
support. The Kinetex PFP column was tested using Method M3 (0.1% TFA, MeOH as
mobile phases) for the separation of quercetin (QUE), isoquercitrin (ISQ), rutin (RUT),
taxifolin (TAX), luteolin (LUT), myricetin (MYR), and ampelopsin (AMP) and their sulfates.
Although the pH of the mobile phase was very low (pH = 2.0, column limit is 1.5) and the
ionization of compounds (sulfates and their parent compounds) should be suppressed,
the separation was not satisfactory in many cases. The width of the peak in the 0.5% of
its height (w0.05 ) ranged from 0.295 to 1.703 (Table 3). The widest peaks were observed in
quercetin and quercetin sulfates (w0.05 0.554–1.703), luteolin sulfates (w0.05 0.655–1.703), and
myricetin sulfates (w0.05 0.542–1.398). The values of w0.05 for the other tested substances
were up to 0.500. No coelution of peaks was observed in the reaction mixtures. The longest
retention time was 20.010 min (luteolin), but for most of the analyzed substances, it ranged
from 6 to 16 min.
Int. J. Mol. Sci. 2022, 23, 5743 8 of 17

Table 3. Comparison of retention times and peak widths of selected analytes using different columns
and methods.

Stationary
Phase PFP a ZICpHILIC b C18 C18-Polar

Method c M1 M2 M4 M5 M6 M7
d e f e f e f e f e f e f
Analyte tR w0.05 tR w0.05 tR w0.05 tR w0.05 tR w0.05 tR w0.05
[min]
DHSB 23.101 0.329 - - 1.160 0.251 7.293 0.146 7.039 0.177 9.032 0.143
DHSB-S 17.385 0.347 - - 3.636 0.849 6.914 0.189 6.771 0.608 7.904 0.234
DHSB-SS 8.280 0.525 - - 8.853 0.588 6.097 0.392 3.180 1.701 8.490 0.146
DHSCH 17.010 0.425 - - 2.025 1.209 6.691 0.108 6.957 0.101 7.573 0.125
DHSCH-S 12.060 0.576 - - 5.448 0.465 6.258 0.177 6.771 0.478 6.768 0.322
12.449, 0.299, g
SCH - - 3.047 1.210 6.002 n.d. 5.753 0.156 7.413 0.132
13.285 i 0.312
6.176, g
SCH-S 0.338 - - 6.332 0.540 5.552 n.d. 4.985 0.393 6.313 0.395
6.654 h
15.202, g 6.480, g 4.141, g
SB n.d. - - 1.094 0.647 i n.d. n.d. 7.410 0.186
15.521 i 6.514 i 4.225 i
13.728, 2.470, 6.487,
0.287 3.007, g g
SB-S - - n.d. n.d. 3.165 0.981 6.555 0.186
14.479 h 0.415
4.365 j 6.579 h
CAF 5.150 0.238 12.527 0.356 2.540 0.660 4.954 0.102 3.087 0.211 5.987 0.270
4.326, g
CAF-S 3.720 0.267 9.233 0.671 4.194 0.528 n.d. 5.847 2.555 5.366 1.040
4.494 j
PRO 3.721 0.270 7.213 0.337 3.184 0.787 2.816 0.410 1.822 0.237 3.653 0.471
PRO-S 2.990 0.225 5.541 0.403 6.255 0.558 2.104 0.276 4.697 2.579 2.500 0.920
THB 3.568 0.246 6.240 0.368 4.456 0.638 1.944 0.175 2.058 0.259 4.122 0.383
2.693, 0.329, k l
THB-S 4.949 0.317 n.d. - n.d. - 0.718 0.050 2.966 0.582
3.033 j 0.266
CAT 4.578 0.256 7.453 0.350 1.257 0.270 2.920 0.356 2.317 0.246 4.331 0.403
CAT-S 3.240 0.207 4.938 0.386 n.d. k 4.290 n.d. g 5.109 2.564 7.627 0.305
MeCAT 5.978 0.335 11.944 0.508 0.963 n.d. g 4.946 0.226 3.769 0.300 6.302 0.261
MeCAT-S 4.112 0.249 7.910 0.524 1.049 0.314 4.362 j n.d. g 6.578 2.546 5.536 0.507
PG 2.906 0.205 4.004 0.235 4.641 0.430 1.180 0.288 1.039 0.197 1.641 0.310
PG-S 2.503 0.177 3.339 0.266 9.500 0.732 1.465 0.567 2.683 1.534 1.265 0.308
pNP 11.528 0.338 20.542 0.385 0.984 0.273 5.609 0.169 5.031 0.221 7.274 0.278
pNP-S 4.930 0.457 10.401 0.498 1.494 0.600 5.216 0.900 0.796 0.140 5.567 0.621
a Kinetex pentafluorophenyl, b hydrophilic interaction chromatography, c for details on the individual methods,
see Table 2, d full names and structures of the analytes are shown at Figures 1–3, e retention time, f the width of
the peak in 5% of its height, g the peak shape did not allow the determination of w0.05 , h separation of sulfated
stereoisomers A and B, i partial separation of stereoisomers A and B, j partial separation of sulfated regioisomers,
k the compound was decomposed during the analysis, only the parent compound without sulfate was detected,

l the compound was not caught on the column and eluted with a dead volume. Dark green means w0.05 < 0.300,
light green means 0.300 < w0.05 < 0.500, and red means w0.05 > 0.500.

We also tested two C18 columns, one monolithic (Chromolith RP18e, Method M6), and
one specially designed for increased retention of polar compounds (Luna Omega Polar C18,
Method M7, Table 2). The mobile phase was the same in both cases (acetonitrile 5% and 80%,
water, 0.1% formic acid, pH 2.7; the pH limits of the columns are 2.0 and 1.5, respectively).
The Luna Omega Polar C18 stationary phase is a combination of a universal C18 ligand
and a polar modified surface that provides improved polar retention and aqueous stability.
Due to the smaller particle size of the stationary phase (3 µm) and the associated higher
backpressure, the flow rate was only 0.4 mL/min. In contrast, the monolithic C18 column
has much higher permeability and porosity, so the flow rate was 1 mL/min.
Nevertheless, for most analytes, we observed a lower w0.05 of the peaks using this C18
Polar column compared to the monolithic column, or the widths of the peaks were very
similar on both columns (e.g., 2,3-dehydrosilychristin DHSCH, caffeic acid CAF or p-NP).
The width of the peaks was very good for 2,3-dehydrosilybin DHSB, 2,3-dehydrosilybin-20-
O-sulfate DHSB-S, and 2,3-dehydrosilybin-7,20-di-O-sulfate DHSB-SS, further for DHSCH,
silychristin SCH, silybin SB, caffeic acid CAF, 4-methylcatechol MeCAT, and p-NP; good
for 2,3-dehydrosilychristin-19-O-sulfate DHSCH-S, silychristin-19-O-sulfate SCH-S, proto-
catechuic acid PRO, 2,3,4-trihydroxybenzoic acid THB, catechol CAT, catechol-1-O-sulfate
CAT-S, phloroglucinol PG, and its sulfate. Broader peaks were detected for five compounds,
namely CAF-S, PRO-S, THB-S, MeCAT-S, and p-NP-S. In contrast, eight broad peaks were
detected in the monolithic C18 column, most of which were sulfates (DHSB-S, DHSB-SS,
SB-S, CAF-S, PRO-S, CAT-S, MeCAT-S, and PG-S). With the monolithic column, coelution of
the peaks was observed in the case of p-NP-S and THB-S in the respective reaction mixtures.
Int. J. Mol. Sci. 2022, 23, 5743 9 of 17

The retention times of the eluted compounds were up to 7 min. With the Polar C18 column,
coelution of the peaks was observed only in the case of p-NP-S and MeCAT-S. The retention
times of the eluted compounds were up to 9 min. Thus, when we compare the two C18
columns, the separation on the C18 Polar column shows narrower peaks and also a lower
consumption of the mobile phase due to the lower flow rate (0.4 mL/min). In addition,
this method is directly applicable in conjunction with a mass detector. The method with
the monolithic column (M6) would require further modification in case of connection with
the mass detector (flow rate 1 mL/min). The comparison of HPLC chromatograms of
individual compounds is in Supplementary Material (Figures S13–S51).

2.4. Separation with Buffer in the Mobile Phase

As can be seen in the example of the separations of polyphenol sulfates on the PFP
column and the C18 columns mentioned above, it is clear that in some cases, the mere
acidification of the mobile phase is not sufficient to deionize ionizable compounds (broad
peaks). Fine-tuning of the separation of highly polar sulfated polyphenols can be achieved
by changes in the mobile phase, e.g., by choice of buffer, and thus consistent control of pH.
The most commonly used buffers for HPLC with UV detection are acetate and phosphate.
Since phosphate buffer is not compatible with MS detection, we chose ammonium acetate
buffer with a pH of 3.8, and the buffer strength was set at 10 mM. All methods tested with
10 mM buffer were also tested with 5 mM concentration on several samples, but the width
of the peaks was broader than with 10 mM buffer in all cases (data not shown).
Separation of quercetin (QUE), ampelopsin (AMP), luteolin (LUT), myricetin (MYR),
isoquercitrin (ISQ), rutin (RUT), taxifolin (TAX), and their respective sulfates on PFP column
using 10 mM ammonium acetate buffer and methanol as mobile phases (Method M1)
showed good separation of most compounds (0.300 < w0.05 < 0.500). Only in two cases (QSS
and AMP) was this range slightly exceeded (0.642 and 0.519, respectively). Moreover, the
retention time of most compounds was shorter than in Method M3 (Table 3). No coelution
of peaks was observed in the reaction mixtures. When we compare the separation of these
compounds on the same PFP column, it is clear that we obtained better or equal separation
with the acetate buffer method (Method M1) than with the method without acetate buffer
(Method M3) in all cases except for ampelopsin (w0.05 0.519 versus 0.314, Table 4).

Table 4. Comparison of retention times and peak widths of selected analytes using PFP column with
(M1) and without buffer (M3).

Method M1 a M3 b
Analyte c tR d [min] w0.05 e [min] tR d [min] w0.05 e [min]
QUE 16.127 0.385 17.470 0.554
QUE-S 12.139 0.397 15.084 0.632
QUE-SS 6.014 0.642 11.084 1.703
AMP 5.480 0.519 6.062 0.314
AMP-S 6.007 0.352 7.449 0.437
LUT 17.916 0.427 20.010 0.422
LUT-S 13.340 0.377 16.741 0.501
LUT-SS 12.830 0.362 13.595 1.426
MYR 12.716 0.390 14.014 0.410
M-S 8.566 0.478 15.877 0.542
M-SS 4.602 0.400 12.497 1.398
ISQ 9.397 0.344 9.615 0.312
ISQ-S 6.681 0.311 8.247 0.381
RUT 8.869 0.342 9.156 0.322
RUT-S 6.055 0.261 7.175 0.287
TAX 7.380 0.285 7.767 0.466
TAX-S 5.582 0.321 7.557 0.368
pNP 11.528 0.338 13.414 0.367
pNP-S 4.930 0.457 6.751 0.295
aWith ammonium acetate buffer, b with 0.1% TFA (for details, see Table 2), c full names and structures of the
analytes are shown in Figures 1–3, d retention times, e width of the peak in 5% of its height; dark green means
w0.05 < 0.300, light green means 0.300 < w0.05 < 0.500, and red means w0.05 > 0.500.
Int. J. Mol. Sci. 2022, 23, 5743 10 of 17

In addition, we tested the separation of other polyphenolic compounds and their

sulfates on this column so that we can compare it with other stationary phases used in this
work. The separation of CAF, CAF-S, PRO, PRO-S, THB, THB-S, CAT, CAT-S, MeCAT-S,
and PG-S was very good. The separation of DHSB-S, DHSCH, SCH, SCH-S, SB-S, MeCAT,
p-NP, and p-NP-S was good; only the peaks of DHSB-SS and DHSCH-S were slightly
broader (w0.05 was 0.525 and 0.576, respectively). Retention times of analytes ranged from
2 to 23 min; more than half of them were less than 10 min. No coelution of peaks in reaction
mixtures was observed. From our previous unpublished experiments, we know that this
gradient in method M1 is too fast for many phenolic acids, benzoic acids, and their sulfates.
Therefore, we tried to modify this method. We reduced the initial concentration of mobile
phase B from 40 to 20% and the final concentration to 50% (instead of the original 72%),
and the length of the gradient was maintained (Method M2). However, we did not observe
any significant improvement in w0.05 , only the retention times were longer compared with
Method 1.
Separation of phenolic compounds and their sulfates on the monolithic C18 column
was also performed in 10 mM ammonium acetate buffer as the mobile phase (Method M5).
The separation of many compounds was very good or good using this column (DHSB and
its sulfates, DHSCH, DHSCH-S, CAF, PRO, PRO-S, THB, CAT, MeCAT, PG, and p-NP).
However, for several compounds (SCH, SCH-S, SB, SB-S, CAF-S, CAT-S, MeCAt-S), the
width of the peaks could not be determined because of their unusual shape (double hunch,
see Section 2.5). The peak of THB-S was not caught on the stationary phase and eluted with
the dead volume. The retention times of all compounds were up to 7 min. Coelution of
p-NP and SCH-S was observed in the reaction mixture.
The Zic-pHILIC column was designed by the manufacturer for difficult separations
of polar hydrophilic compounds. It is a polymer-based column with densely bound
zwitterionic functional groups with a charged equilibrium of 1:1 and the lowest pH stability
of 2. The mobile phases were acetonitrile and 10 mM ammonium acetate, both acidified
with formic acid (pH 3.8), and the compounds were separated by gradient elution (Method
M4). Almost all substances analyzed showed broad peaks except for DHSB, DHSCH-S,
CAT-S, MeCAT-S, PG, and p-NP. Retention times were not measured for THB-S and CAT-S
because the sulfated substance is likely degraded to the parent substance on the column.
Retention times ranged from 0.9 to 9.5 min. Coelution of p-NP and SB, p-NP-S, and CAT was
observed in the reaction mixture. The comparison of HPLC chromatograms of individual
compounds is in Supplementary Material (Figures S19–S51).

2.5. Separation of Regioisomers and Stereoisomers

NMR analyses have shown that in some cases, multiple regioisomers are formed in
enzymatic sulfation reactions, as in the case of QUE-S, QUE-SS, TAX-S, CAF-S, MeCAT-S,
PRO-S, and THB-S. Separation of these regioisomers was achieved on a monolithic C18
column (Method M5) in CAF-S (peaks at 4.326 min and 4.494 min, resolution 1.251) and
in MeCAT-S (double hunch peak at 4.362 min, resolution 0.540). The separation of two
THB-S regioisomers was observed in Method M1 (peaks at 2.827 min and 3.033 min, reso-
lution 1.944). The separation of two sulfated regioisomers was previously published on
a C18 UHPLC column with MS/MS detection (3-phenylpropionic acid 40 -O-sulfate (RT
4.92 min) and 3-phenylpropionic acid 30 -O-sulfate (RT = 5.22 min) or caffeic acid-40 -O-
sulfate (RT = 3.97 min) and caffeic acid-30 -O-sulfate) [2]. Baseline separation of disulfate
regioisomers of myricetin was reported on the PFP column (mobile phase 0.1% trifluo-
roacetic acid in water and methanol) [8].
Since silybin is a mixture of two diastereoisomers, silybin A and B (in a ratio of
approximately 1:1), we observed the separation of these two diastereoisomers in methods
M1, M5, and M6. Sulfated diastereoisomers of silybin were separated by Methods M1 and
M5. The resolution of the peaks was 1.365 (M1), 0.2 (M5), and 0.02 (M6). In the case of
Method M4, we observed even three peaks (partially separated), and all of them have the
same absorption maximum (286 nm). In this case, it is probably peak cleavage due to the
 acid in water and methanol) [8].
Since silybin is a mixture of two diastereoisomers, silybin A and B (in a ratio of ap-
proximately 1:1), we observed the separation of these two diastereoisomers in methods
M1, M5, and M6. Sulfated diastereoisomers of silybin were separated by Methods M1 and
M5. The resolution of the peaks was 1.365 (M1), 0.2 (M5), and 0.02 (M6). In the case of
Int. J. Mol. Sci. 2022, 23, 5743 11 of 17
Method M4, we observed even three peaks (partially separated), and all of them have the
same absorption maximum (286 nm). In this case, it is probably peak cleavage due to the
unsuitability of the stationary phase for the separation of polyphenolic substances, as can
unsuitability
be seen from of thelarge
the stationary phase for
peak width of the
mostseparation of polyphenolic
of the compounds substances,
examined. as cansi-
Although be
seen from the large peak width of most of the compounds examined. Although
lychristin is also a mixture of two diastereoisomers A and B, we observed their separation silychristin
is also
only onathemixture of two diastereoisomers
PFP column A and
(M1). The separation of B, we observed
sulfated their diastereoisomers
silychristin separation only onwasthe
PFP column (M1). The separation of sulfated silychristin diastereoisomers
observed with the PFP column and Method M1 (Table 3). The separation of diastereoiso- was observed
with of
mers thesilychristin
PFP column and Method
or silybin M1 (Table
at the reverse 3). is
phase The separation
described of diastereoisomers
in many of
papers, e.g., [25–
silychristin or silybin at the reverse phase is described in many papers, e.g., [25–27], but
27], but the separation of sulfated diastereoisomers have not been described so far.
the separation of sulfated diastereoisomers have not been described so far.
2.6. Selection of the Best Method
2.6. Selection of the Best Method
In
In this study,we
this study, wecompared
compared four
four types
types of stationary
of stationary phases,
phases, namely namely pentafluoro-
pentafluorophenyl,
phenyl, ZICpHILIC, monolithic C18, and C18 stationary phases,
ZICpHILIC, monolithic C18, and C18 stationary phases, with treatment for better with treatment for better
polar
polar
retention. We combined these stationary phases with either a mobile phase withoutwithout
retention. We combined these stationary phases with either a mobile phase a buffer
aorbuffer
with aorbuffer.
with aThe buffer. The combination
combination of pentafluorophenyl
of pentafluorophenyl stationary stationary
phase and 10 phase
mMand 10
acetate
mM acetate buffer/methanol
buffer/methanol in gradientin gradient
elution elution
proved to beproved to be
the best. theresulted
This best. This resulted
in sharp in
peaks
sharp peaks for almost all test compounds without tailing and very good
for almost all test compounds without tailing and very good separation of all components separation of all
components
in the mixture in the mixture
for up to 20 for
minup to 20 min
(Figure (Figure
5). The other5). The other columns/mobile
columns/mobile phases
phases tested were
tested were only suitable for some of the compounds analyzed. The
only suitable for some of the compounds analyzed. The retention times and peak widths retention times and
peak widths of all compounds tested are shown in Tables 3 and 4.
of all compounds tested are shown in Tables 3 and 4. The peak shapes and comparisonsThe peak shapes and
comparisons of HPLC chromatograms
of HPLC chromatograms of individualofcompounds
individual compounds
in all methodsin all methods
tested can betested
foundcanin
be found in Supplementary
Supplementary Materials
Materials (Figures (Figures S13–S51).
S13–S51).

Figure
Figure5.5.HPLC
HPLCchromatogram—an example of
chromatogram—an example oftypical
typicalcomposition
compositionofofthe
theenzymatic
enzymatic reaction
reaction mix-
mixture
ture
to betoanalyzed:
be analyzed: p-NP,
p-NP, p-NP-S,
p-NP-S, parent
parent compound
compound (CAT),
(CAT), andsulfate
and its its sulfate (CAT-S),
(CAT-S), MethodMethod
M1. M1.

2.7.Method
2.7. MethodValidation
Validation
Dueto
Due to the
the large
large number
number of
of samples
samplesmeasured
measuredand andcolumns
columnstested,
tested,one
onerepresentative
representa-
from the group of sulfated phenols, phenolic acids, flavonoids, and flavonolignans, namely
tive from the group of sulfated phenols, phenolic acids, flavonoids, and flavonolignans,
4-methylcatechol sulfate (MeCAT-S), caffeic acid sulfate (CAF-S), ampelopsin sulfate (AMP-
S), and silychristin sulfate (SCH-S), was selected for the validation of Method M1 (the
most universal PFP column and mobile phase for all samples tested). The linearity, limit
of detection, the limit of quantification, precision, accuracy, recovery and repeatability are
summarized in Table 5.
Int. J. Mol. Sci. 2022, 23, 5743 12 of 17

Table 5. The linearity, limit of detection (LOD), limit of quantification (LOQ), intermediate precision,
repeatability, accuracy, and recovery for four representatives of sulfated phenol (MeCAT-S), phenolic
acid (CAF-S), flavonoids (AMP-S), and flavonolignans (SCH-S).

LOD LOQ Repeatability Intermediate Accuracy Recovery

Sample Regression Equation R2 [%]a Precision [%] a
[mM] [mM] [%] b [%]
MeCAT-S y = 161,221 × c + 17,188 0.9999 0.032 0.108 1.55 2.18 1.3 104
CAF-S y = 26,266 × c + 4120 0.9998 0.560 1.680 3.63 8.13 2.2 104
AMP-S y = 96,081 × c 0.9977 0.061 0.202 1.74 6.30 2.4 103
SCH-S y = 127,596 × c + 27,799 0.9999 0.340 1.020 1.78 9.04 3.3 105
R2 correlation coefficient; a expressed as relative standard deviation, n = 6; b expressed as relative standard
deviation, n = 3.

The calibration curves were linear in the range from 0.625 to 50 mM for all analytes.
The correlation coefficients were greater than 0.9997 in all cases (except AMP-S, 0.9977),
demonstrating a high degree of correlation and good linearity of the method. LOD and LOQ
ranged from 0.032 to 1.680 mM. This indicates that our method has adequate sensitivity.
The ranges of %RSD parameters for repeatability (intra-day precision) and intermediate
(inter-day) precision were 1.55 to 3.63 and 2.18 to 9.04, respectively. The accuracy ranges
were from 1.3 (MeCAT-S) to 3.3% (SCH-S), and recoveries were in the range of 103–104%
for all tested samples. Sample stability was evaluated by storing unprocessed samples at
ambient temperature up to 24 h and freeze/thaw cycles after three cycles at −18 ◦ C. The
experiments indicated that all four analytes tested were stable in the period of 24 h, as the
recoveries ranged between 97–102% for SCH-S, 94–104% for CAF-S, 98–103% for AMP-S,
and 97–102% for MeCAT-S.

3. Materials and Methods

3.1. Material
Acetonitrile, methanol, formic acid (all VWR chemicals, Stříbrná Skalice, Czech Re-
public analytical grade), deionized water (Ultrapure, Watrex, Prague, Czech Republic),
ammonium acetate (Lach-Ner, Neratovice, Czech Republic), ampelopsin (Herb Nutritionals,
Shanghai, China), 3,4-dihydroxycinamic acid (caffeic acid), p-nitrophenol sulfate, 2,3,4-
trihydroxybenzoic acid, 3,4-dihydroxybenzoic (protocatechuic) acid, catechol (all Acros
Organics, Thermo Fisher Scientific, Waltham, MA, USA), 4-methylcatechol (Aldrich, Merck
KGaA, Darmstadt, Germany), quercetin (Sigma, Merck KGaA, Darmstadt, Germany),
phloroglucinol (Alfa Aesar, Haverhill, MA, USA), p-nitrophenol, luteolin, and myricetin
(abcr GmbH, Karlsruhe, Germany). Standards of silybin, 2,3-dehydrosilybin, and sily-
christin were prepared and fully characterized in the Laboratory of Biotransformation,
Institute of Microbiology, Prague, Czech Republic [28–30].
The description of the preparation and full characterization (NMR, HRMS, and HPLC)
of the following sulfates have already been published: silybin A-20-O-sulfate and silybin
B-20-O-sulfate (SB-S, 50:50) [10], silychristin-19-O-sulfate (SCH-S), 2,3-dehydrosilybin-20-
O-sulfate (DHSB-S), 2,3-dehydrosilychristin-19-O-sulfate (DHSCH-S) [13], quercetin-30 -
O-sulfate and quercetin-40 -O-sulfate (QUE-S, 75:25) [11,12,19], rutin-40 -O-sulfate (RUT-S),
taxifolin-40 -O-sulfate and taxifolin-30 -O-sulfate (TAX-S, 80:20), isoquercitrin-40 -O-sulfate
(ISQ-S) [11,19], ampelopsin-40 -O-sulfate (AMP-S), luteolin-30 -O-sulfate (LUT-S), luteolin-7,
30 - and 7, 40 -di-O-sulfates (LUT-SS), myricetin-40 -O-sulfate (MYR-S), myricetin-di-7, and
40 -O-sulfate (MYR-SS) [8].

3.2. Preparation of the Enzyme

The aryl sulfotransferase from Desulfitobacterium hafniense used for the sulfation was
prepared as described previously in our works [11,12].
Int. J. Mol. Sci. 2022, 23, 5743 13 of 17

3.3. General Method for the Preparation of Sulfates

The substrate (MeCAT, PRO, THB, CAF, CAT, PG; 200 mg of each, 1 eq) was dissolved
in 5 mL of acetone in a flask and then p-NPS (25 mg/mL, 1 or 2 eq in 100 mM Tris-glycine
buffer pH 8.9), 24 mL of Tris-glycine buffer, and 2 mL of AST enzyme (360 U/mL) were
added. The reaction mixture was then incubated under an inert atmosphere (Ar) at 30 ◦ C
for 5 h. The monitoring of the reaction was performed using TLC (mobile phase ethyl
acetate/chloroform/trifluoroacetic acid, 16/1/0.01). After incubation, the reaction mixture
was heated up to 95 ◦ C to terminate the enzymatic reaction and stored at −20 ◦ C until
purification.

3.4. Purification of Sulfates

In the case of MeCAT, PRO, THB, CAF, and CAT, the reaction mixture was partially
evaporated in a rotary evaporator to remove acetone from the mixture. The pH was
adjusted to 7.5–7.7 using formic acid, and then the mixture was extracted with ethyl
acetate (3 × 50 mL) to remove p-nitrophenol (control by TLC, mobile phase ethyl ac-
etate/chloroform/trifluoroacetic acid, 16/1/0.01). The aqueous phase was then evapo-
rated, dissolved in 2–5 mL of 80% methanol, centrifuged (5000 × rpm, 20 min), and loaded
onto a Sephadex LH-20 column (GE Healthcare Bio-Sciences, Uppsala, Sweden; 30 g of
dry weight, 3 cm i.d.) with 80% methanol as a mobile phase (0.2 mL/min). The elu-
tion time was usually 2 days, and the fraction detection was performed by TLC (ethyl
acetate/chloroform/trifluoroacetic acid, 16/1/0.01). Purification of the phloroglucinol re-
action mixture was performed using preparative HPLC (Section 3.5) using an ASAHIPAK
GS-310 20F column (Shodex, Munich, Germany). The reaction mixture (100 mg) was dis-
solved in 1 mL of 50% methanol, filtered, and injected onto the column (5 mL/min, 25 ◦ C,
detection at 254 and 369 nm). The fractions containing the desired product were then
joined, fully evaporated, and lyophilized. Low purity fractions were re-purified using the
same methodology.

3.5. Preparative HPLC

The preparative HPLC (Shimadzu, Kyoto, Japan) system consisted of an LC-8A high-
pressure pump with an SPD-20A dual-wavelength detector (with preparative cell), FRC-10A,
and fraction collector. The system was connected to a PC using a CBM20A command module
and controlled by the LabSolution 1.24 SPI software suite supplied with the instrument.

3.6. Mass Spectrometry (MS)

The samples were dissolved in MeOH and introduced into the mobile phase flow
(MeOH/H2O 4:1; 100 µL/min) using a 2 µL loop. Spray voltage, capillary voltage, tube lens
voltage, and capillary temperature were 4.0 kV, −16 V, −120 V, and 275 ◦ C, respectively.

3.7. Nuclear Magnetic Resonance (NMR)

NMR spectra were recorded on a Bruker Avance III 600 MHz and 400 MHz spectrome-
ters at 30 ◦ C in dimethylsulfoxide (DMSO-d6 ); residual solvent signal (δH 2.499 ppm, δC
39.46 ppm) served as an internal standard. NMR experiments: 1 H NMR, 13 C NMR, gCOSY,
gHSQC, and gHMBC were performed using the standard manufacturer’s software. The
position of sulfate attachment was determined using typical changes in chemical shifts of
the attached and adjacent carbons (compared with starting acceptors) as described in [11].

3.8. Analytical HPLC System

The Shimadzu Prominence LC analytical system comprised Shimadzu CBM-20A
system controller, Shimadzu LC-20AD binary HPLC pump, Shimadzu CTO-10AS column
oven, Shimadzu SIL-20ACHT cooling autosampler, and Shimadzu SPD-20MA diode array
detector (Shimadzu, Kyoto, Japan).
Int. J. Mol. Sci. 2022, 23, 5743 14 of 17

3.9. Analytical Columns and Mobile Phases

Kinetex PFP column (150 × 4.6 mm, 5 µm), guard column PFP (4 × 3 mm, 5 µm),
both Phenomenex (USA); Method M1: mobile phase A = 10 mM ammonium acetate, 0.1%
HCOOH, pH 3.8; mobile phase B = 100% MeOH; gradient elution: 0 min 40% B, 0–20 min
40–72% B, 20–21 min 72–40% B, 21–24 min 40% for equilibration of the column; flow
rate 0.6 mL/min, 45 ◦ C, PDA detection (200–400 nm), the wavelength of the absorption
maximum of the respective compound was extracted. The method with the same mobile
phases, flow rate, temperature, and detection but a different gradient was used for the
analysis of phenolic acids PRO, THB, CAF, and also for CAT, MeCAT, PG, respectively.
(Method M2): 0 min 20% B, 0–20 min 20–50% B, 20–21 min 50–20% B, 21–24 min 20% B for
equilibration of the column. Another method (Method M3) that has also been tested with
this column: mobile phase A = 0.1% TFA (pH = 2.0), B = 100% MeOH; gradient elution
0 min 40% B, 0–25 min 40–80% B, 25–26 min 80–40% B, 26–28 min 40% for equilibration of
the column; flow rate 0.6 mL/min, 45 ◦ C.
ZicpHILIC column (100 × 2.1 mm, 5 µm), guard column ZicHILIC (20 × 2.1 mm,
5 µm), both Merck (DE); Method M4: mobile phase A = 100% acetonitrile, 0.1% HCOOH;
mobile phase B = 10 mM ammonium acetate, 0.1% HCOOH, pH 3.8; gradient elution:
0 min 5% B, 0–7.5 min 5–20% B, 7.5–10 min 20% B, 10–12 min 20–5% B, 12–15 min 5% B,
15–17 5% B for equilibration of the column; flow rate 0.4 mL/min, 25 ◦ C, PDA detection
(200–400 nm), the wavelength of the absorption maximum of the respective compound
was extracted.
Chromolith RP 18e column (100 × 3 mm, monolith), guard column Chromolith RP
18-e (5 × 4.6 mm, monolitith), both Merck (DE); Method M5: mobile phase A = 10 mM
ammonium acetate, 0.1% HCOOH, pH 3.8, mobile phase B = 100% MeOH; gradient elution:
0–2 min 5% B, 2–7 min 5–90% B, 7–8 min 90%B, 8–11 min 90–5% B, 11–14 min 5% B for
equilibration of the column; flow rate 1 mL/min, 25 ◦ C, PDA detection (200–400 nm),
the wavelength of the absorption maximum of the respective compound was extracted.
Another method (Method M6) has also been tested with this column: mobile phase A = 5%
acetonitrile, 0.1% HCOOH, B = 80% acetonitrile, 0.1% HCOOH; gradient elution 0–5 min
0–30% B, 5–7 min 30–0% B, 7–9 min 0% B for equilibration of the column; flow rate
1.0 mL/min, 25 ◦ C.
Luna Omega Polar C18 column (100 × 2.1 mm, 3 µm), guard column Polar C18 for
2.1 mm ID, both Phenomenex (USA); Method M7: mobile phase A = 5% acetonitrile, 0.1%
HCOOH, mobile phase B = 80% acetonitrile, 0.1% HCOOH; gradient elution: 0–7 min
0–90% B, 7–8 min 90% B, 8–11 min 90–0% B, 11–14 min 0% B for equilibration of the column;
flow rate 0.4 mL/min, 45 ◦ C, PDA detection (200–400 nm), the wavelength of the absorption
maximum of the respective compound was extracted.
All of the above methods, where 10 mM ammonium acetate was used in the mobile
phase, were also tested with 5 mM ammonium acetate under the same conditions. An
overview of the methods used is in Table 2.

3.10. Sample Preparation

Samples of individual sulfates were dissolved in water or parent compounds in
MeOH/water (1/1, v/v) (1 mg/mL), centrifugated, and injected (1 µL). The reaction mix-
tures (10 µL) were diluted by water (50 µL), centrifuged, and injected (1 µL).

3.11. Method Validation

Four representatives from the group of phenol, phenolic acid, flavonoid, and flavono-
lignan sulfates, namely 4-methylcatechol sulfate (MeCAT-S), caffeic acid sulfate (CAF-S),
ampelopsin sulfate (AMP-S), and silychristin sulfate (SCH-S), were selected for validation
on the PFP column (the most universal column for all samples tested, Method M1, see
Table 2).
Int. J. Mol. Sci. 2022, 23, 5743 15 of 17

3.11.1. Linearity
Linearity was evaluated by measuring five concentrations of each analyte in duplicates.
The concentrations of the analytes prepared in the volumetric flasks were 50, 25, 12.5, 6.25,
and 0.625 mM. The results were examined for a linear relationship by plotting the peak
area and the corresponding concentrations of the analyte, followed by linear least squares
regression and calculation of the slope and correlation coefficient.

3.11.2. Limit of Detection (LOD) and Limit of Quantification (LOQ)

The values of LOQ and LOD were determined according to the following equations:
LOD = 3 × noise/slope of the corresponding calibration curve, LOQ = 10 × noise/slope of
the corresponding calibration curve.

3.11.3. Precision and Repeatability

Intra-day precision (repeatability) and inter-day precision (intermediate precision)
were calculated by analyzing four selected samples at the concentration of 12.5 mM. Results
were expressed as the relative standard derivation (RSD). Repeatability measurement was
performed on the same day in six replicates; the intermediate precision was measured on
six different days.

4. Conclusions
We have developed a robust HPLC analytical method suitable for the separation of
enzymatic sulfation reaction mixtures of flavonoids, dehydroflavonoids, phenolic acids,
and catechols with PDA detection. This method is based on the combination of pentafluo-
rophenyl stationary phase and 10 mM acetate buffer/methanol in gradient elution. More-
over, the low flow rate (0.6 mL/min) and the absence of phosphate buffer and/or ion-
pairing reagents in the mobile phase make the method directly applicable in combination
with mass detection. Last but not least, four authentic standards of 2,3,4-trihydroxybenzoic
acid sulfates, catechol sulfate, 4-methylcatechol sulfate, and phloroglucinol sulfate were
prepared in this work.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10

.3390/ijms23105743/s1.
Author Contributions: Conceptualization, L.P., V.K. and K.V.; data curation, K.K.; formal analysis,
L.P., A.H. and H.P.; funding acquisition, L.P. and K.V.; investigation, L.P., K.K., K.B., A.H., B.P. and
H.P.; methodology, L.P.; project administration, K.V.; resources, L.P.; supervision, L.P., V.K. and K.V.;
validation, L.P.; visualization, A.H.; writing—original draft, L.P.; writing—review and editing, L.P.,
K.K., K.B., A.H., B.P., H.P., V.K. and K.V. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the Czech Science Foundation, grant number 19-00043S, and
Czech Health Research Council, grant number NU21-02-00135.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in the article or supple-
mentary material.
Conflicts of Interest: The authors declare no conflict of interest.
Int. J. Mol. Sci. 2022, 23, 5743 16 of 17

Abbreviations

AMP ampelopsin
AMP-S ampelopsin-40 -O-sulfate
CAF caffeic acid
CAF-S caffeic acid 3- and 4-O-sulfate (69:31)
CAT catechol
CAT-S catechol-O-sulfate
DHSB 2,3-dehydrosilybin
DHSB-S 2,3-dehydrosilybin-20-O-sulfate
DHSB-SS 2,3-dehydrosilybin-7,20-di-O-sulfate
DHSCH 2,3-dehydrosilychristin
DHSCH-S 2,3-dehydrosilychristin-19-O-sulfate
ISQ isoquercitrin
ISQ-S isoquercitrin-40 -O-sulfate
LUT luteolin
LUT-S luteolin-30 -O-sulfate
LUT-SS luteolin-7,30 - and 7, 40 -di-O-sulfates (82:12)
MeCAT 4-methylcatechol
MeCAT-S 4-methylcatechol-1- and 2-O-sulfate (64:36)
MYR myricetin
MYR-S myricetin-40 -O-sulfate
MYR-SS myricetin-7,40 -di-O-sulfate
p-NP p-nitrophenol
p-NPS p-nitrophenyl sulfate
PG phloroglucinol
PG-S phloroglucinol-O-sulfate
PRO protocatechuic acid
PRO-S protocatechuic acid 3- and 4-O-sulfates (70:30)
QUE quercetin
QUE-S quercetin-30 - and 40 -O-sulfate (75:25)
QUE-SS quercetin- 30 ,40 -, 7,30 - and 7, 40 -di-O-sulfate
RUT rutin
RUT-S rutin-40 -O-sulfate
SB silybin A and B (50:50)
SB-S silybin A-20-O-sulfate and silybin B-20-O-sulfate (50:50)
SCH silychristin A and B (90:10)
SCH-S silychristin-19-O-sulfate
THB 2,3,4-trihydroxybenzoic acid
TX taxifolin
TX-S taxifolin-40 - and 30 -O-sulfate (80:20)

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